U.S. patent application number 13/714921 was filed with the patent office on 2014-01-09 for isolation and use of solid tumor stem cells.
This patent application is currently assigned to The Regents of the University of Michigan. The applicant listed for this patent is Muhammad Al-Hajj, Michael F. Clarke, Sean J. Morrison, Max S. Wicha. Invention is credited to Muhammad Al-Hajj, Michael F. Clarke, Sean J. Morrison, Max S. Wicha.
Application Number | 20140011274 13/714921 |
Document ID | / |
Family ID | 46328681 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011274 |
Kind Code |
A1 |
Clarke; Michael F. ; et
al. |
January 9, 2014 |
ISOLATION AND USE OF SOLID TUMOR STEM CELLS
Abstract
A small percentage of cells within an established solid tumor
have the properties of stem cells. These solid tumor stem cells
give rise to both more tumor stem cells and to the majority of
cells in the tumor that have lost the capacity for extensive
proliferation and the ability to give rise to new tumors. Thus,
solid tumor heterogeneity reflects the presence of tumor cell
progeny arising from a solid tumor stem cell. We have developed a
xenograft model in which we have been able to establish tumors from
primary tumors via injection of tumors in the mammary gland of
severely immunodeficient mice. These xenograft assay have allowed
us to do biological and molecular assays to characterize clonogenic
solid tumor stem cells. We have also developed evidence that
strongly implicates the Notch pathway, especially Notch 4, as
playing a central pathway in carcinogenesis.
Inventors: |
Clarke; Michael F.; (Palo
Alto, CA) ; Morrison; Sean J.; (Ann Arbor, MI)
; Wicha; Max S.; (Ann Arbor, MI) ; Al-Hajj;
Muhammad; (Eagleville, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Clarke; Michael F.
Morrison; Sean J.
Wicha; Max S.
Al-Hajj; Muhammad |
Palo Alto
Ann Arbor
Ann Arbor
Eagleville |
CA
MI
MI
MI |
US
US
US
US |
|
|
Assignee: |
The Regents of the University of
Michigan
Ann Arbor
MI
|
Family ID: |
46328681 |
Appl. No.: |
13/714921 |
Filed: |
December 14, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12979991 |
Dec 28, 2010 |
8357491 |
|
|
13714921 |
|
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|
11788489 |
Apr 20, 2007 |
|
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|
12979991 |
|
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|
11529869 |
Sep 29, 2006 |
7850961 |
|
|
11788489 |
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|
10343692 |
Aug 25, 2003 |
7115360 |
|
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PCT/US01/24243 |
Aug 2, 2001 |
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11529869 |
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Current U.S.
Class: |
435/366 |
Current CPC
Class: |
C12N 5/0695 20130101;
C12N 2503/00 20130101; C12N 5/0693 20130101; G01N 33/57492
20130101; G01N 33/5011 20130101 |
Class at
Publication: |
435/366 |
International
Class: |
C12N 5/09 20060101
C12N005/09 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under
CA075136 awarded by the National Institute of Health. The
government has certain rights in the invention.
Claims
1-44. (canceled)
45. An enriched population of human solid tumor stem cells obtained
from a solid tumor of epithelial origin, wherein the solid tumor
stem cells: (a) express CD44; (b) are enriched at least two-fold
compared to unfractionated tumor cells; and (c) are tumorigenic;
wherein the solid tumor is a primary tumor or a xenograft tumor
established from a primary tumor.
46. The enriched population of claim 45, wherein the population is
enriched at least 5-fold.
47. The enriched population of claim 45, wherein the population is
enriched at least 10-fold.
48. The enriched population of claim 45, wherein the solid tumor
stem cells further express epithelial specific antigen (ESA).
49. The enriched population of claim 45, wherein the solid tumor
stem cells are from a solid tumor selected from the group
consisting of colon carcinoma, pancreatic cancer, breast cancer,
ovarian cancer, prostate cancer, squamous cell carcinoma, cervical
cancer, lung carcinoma, small cell lung carcinoma, bladder
carcinoma, and melanoma.
50. A cellular composition comprising the enriched population of
human solid tumor stem cells of claim 45.
51. A method of enriching for a population of solid tumor stem
cells, the method comprising: (a) obtaining an initial mixture of
solid tumor stem cells and solid tumor cells from a human solid
tumor of epithelial origin, wherein the solid tumor is a primary
tumor or a xenograft tumor established from a primary tumor; and
(b) separating a fraction of solid tumor stem cells from the
mixture of solid tumor stem cells and solid tumor cells, wherein
the solid tumor stem cells: (i) express CD44; (ii) are enriched at
least two-fold compared to the initial unfractionated mixture of
solid tumor stem cells and solid tumor cells; and (iii) are
tumorigenic.
52. A method of enriching for a population of CD44+solid tumor stem
cells, the method comprising: (a) dissociating a human solid tumor
into an initial mixture of solid tumor stem cells and solid tumor
cells, wherein the solid tumor is a primary tumor or a xenograft
tumor established from a primary tumor; (b) contacting the
dissociated cells with a first reagent that binds CD44; and (c)
selecting cells that bind to the first reagent, wherein the
selected cells are: (i) CD44+; (ii) enriched at least two-fold
compared to the initial unfractionated mixture of tumor cells; and
(iii) tumorigenic.
53. The method of claim 52, wherein the first reagent is an
antibody.
54. The method of claim 52, wherein the first reagent is conjugated
to a fluorochrome or magnetic particles.
55. The method of claim 52, wherein the selecting is performed by
flow cytometry, fluorescence activated cell sorting (FACS) sorting,
panning, affinity chromatography, or magnetic selection.
56. The method of claim 52, wherein the dissociated cells are
contacted with at least 1 additional reagent, at least 2 additional
reagents, at least 3 additional reagents, or at least 5 additional
reagents.
57. The method of claim 52, wherein the additional reagent(s) binds
human ESA, CD3, CD2, CD10, CD14, CD16, CD31, CD45, CD140b, CD64,
CD24, and/or a mouse cell marker.
58. The method of claim 52, wherein the solid tumor is selected
from the group consisting of colon carcinoma, pancreatic cancer,
breast cancer, ovarian cancer, prostate cancer, squamous cell
carcinoma, cervical cancer, lung carcinoma, small cell lung
carcinoma, bladder carcinoma, and melanoma.
59. A method of obtaining a cellular composition comprising an
enriched population of human CD44+CD24-/low solid tumor stem cells,
the method comprising: (a) obtaining an initial mixture of solid
tumor stem cells and solid tumor cells from a human breast,
ovarian, or prostate solid tumor, wherein the solid tumor is a
primary tumor or a xenograft tumor established from a primary
tumor; (b) separating a fraction of solid tumor stem cells from the
mixture of solid tumor stem cells and solid tumor cells, wherein
the solid tumor stem cells are: (i) CD44+; (ii) CD24-/low; (iii)
enriched at least two-fold compared to the initial unfractionated
mixture of solid tumor stem cells and solid tumor cells; and (iv)
tumorigenic.
60. The method of claim 59, wherein the separating is performed by
flow cytometry, fluorescence activated cell sorting (FACS),
panning, affinity chromatography, or magnetic selection.
61. The method of claim 59, further comprising: (c) isolating the
separated solid tumor stem cells.
62. The method of claim 59, wherein the solid tumor stem cells are
enriched at least five to six-fold compared to the initial
unfractionated mixture.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation of U.S.
application Ser. No. 12/979,991, filed Dec. 28, 2010, which is a
Continuation of U.S. application Ser. No. 11/788,489, filed Apr.
20, 2007, now abandoned, which is a Continuation of U.S.
application Ser. No. 11/529,869, filed Sep. 29, 2006, which is now
U.S. Pat. No. 7,850,961, which is a Continuation of U.S.
application Ser. No. 10/343,692, filed Aug. 25, 2003, now U.S. Pat.
No. 7,115,360, which is a U.S. National Entry of International
Application PCT/US01/24243 filed Aug. 2, 2001, is a Continuation of
U.S. application Ser. No. 09/920,517 filed Aug. 1, 2001, now U.S.
Pat. No. 6,984,522, which claims priority to U.S. Provisional
Application 60/240,317, filed Oct. 13, 2000 and U.S. Provisional
Application 60/222,794, filed Aug. 3, 2000, all of which are herein
incorporated by reference.
TECHNICAL FIELD
[0003] This invention relates to the diagnosis and treatment of
cancer.
BACKGROUND ART
[0004] Cancer remains the number two cause of mortality in this
country, resulting in over 500,000 deaths per year. Despite
advances in detection and treatment, cancer mortality remains high.
Despite the remarkable progress in understanding the molecular
basis of cancer, this knowledge has not yet been translated into
effective therapeutic strategies.
[0005] In particular, breast cancer is the most common cancer in
American women, with approximately one in nine women developing
breast cancer in their lifetime. Unfortunately, metastatic breast
cancer is still an incurable disease. Most women with metastatic
breast cancer succumb to the disease.
[0006] Traditional modes of therapy (radiation therapy,
chemotherapy, and hormonal therapy), while useful, have been
limited by the emergence of treatment-resistant cancer cells.
Clearly, new approaches are needed to identify targets for treating
metastatic breast cancer and cancer generally.
DISCLOSURE OF THE INVENTION
[0007] The invention is based upon the discovery that a small
percentage of cells within an established solid tumor have the
properties of "stem cells". These solid tumor "stem" cells give
rise both to more solid tumor stem cells and to the majority of
cells in the tumor, cancer cells that have lost the capacity for
extensive proliferation and the ability to give rise to new tumors.
Thus, solid tumor cell heterogeneity reflects the presence of a
variety of tumor cell types that arise from a solid tumor stem
cell.
[0008] The previous failure of cancer therapies to significantly
improve outcome has been due in part to the failure of these
therapies to target the solid tumor stem cells within a solid tumor
that have the capacity for extensive proliferation and the ability
to give rise to all other solid tumor cell types. This invention
provides a way that anti-cancer therapies can be directed, both
generally and now specifically directed, against the solid tumor
stem cells. The directed anti-cancer therapies of the invention
thus result in much more effective and durable therapeutic
responses.
[0009] By the methods of the invention, one can characterize the
phenotypically heterogeneous populations of cells within a solid
tumor. Populations of cells obtained from the solid tumor are
isolated and structurally characterized using Fluorescence
Activated Cell Sorting (FACS). In particular, one can identify,
isolate, and characterize a phenotypically distinct cell population
within a tumor having the stem cell properties of extensive
proliferation and the ability to give rise to all other tumor cell
types. Solid tumor stem cells are the truly tumorigenic cells that
are capable of re-establishing a tumor following treatment.
[0010] The invention provides in vivo and in vitro assays of solid
tumor stem cell function and cell function by the various
populations of cells isolated from a solid tumor. The invention
provides methods for using the various populations of cells
isolated from a solid tumor (such as a population of cells enriched
for solid tumor stem cells) to identify factors influencing solid
tumor stem cell proliferation, to analyze populations of cells
isolated from solid tumors for gene expression patterns or protein
expression patterns, to identify new anti-cancer drug targets, to
predict the sensitivity of tumors from individual patients to
existing anti-cancer treatment regimens, to model anti-cancer
treatment, to test new therapeutic compounds, to identify and test
new diagnostic markers, to treat tumors, to produce genetically
modified solid tumor stem cells, and to prepare cDNA libraries and
microarrays of polynucleotides and polypeptides from solid tumor
stem cells.
[0011] The invention provides a method for consistently growing
solid tumor cells in vivo. The invention also provides a method to
grow solid tumor cells that are in single cell suspension or in
small aggregates. Moreover, the invention provides a chimeric
animal (a xenograft model) in which tumors can be established from
solid tumor primary cells and in which the tumors derived from
these solid tumor cells can be tested. Furthermore, the invention
provides tumor banks large enough to perform substantial numbers of
bioassays derived from single solid tumor stem cells.
[0012] In its several aspects, the invention usefully provides
methods for screening for anti-cancer agents; for the testing of
anti-cancer therapies; for the development of drugs targeting novel
pathways; for the identification of new anti-cancer therapeutic
targets; the identification and diagnosis of malignant cells in
pathology specimens; for the testing and assaying of solid tumor
stem cell drug sensitivity; for the measurement of specific factors
that predict drug sensitivity; and for the screening of patients
(e.g., as an adjunct for mammography). The invention can be used as
a model to test patients' tumor sensitivity to known therapies; as
a model for identification of new therapeutic targets for cancer
treatment; as a system to establish a tumor bank for testing new
therapeutic agents for treatment of cancer; and as a system to
identify the tumorigenic cancer cells. Also, the invention provides
synergy between the methods of the invention and breast cancer
genomic databases, for an improved anti-cancer drug discovery.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIGS. 1A and 1B show two models of solid tumor
heterogeneity. In the classic model (FIG. 1A), mutations or
environmental differences cause tumor cells to adopt a variety of
different phenotypes. Environmentally determined differences in
phenotype, represented by white, green, and red cells, may be
reversible while mutationally determined changes in phenotype,
represented by purple cells, may not be reversible. Many cells with
a variety of different phenotypes are thought to have the potential
to proliferate extensively and form new tumors. The tumor stem cell
model (FIG. 1B) is distinguished by having only a minor population
of tumor cells that are tumorigenic. These tumor stem cells are
characterized by indefinite proliferative potential, the ability to
form new tumors, and the ability to give rise to heterogeneous
non-tumorigenic cancer cells that typically form the bulk of a
tumor.
[0014] FIGS. 2A and 2B are a set of FACS plots of breast cancer
tumor cells. Mice were implanted with primary breast cancer tumor
cells removed from two human patients. Resultant tumors were
removed from the mouse and single cell suspensions were made. Cells
were stained with anti-CD44-PE, anti-520C9-APC, anti-mouse H2K-FITC
(which stains infiltrating mouse cells) and Propidium Iodide (PI,
which stains dead cells). FIGS. 2A and 2B show FACS plots of tumor
cells obtained from mice implanted with primary breast cancer tumor
cells removed from human Patient (1) and human Patient (2),
respectively. Live, human CD44.sup.+ and human CD44.sup.- cells
were isolated and used for in vitro and in vivo studies.
[0015] FIGS. 3A-3C are a set of FACS plots showing the expression
of CD24 by malignant breast cells. Cells were isolated and stained
as described in FIG. 2. Mouse cells and dead cells were gated out
of the analysis. The FIGS. 3A, 3B and 3C show FACS plots of cells
from three breast cancer tumors are shown obtained from mice
implanted with Tumor(1), Tumor(2) and Tumor(3), respectively. Note
that cells from all three tumors have a similar phenotype.
[0016] FIG. 4 is a set of FACS plots showing an analysis of tumors
arising from the CD24.sup.- cell population from human breast
cancers. According to the solid tumor stem cell model, the
CD24.sup.- cells give rise to tumors that contain both CD24.sup.+
and CD24.sup.- cells. Accordingly, secondary transplants were
performed using B38.1.sup.+CD24.sup.- cells (FIG. 4A). The
resultant tumors were removed and the cells were re-analyzed with
respect to B38.1 and CD24 expression. As predicted by the stem cell
model, cells obtained from a tumor arising from transplanted
B38.1.sup.+CD24.sup.- cells were heterogeneous with respect to
expression of both B38.1 and CD24 (FIG. 4B). The marker expression
pattern of the cells isolated from the tumor initiated by the
B38.1.sup.+CD24.sup.- cells was similar to that of the original
tumor (FIG. 4).
[0017] FIG. 5 is a FACS plot showing an analysis of Notch 4
expression. Cells were isolated from a mouse xenograft tumor (see.
below) and stained with antibodies. Malignant cells were analyzed
for expression of B38.1 and Notch 4. Mouse cells and dead cells
were gated out of the analysis.
[0018] FIG. 6 shows the fractionation of breast cancer cells based
upon CD44 expression. Tumor T1 cells (FIG. 6A, FIG. 6C, and FIG.
6E) and Tumor 1'2 cells (FIG. 6B, FIG. 6D, and FIG. 6F) were
stained with anti-CD44-PE, anti-mouse H2K-FITC and the viability
dye 7AAD. Flow cytometry was used to isolate live, human (H2K-)
cells that were either CD44.sup.+ (FIG. 6C, FIG. 6D) or CD44.sup.-
(FIG. 6E, FIG. 6F). Dead cells (7AAD) were eliminated from all
analyses. FIG. 6A and FIG. 6B are dot plots of the unfractionated
T1 and T2 cells showing CD44 and H2K expression as indicated. Plots
showing the isolated CD44.sup.+ (FIG. 6C, FIG. 6D) and CD44.sup.-
(FIG. 6E, FIG. 6F) populations depict reanalyses of cells that had
been isolated by flow-cytometry. These cells were injected into the
mammary fat pads of mice to examine their tumorigenicity. TABLES 1
and 3 show that the CD44.sup.+ cells but not the CD44.sup.- cells
were tumorigenic.
[0019] FIG. 7 shows the isolation of tumorigenic cells. Flow
cytometry was used to isolate subpopulations of Tumor T1 (FIG. 7A,
FIG. 7D, and FIG. 7G), Tumor 2 (FIG. 7B, FIG. 7E, and FIG. 7F) or
Tumor T5 cells (FIG. 7C, FIG. 7F, and FIG. 7I) that were tested for
tumorigenicity in NOD/SCID mice. T1 and T2 cells had been passaged
once in NOD/SCID mice while T5 cells were obtained from material
that had been frozen immediately after removal from a patient.
Cells were stained with anti-B38.1-APC, anti-CD44-PE,
anti-CD24-FITC, anti-LINEAGE-Cytochrome, anti-mouse-H2K-Cytochrome
(T1 and T2 cells only), and 7AAD. Dead cells (7AAD), mouse cells
(H2K) and LINEAGE.sup.+ cells were eliminated from all analyses.
Each dot plot depicts the CD24 and C044 staining patterns of live
human B38.1.sup.+CD44.sup.+LINEAGE.sup.- cells. FIG. 7A, FIG. 7B,
and FIG. 7C show unfractionated tumor cells.
B38.1.sup.+CD44.sup.+LINEAGE.sup.- cells that were either
CD24.sup.-/lo (FIG. 7G, FIG. 7H, FIG. 7I) or CD24.sup.+. (FIG. 7D,
FIG. 7E, FIG. 7F) were isolated from these tumor cells by
flow-cytometry. FIGS. 7D-7I depict reanalyses of these sorted
populations, which were subsequently injected into the mammary fat
pads of NOD/SCID mice to test tumorigenicity. FIG. 7J shows a
representative tumor in a mouse at the
B38.1.sup.+CD44.sup.+CD24.sup.-/lo LINEAGE.sup.- injection site,
but not at the B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.-
injection site. Histology performed on tissue from the CD24.sup.+
(FIG. 7K, 20.times. objective magnification) and C024.flo (FIG. 7L,
40.times. objective magnification) injection sites exhibited normal
mouse tissue and malignant cells respectively.
[0020] FIG. 8 shows the enrichment of tumorigenic cells based upon
ESA expression. Flow cytometry was used to isolate subpopulations
of Tumor T1 cells that were tested for tumorigenicity in NOD/SCID
mice. T1 cells had been passaged once in NOD/SCID mice. Cells were
stained with anti-B38.1-APC, anti-CD24-PE, anti-ESA-FITC,
anti-LINEAGE-Cytochrome, anti-mouse-H2K-Cytochrome (T1), and 7AAD.
Dead cells (7AAD+, mouse cells (H2K+) and LINEAGE+ cells were
eliminated from the analysis.
[0021] FIG. 9 is the results of an in vitro clonogenic assay. Flow
cytometry was used to isolate tumorigenic cell or the rest of the
non-tumorigenic neoplastic (non-tumorigenic cells) as described.
The cells were placed in tissue culture medium containing soluble
Delta for the indicated number of days. The tumorigenic and
non-tumorigenic xenograft Tumor 1 (T1) (FIG. 9A), Tumor 4 (T4)
(FIG. 9B) or primary patient (FIG. 9C) cells are shown at the
indicated time after being placed in tissue culture. T4 cells were
stained with Papanicolaou stain and examined under light microscopy
(100.times. objective). Note that both the non-tumorigenic (FIG.
9D) and tumorigenic (FIG. 9E) populations consist of neoplastic
cells with large nuclei and prominent nucleoli. Note that the
number of cells that attached to the tissue culture plate is
similar in both populations, but that the tumorigenic population
always gave rise to colonies. Non-tumorigenic populations do not
give rise to established colonies (or only for brief periods, about
2-6 days).
[0022] FIG. 10 is a set of dot plots showing the phenotypic
diversity in tumors arising from B38.1.sup.+CD44.sup.+CD24.sup.-/lo
LINEAGE.sup.- cells. The dot plots depict the CD24 and CD44
staining patterns of live human LINEAGE.sup.- cells from Tumor T1
(FIG. 10A-FIG. 10C) or Tumor T2 (FIG. 10-FIG. 10F). FIG. 10A and
FIG. 10D show unfractionated T1 or T2 cells obtained from tumors
that had been passaged once in NOD/SCID mice.
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells from T1 (FIG.
10B) or T2 (FIG. 10E) were isolated as described in FIG. 2, above.
The B38.1.sup.+CD44.sup.+CD24.sup.-/lo LINEAGE.sup.- populations
reanalyzed in FIG. 10B and FIG. 10E were injected into the mammary
fat pads of NOD/SCID mice. FIG. 10C and FIG. 10F depict analyses of
the tumors that arose from these
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells. Note that in
both cases, the B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
cells formed tumors that contained a phenotypically diverse
population of cells similar to that observed in the original
tumor.
[0023] FIGS. 11A-11E show the expression of HER2/neu and EGF-R.
Flow cytometry was used to isolate subpopulations of Tumor T1 cells
that had been passaged once in NOD/SCID mice. Cells were stained
with, in FIG. 11A, anti-EGF-R-PE, anti-B38.1-APC, anti-CD24-FITC,
anti-LINEAGE-Cytochrome, anti-mouse-H2K-Cytochrome, and 7AAD or, in
FIG. 11B anti-HER2/neu-FITC, anti-B38.I-APC, anti-CD24-PE,
anti-LINEAGE-Cytochrome, anti-mouse-H2K-Cytochrome, and 7AAD. Dead
cells (7AAD.sup.+), mouse cells (H2K.sup.+) and LINEAGE.sup.+ cells
were eliminated from all analyses. The histogram in FIG. 11A
depicts the EGF-R expression of the unstained cells (dotted line),
B38.1.sup.+CD24.sup.- LINEAGE.sup.- tumorigenic population (shaded)
and the B38.1.sup.+CD24.sup.+LINEAGE.sup.- non-tumorigenic (solid
line) population. The histogram in FIG. 11B shows HER2/neu
expression of the unstained cells (dotted line),
B38.1.sup.+CD24.sup.- LINEAGE.sup.- tumorigenic population (shaded)
and the B38.sup.+CD24.sup.+LINEAGE.sup.- non-tumorigenic (solid
line) population. RT-PCR was performed using nested primers to
detect EGF-R (FIG. 11C and FIG. 11D) or to detect HER2/neu (FIG.
11E). One cell per sample in panels FIG. 11D and FIG. 11E, or ten
cells per sample in panel FIG. 11C, were analyzed. EGF-R is
expressed at lower levels in tumorigenic cells than in
non-tumorigenic cells at both the protein (FIG. 11A) and mRNA
levels (FIG. 11C, FIG. 11D).
[0024] FIG. 12 is a photomicrograph of breast cancer cells placed
in tissue culture after exposure to an anti-Notch 4 antibody. Cells
were incubated on ice for one hour in HBSS with or without soluble
Delta but no anti-Notch 4 antibody, with anti-Notch 4 antibody, or
with anti-Notch 4 antibody that had been preincubated with the
peptide used to generate the antibody. The number of colonies that
formed in the triplicate experiments is shown. Soluble Delta was
added to the culture. Fc-control medium without soluble Delta was
added to the culture. Symbols: Ab=the anti-Notch 4 antibody;
Block=the peptide used to generate the anti-Notch 4 antibody.
[0025] FIG. 13 is a schematic diagram of B38.1.sup.+ cells within a
tumor. The breast cancer stem cells from multiple patients are
B38.1.sup.+. To successfully treat a cancer with a gene therapy
approach, these cells can be targeted with a vector.
[0026] FIG. 14 is a description of the method for obtaining the
bi-specific conjugate and the chemical modifications introduced in
the antibodies.
[0027] FIG. 15 is a strategy for re-targeting Adenovirus. The LaZ
virus can infect most of the cells from a tumor. After the LaZ
virus is incubated only with the anti-fiber antibody, the LaZ virus
loses ability to infect all of the cells. After the LaZ virus is
incubated with the bi-specific conjugate, the B38.1 moiety of the
molecule allows the attachment of the virus to the B38.1.sup.+
cells, so only these cells are infected.
[0028] FIG. 16 shows the targeting of breast cancer stem cells with
the bi-specific antibody. Different cell lines were infected with
AdLaZ, which is an El-deleted Adenovirus that expresses the
.beta.-galactosidase gene (gray columns, control for virus
infection). In some cases, the virus was incubated with the
anti-fiber antibody for 30 min before infection (yellow columns).
In other cases, the virus was incubated with the bi-specific
conjugate (green columns). After 24 hr of infection, the monolayers
were fixed and incubated with X-Gal-contained buffer. The infected
cells are blue, and the graphic shows the percentage of blue cells
obtained, relative to the control infection (i.e., the reduction or
increase in infectivity of the virus after incubation with the
different antibodies).
[0029] FIG. 17 shows that the bispecific antibody can target an
adenovirus vector to breast cancer stem cells. The columns
represent the absolute number of infected cells per field. Gray:
the indicated cells infected with the control adenovirus. Yellow:
the indicated cells were infected with the adenovirus that had been
incubated with the anti-fiber antibody. Green: the indicated cells
were incubated with the bi-specific conjugate antibody. Red: cells
were infected with the virus that had been incubated with the
bi-specific conjugate antibody, but the cells were pre-treated with
an excess of B38.1 antibody.
[0030] FIG. 18 is a photograph of some of the cell monolayers after
X-Gal staining. The infected cells appear like dark dots in this
black and white picture (the .beta.-galactosidase gene of the LaZ
virus has a nuclear localization signal. The staining is in the
nuclei of the cells.
[0031] FIG. 19 is an analysis of different populations of cells in
a breast cancer. ESA.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
cells breast cancer stem cells (FIG. 19A) and
ESA.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- non-tumorigenic cells
(FIG. 19B) were obtained as described in FIG. 8. The cells were
stained with Hoechst 33342 as described by Eaves and colleagues
(Glimm H et al., Blood. 96(13): 4185-93 (2000)). The histogram for
the breast cancer stem cells is shaded. Note that the breast cancer
stem cells and the non-tumorigenic cells are distributed in all
phases of the cells cycle.
[0032] FIG. 20 is a further analysis of different populations of
cells in a breast cancer. CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
cells breast cancer stem cells and non-tumorigenic
CD44.sup.+CD24.sup.+ LINEAGE.sup.- non-tumorigenic cells were
obtained as described in FIG. 7. The cells were stained with
Rhodamine 123 as described by Spangrude et al., Blood
85(4):1006-16, 1995. The histogram for the breast cancer stem cells
is shaded. Note that the breast cancer stem cells tend to stain
less intensely with Rhodamine 123.
[0033] FIG. 21 is an analysis of as cites fluid for ovarian cancer
stem cells. Cells were stained with anti-B38.1-APC, anti-CD44-PE,
anti-CD24-FITC, anti-Lineage-Cytochrome and 7AAD. Dead cells
(7AAD.sup.+), and LINEAGE.sup.- cells were eliminated from the
analyses. Note that there is a distinct
CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population of cells that
resembles the breast cancer stem cells.
[0034] FIG. 22 is an analysis of sarcoma cells for solid tumor stem
cells. P1 sarcoma cells growing in the xenograft model were stained
with anti-B38.1-APC, anti-CD44-PE, anti-CD24FITC,
anti-LINEAGE-Cytochrome, anti-H2K-Cytochrome and 7AAD. Dead cells
(7AAD.sup.+), LINEAGE.sup.+ cells and mouse cells were eliminated
from the analyses. Note that the lineage cocktail in this analysis
did not include CD10, CD31 or CD140b. Also note that there is a
distinct CD44.sup.+CD24.sup.-/lo LINEAGE.sup.- population of
cells.
MODES FOR CARRYING OUT THE INVENTION
[0035] Stem Cells and Solid Tumor Heterogeneity Models.
[0036] Solid tumors are composed of heterogeneous cell populations.
For example, breast cancers are a mixture of cancer cells and
normal cells, including mesenchymal (stromal) cells, inflammatory
cells, and endothelial cells. Classic models hold that
phenotypically distinct cancer cell populations all have the
capacity to proliferate and give rise to a new tumor (FIG. 1A). In
the classical model, tumor cell heterogeneity results from
environmental factors as well as ongoing mutation within cancer
cells resulting in diverse populations of tumorigenic cells and all
populations of cells would have similar tumorigenic potential.
(Pandis et al, Genes, Chromosomes &Cancer 12: 122-129 (1998);
Kuukasjarvi et al., Cancer Res. 57: 1597-1604 (1997); Bonsing et
al., Cancer 71: 382-391 (1993); Bonsing et al., Genes Chromosomes
& Cancer 82: 173-183 (2000); Beennan H et al., Cytometry 12(2):
147-54 (1991); Aubele M & Werner M, Analyt. Cell. Path. 19: 53
(1999); Shen L et al., Cancer Res. 60: 3884 (2000).).
[0037] This invention is based upon an alternative model of solid
tumor cell heterogeneity, in which a solid tumor results from a
"solid tumor stem cell" (or "cancer stem cell" from a solid tumor)
and the subsequent chaotic development of the solid tumor stem
cell. In this stem cell model (FIG. 1B), solid tumors contain a
distinct, limited (or possibly rare) subset of cells that share the
properties of normal "stem cells", in that they proliferate
extensively or indefinitely and that they efficiently give rise to
additional solid tumor stem cells. Within an established solid
tumor, most cells have lost the ability to proliferate extensively
and form new tumors, but solid tumor stem cells proliferate
extensively and give rise to additional solid tumor stem cells as
well as to other tumor cells that lack tumorigenic potential. It is
this solid tumor stem cell population that proliferates and
ultimately proves fatal.
[0038] To distinguish between these models, the deficiencies of the
previous clonogenic assays (see, below) must be overcome. To prove
the existence of a consistent stem cell population rather that a
constant low probability of tumorigenicity in any cell type, one
must be able to purify the stem cells and show that they are highly
enriched for tumorigenicity, while the remainder of the neoplastic
cells are depleted of such activity. The invention provides this
ability.
[0039] The ability to isolate and analyze cell populations within a
solid tumor, based upon structural features of the solid tumor stem
cells, described herein, allows one skilled in the art of oncology
or stem cell biology to distinguish between the two models shown in
FIG. 1. By this invention, solid tumor stem cells and cell
populations from solid tumors have been isolated and analyzed.
Moreover, these solid tumor stem cells have very high or unlimited
proliferative potential, and thus represent the truly tumorigenic
population. According to the solid tumor stem cell model and the
results provided below (see, EXAMPLES), these tumorigenic cells are
the clonogenic cells of solid tumors.
[0040] During normal animal development, cells of most or all
tissues are derived from normal precursors, called stem cells
(Morrison et al., Cell 88(3): 287-98 (1997); Morrison et al., Curr.
Opin. Immunol. 9(2): 216-21 (1997); Morrison et al., Annu. Rev.
Cell. Dev. Bioi. 11: 35-71 (1995)). The term "stem cell" is known
in the art to mean (1) that the cell is a cell capable of
generating one or more kinds of progeny with reduced proliferative
or developmental potential; (2) that the cell has extensive
proliferative capacity; and (3) that the cell is capable of
self-renewal or self-maintenance (see, Potten et al., Development
110: 1001 (1990); U.S. Pat. Nos. 5,750,376, 5,851,832, 5,753,506,
5,589,376, 5,824,489, 5,654,183, 5,693,482, 5,672,499, and
5,849,553, all incorporated by reference). In adult animals, some
cells (including cells of the blood, gut, breast ductal system, and
skin) are constantly replenished from a small population of stem
cells in each tissue. Thus, the maintenance of tissues (whether
during normal life or in response to injury and disease) depends
upon the replenishing of the tissues from precursor cells in
response to specific developmental signals.
[0041] The best-known example of adult cell renewal by the
differentiation of stem cells is the hematopoietic system (see,
U.S. Pat. Nos. 5,061,620, 5,087,570, 5,643,741, 5,821,108,
5,914,108, each incorporated by reference). Developmentally
immature precursors (hematopoietic stem and progenitor cells)
respond to molecular signals to gradually form the varied blood and
lymphoid cell types. Stem cells are also found in other tissues,
including epithelial tissues (see, Slack, Science 287: 1431 (2000))
and mesenchymal tissues. (see, U.S. Pat. No. 5,942,225;
incorporated by reference). In normal breast development, a normal
stem cell gives rise to differentiated progeny to form a normal
ductal system. Kordon & Smith, Development 125: 1921-1930
(1998); see also, U.S. Pat. Nos. 5,814,511 and 5,650,317.
[0042] By this invention, the principles of normal stem cell
biology have been applied to isolate and characterize solid tumor
stem cells. Examples of solid tumors from which solid tumor stem
cells can be isolated or enriched for according to the invention
include sarcomas and carcinomas such as, but not limited to:
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma,
mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma,
colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer,
prostate cancer, squamous cell carcinoma, basal cell carcinoma,
adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma,
papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma,
medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma,
hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, meningioma, melanoma, neuroblastoma, and
retinoblastoma. The invention is applicable to sarcomas (see, FIG.
22) and epithelial cancers, such as ovarian cancers (see, FIG. 21)
and breast cancers (see, EXAMPLES).
[0043] Solid tumor stem cells are defined structurally and
functionally as described herein; using the methods and assays
similar to those described below. Because tumor cells are known to
evolve phenotypically and functionally over time as additional
genetic mutations occur, the solid tumor stem cells may change
phenotypically and functionally over time in an individual patient.
Nevertheless, one can use the method of the invention, employing
the markers disclosed herein, which are consistently useful in the
isolation or identification of solid tumor stem cells in a majority
of patients.
[0044] Also, solid tumor stem cells undergo "self-renewal" and
"differentiation" in a chaotic development to form a tumor, give
rise to abnormal cell types, and may change over time as additional
mutations occur. The functional features of a solid tumor stem cell
are that they are tumorigenic, they give rise to additional
tumorigenic cells ("self-renew"), and they can give rise to
non-tumorigenic tumor cells ("differentiation").
[0045] The developmental origin of solid tumor stem cells can vary
between different types of solid tumor cancers. Solid tumor stem
cells may arise either as a result of genetic damage that
deregulates the proliferation and differentiation of normal stem
cells (Lapidot et al., Nature 367(6464): 645-8 (1994)) or by the
dysregulated proliferation of a normal restricted progenitor or a
normal differentiated cell type. Typically, solid tumors are
visualized and initially identified according to their locations,
not by their developmental origin.
[0046] By contrast, a non-tumorigenic cell from a solid tumor is a
cell from a population that fails to form a palpable tumor upon
transplantation into an immunocompromised mouse, wherein if the
same number of unfractionated, dissociated tumor cells were
transplanted under the same circumstances, the solid tumor stem
cells would form a palpable tumor in the same period of time. Thus
non-tumorigenic cells are depleted for tumor forming activity in an
animal model.
[0047] A "palpable tumor" is known to those in the medical arts as
a tumor that is capable of being handled, touched, or felt.
[0048] Because the tumorigenic changes are intrinsic to solid tumor
stem cells, even after they have been removed from their normal
environment within the tumor, the invention provides several novel
uses:
[0049] (1) by identifying the genes and proteins expressed by solid
tumor stem cells it is possible to identify proteins whose function
is necessary for tumorigenesis and which represent novel drug
targets;
[0050] (2) by purifying solid tumor stem cells based on phenotypic
markers it is possible to study their gene expression patterns and
functions much more directly and efficiently;
[0051] (3) by developing in vitro and in vivo assays of solid tumor
stem cell function it is possible `to more effectively test the
effects of potential therapeutic compounds;
[0052] (4) by identifying markers of solid tumor stem cells it is
possible to more effectively diagnose the presence of malignant
cells (even those that do not depend on rare environmental
characteristics for their ability to make tumors); and
[0053] (5) by isolating solid tumor stem cells from individual
patients and transplanting them into in vitro and in vivo
functional assays it is possible to test the effectiveness of
different drug regimens against them. Thus, it is possible to
predict drug sensitivity and drug resistance.
[0054] The solid tumor stem cells of the model of the invention
differs from the "cancer stem line" provided by U.S. Pat. No.
6,004,528. In that patent, the "cancer stem line" is defined as a
slow growing progenitor cell type that itself has few mutations but
which undergoes symmetric rather than asymmetric cell divisions as
a result of tumorigenic changes that occur in the cell's
environment. This "cancer stem line" hypothesis thus proposes that
highly mutated, rapidly proliferating tumor cells arise largely as
a result of an abnormal environment, which causes relatively normal
stem cells to accumulate and then undergo mutations that cause them
to become tumor cells. U.S. Pat. No. 6,004,528 proposes that such a
model can be used to enhance the diagnosis of cancer. The solid
tumor stem cell model is fundamentally different than the "cancer
stem line" model and as a result exhibits utilities not offered by
the "cancer stem line" model. First, solid tumor stem cells are not
"mutationally spared". The "mutationally spared cancer stem line"
described by U.S. Pat. No. 6,004,528 may be considered a
pre-cancerous lesion, while the solid tumor stem cells described of
this invention are cancer cells that themselves contain the
mutations that are responsible for tumorigenesis. That is, the
solid tumor stem cells ("cancer stem cells") of the invention would
be included among the highly mutated cells that are distinguished
from the "cancer stem line" in U.S. Pat. No. 6,004,528. Second, the
genetic mutations that lead to cancer are largely intrinsic within
the solid tumor stem cells rather than being environmental. The
solid tumor stem cell model predicts that isolated solid tumor stem
cells can give rise to additional tumors upon transplantation (thus
explaining metastasis) while the "cancer stem line" model would
predict that transplanted "cancer stem line" cells would not be
able to give rise to a new tumor, since it was their abnormal
environment that was tumorigenic. Indeed, the ability to transplant
dissociated, and phenotypically isolated human solid tumor stem
cells to mice (into an environment that is very different from the
normal tumor environment), where they still form new tumors,
distinguishes the present invention from the "cancer stem line"
model. Third, solid tumor stem cells likely divide both
symmetrically and asymmetrically, such that symmetric cell division
is not an obligate property. Fourth, solid tumor stem cells may
divide rapidly or slowly, depending on many variables, such that a
slow proliferation rate is not a defining characteristic.
[0055] As described above, solid tumor stem cells can be
operationally characterized by cell surface markers. These cell
surface markers can be recognized by reagents that specifically
bind to the cell surface markers. For example, proteins,
carbohydrates, or lipids on the surfaces of solid tumor stem cells
can be immunologically recognized by antibodies specific for the
particular protein or carbohydrate (for construction and use of
antibodies to markers, see, Harlow, Using Antibodies: A Laboratory
Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1999);
see also, EXAMPLES). The set of markers present on the cell
surfaces of solid tumor stem cells (the "cancer stem cells" of the
invention) and absent from the cell surfaces of these cells is
characteristic for solid tumor stem cells. Therefore, solid tumor
stem cells can be selected by positive and negative selection of
cell surface markers. A reagent that binds to a solid tumor stem
cell is a "positive marker" (i.e., a marker present on the cell
surfaces of solid tumor stem cells) that can be used for the
positive selection of solid tumor stem cells. A reagent that binds
to a solid tumor stem cell "negative marker" (i.e., a marker not
present on the cell surfaces of solid tumor stem cells but present
on the surfaces of other cells obtained from solid tumors) can be
used for the elimination of those solid tumor cells in the
population that are not solid tumor stem cells (i.e., for the
elimination of cells that are not solid tumor stem cells).
[0056] In one embodiment, the discrimination between cells based
upon the detected expression of cell surface markers is by
comparing the detected expression of the cell surface marker as
compared with the mean expression by a control population of cells.
For example, the expression of a marker on a solid tumor stem cell
can be compared to the mean expression of the marker by the other
cells derived from the same tumor as the solid tumor stem cell.
Other methods of discriminating among cells by marker expression
include methods of gating cells by flow cytometry based upon marker
expression (see, Givan A, Flow Cytometry First Principles,
(Wiley-Liss, New York, 1992); Owens M A & Loken M R., Flow
Cytometry Principles for Clinical Laboratory Practice, (Wiley-Liss,
New York, 1995)).
[0057] Solid tumor stem cell positive markers may also be present
on cells other than solid tumor stem cells. Solid tumor stem cell
negative markers may also be absent from cells other than solid
tumor stem cells. While it is rare to identify a single marker that
identifies a stem cell, it has often been possible to identify
combinations of positive and negative markers that uniquely
identify stem cells and allow their substantial enrichment in other
contexts. Morrison et al., Cell 96(5): 737-49 (1999); Morrison et
al., Proc. Natl. Acad. Sci. USA 92(22): 10302-6 (1995); Morrison
& Weissman, Immunity 1(8): 661-73 (1994).
[0058] A "combination of reagents" is at least two reagents that
bind to cell surface markers either present (positive marker) or
not present (negative marker) on the surfaces of solid tumor stem
cells, or to a combination of positive and negative markers (see,
EXAMPLES 7 and 8, TABLE 6). The use of a combination of antibodies
specific for solid tumor stem cell surface markers results in the
method of the invention being useful for the isolation or
enrichment of solid tumor stem cells from a variety of solid
tumors, including sarcomas, ovarian cancers, and breast tumors.
Guidance to the use of a combination of reagents can be found in
PCT patent application WO 011052143 (Morrison & Anderson),
incorporated by reference.
[0059] By selecting for phenotypic characteristics among the cells
obtained from a solid tumor, solid tumor stem cells can be isolated
from any animal solid tumor, particularly any mammalian solid
tumor. It will be appreciated that, taking into consideration
factors such as a binding affinities, that antibodies that
recognize species-specific varieties of markers are used to enrich
for and select solid tumor stem cells. Antibodies that recognize
the species-specific varieties of CD44, B38.1, CD24 and other
markers will be used to enrich for or isolate solid tumor stem
cells from that species (for example, antibody to a mouse CD44 for
mouse solid tumor stem cells, antibody to a monkey B38.1 for monkey
solid tumor stem cells, etc.).
[0060] An Efficient Xenograft Model of Human Breast Cancer.
[0061] The invention provides a xenograft model in which to
establish tumors by the injection of solid tumor cells into a host
animal. The host animal can be a model organism such as nematode,
fruit fly, zebrafish; preferably a laboratory mammal such as a
mouse (nude mouse, SCID mouse, NOD/SCID mouse, Beige/SC1D Mouse),
rat, rabbit, or primate. The severely immunodeficient NOD-SCID mice
were chosen as recipients to maximize the participation of injected
cells. Immunodeficient mice do not reject human tissues, and SCID
and NOD-SCID mice have been used as hosts for in vivo studies of
human hematopoiesis and tissue engraftment. McCune et al., Science
241: 1632-9 (1988); Kamel-Reid & Dick, Science 242: 1706-9
(1988); Larochelle et al., Nat. Med. 2: 1329-37 (1996). In
addition, Beige/SC1D mice also have been used.
[0062] Xenograft tumors have been established from mastectomy
specimens of all the patients that have been tested to date (see,
EXAMPLE 7). Tumors in mice have also been established from
malignant pleural effusions. In addition, tumors have been
established by the subcutaneous injection of cells that have been
obtained from two sarcomas. Furthermore, for all the tumors that we
have attempted, we have been able to make single-cell suspensions
(or suspensions with a few aggregates of cell, such as less than
100; preferably less than 10) and then transfer the tumors. This
xenograft assay is useful for biological and molecular assays to
characterize the tumorigenic, clonigenic solid tumor stem
cells.
[0063] The NOD/SCID or Beige/SC1D mice can be further
immunosuppressed, using VP-16 (see, EXAMPLES 1 and 3), radiation
therapy, chemotherapy, or other immunosuppressive biological
agents.
[0064] This in vivo assay is particularly advantageous for the
better understanding of breast cancer and development of new
treatments for this disease. Until now, it has been impossible to
do biological and molecular studies involving primary breast
cancer. Such studies have been limited to cell lines.
Unfortunately, it is well known that the many of the fundamental
properties of breast cancer cells change in tissue culture. Fenhall
et al., British J Cancer 81: 1142-1149 (1999). This latter problem
only worsens with continued culturing of the cells.
[0065] By contrast, using the method of the invention, breast
cancer cells (preferably enriched for breast cancer stem cells) are
injected into immunocompromised mice, to grow the tumor. In one
embodiment, the cells are injected either into the mammary fat pads
of mice or subcutaneously into the mice. Furthermore, tumors can be
established from single-cell suspensions (or suspensions with a few
aggregates of cell, such as less than 100; preferably less than 10)
and then the tumors transferred to other mice.
[0066] The enrichment of solid tumor stem cells and the isolation
of solid tumor stem cells distinguishes the present invention from
the "primary bioassay of human tumor stem cells" referred to in
U.S. Pat. No. 4,411,990 (see also, Hamburger et al., Blood 47: 995
(1976); Salmon et al., AACR Abstracts 19: 231, Abstract No. 922
(1978)). In previous tissue culture assays, only a small proportion
of the tumor cells were able to form colonies in an in vitro
clonogenic assay, and large numbers of cells (such as myeloma and
hematopoietic cells) were typically needed to be transplanted to
form tumors in vivo. Ogawa M et al., Cancer Research. 31(12):
2116-2119 (1971); Ogawa M et al., Cancer Research 33(12):
3172-3175, 1973. Salmon S E & Hamburger A W, Science 197:
461-463 (1977). Schlag P & Flentje D, Cancer Treatment Reviews
11 Suppl A:131-7 (1984). This led to the hypothesis that only a
small number of tumor cells are actually tumorigenic. However,
because of technical limitations, this tumorigenic fraction of
cells could not be isolated from non-tumorigenic cells and
therefore it could not be proven that there were intrinsically
different subsets of tumor cells, some with substantial
proliferative potential and others with limited potential. That is,
unless the tumorigenic cells can be purified and distinguished from
the non-tumorigenic cells it remains possible that all tumor cells
have a similar low probability of exhibiting clonogenic activity in
any assay. Moreover, without the ability to identify and isolate
the tumorigenic fraction of cells (the tumor stem cells) U.S. Pat.
No. 4,411,990 lacks the utilities described in this invention. For
example, without markers to isolate the tumorigenic cells it is not
possible to study their gene expression patterns, or their
expression of diagnostic markers, or their response to therapeutic
agents. Several technical problems prevented prior inventions from
isolating tumorigenic cells or tumor stem cells. First, in vitro
assays resulted in some initial colony formation, but usually the
cells stopped proliferating and could not be grown continuously in
culture. Salmon, S. B. & Hamburger A W, Science 197: 461-463
(1977); Schlag Pet al., Cancer Treatment Reviews. 11 Suppl A:13I-7,
(1984); Salmon S E, Recent Results in Cancer Research 94: 8 (1984).
Also, cells from many tumors failed to form colonies in vitro at
all. Carney D N et al., Stem Cells 1: 149-164 (1981). Similarly,
dissociated cells isolated from most solid tumors rarely formed
tumors in immunodeficient mouse models. Sakakibara T et al., Cancer
J. Si. Am 2: 291-300 (1996); Mueller B & Reisfeld R A, Cancer
Metastasis Rev. 10: 193-200, (1991). The observation that only
particular clones of immortalized tissue culture cancer cell lines
were capable of forming tumors in the in vivo models further
illustrates this problem (Hamilton T C et al., Cancer Research
44(11): 5286-90 (1984)). Thus, the limitations in the assays made
it impossible to determine whether the colonies arose from stem
cells that had lost their capacity to proliferate in vitro, from
non-tumorigenic cells that had limited proliferative potential, or
whether the small number of cells able to form colonies in vitro
was due to a "stem cell" population within the tumor or due to a
rare cell that could proliferate in vitro. Furthermore, it was not
possible to distinguish phenotypically different populations of
cells: prior to this invention, very limited use was made of
techniques like flow-cytometry to separate and analyze
phenotypically distinct populations of solid tumor cells by
flow-cytometry. Indeed, the clonogenic assays used in the prior art
did not predict the behavior of an individual patient's tumor and
fell out of favor. Von Hoff D D et al., Cancer. 67(1): 20-7 (1991);
Federico M et al., Gynecologic Oncology. 55(3 Pt 2): 8156-63
(1994). Thus, the limitations in the cell separation techniques,
and the assays used in the prior art made it impossible for them to
purify tumorigenic cells. Therefore, it was impossible to prove the
existence of hypothetical tumor stem cells.
[0067] Role of Notch in Breast Cancer.
[0068] The Notch family of receptors has been implicated in stem
cell development and differentiation (see, Morrison et al., Cell
101(5): 499-510 (2000); Artavanis-Tsakonas et al., Science 284: 770
(1999); and Artavanis-Tsakonas et al., Science 268: 225-232 (1995);
U.S. Pat. No. 6,090,922, incorporated by reference). Notch was
originally identified in Drosophila through loss-of-function
mutations that produced too many neurons at the expense of other
cell types. Poulson, Proc. Natl. Acad. Sci. USA 23: 133 (1937). In
all animal models tested, mutations in the Notch receptor result in
developmental abnormalities. In C. elegans, Notch is required for
germ line stem cell self-renewal. Berry et al., Development 124(4):
925-36 (1997). In rats, Notch regulates neural crest stem cell
differentiation. Morrison et al., Cell 101(5): 499-510 (2000).
Transient Notch activation initiates an irreversible switch from
neurogenesis to gliogenesis by neural crest stem cells.
[0069] Because neighboring cells can express Notch receptors and
ligands, one cell can affect the fate of a neighboring cell by
activating Notch signaling in the neighboring cell.
[0070] Proteins with knife-edge names such as Jagged (Shimizu et
al., Journal of Biological Chemistry 274(46):32961-9 (1999);
Jarriault et al., Molecular and Cellular Biology 18:7423-7431
(1998)), Serrate, and Delta (and variants of each, such as Delta1,
Delta2, Delta3, Delta4, and Jagged2, LAG-2 and APX-1 in C.
elegans), bind to the Notch receptor and activate a downstream
signaling pathway that prevents neighboring cells from becoming
neural progenitors. A recently identified ligand is D114, a Notch
ligand of the Delta family expressed in arterial endothelium.
Shutter et al., Genes Dev 14(11): 1313-8 (2000).
[0071] Notch ligands may bind and activate Notch family receptors
promiscuously. The expression of other genes, like Fringe family
members (Panin et al., Nature 387(6636): 908-912 (1997)), may
modify the interactions of Notch receptors with Notch ligands. Numb
family members may also modify Notch signaling intracellularly.
[0072] Ligand binding to Notch results in activation of a
presenilin-1-dependent gamma-secretase-like protein that cleaves
Notch. De Strooper et al., Naltlre 398: 518-522 (1999), Mumm et
al., Molecular Cell. 5: 197-206 (2000). Cleavage in the
extracellular region may involve a furin-like convertase. Logeat et
al., PNAS USA 95: 8108-8112 (1998). The intracellular domain is
released and transactivates genes by associating with the DNA
binding protein RBP-J. Kato et al., Development 124: 4133-4141
(1997). Notch 1, Notch 2 and Notch 4 are thought to transactivate
genes such as members of the Enhancer of Split (HES) family, while
Notch 3 signaling may be inhibitory. Beatus et al., Development
126: 3925-3935 (1999). Finally, secreted proteins in the Fringe
family bind to the Notch receptors and modify their function. Zhang
& Gridley, Nature 394 (1998).
[0073] In mammals, there are four known Notch family members. Notch
4 is the human ortholog of the mouse int-3 oncogene that plays a
role in breast cancer in mice. Gallahan et al., Cancer Res. 56(8):
1775-85 (1996); Uyttendaele et al., Development 2122: 251 (1996);
Imatani & Callahan, Oncogene 19(2): 223-31 (2000).
[0074] The invention is based upon the discovery that Notch 4 plays
a role both in normal human breast development and in
tumorigenesis. Within an individual tumor, only a small
subpopulation of tumorigenic cells expresses high levels of Notch
4. An antibody that recognizes Notch 4 blocks the growth of breast
cancer tumor cells in vitro and in vivo (see, EXAMPLES 2, 5, 12 and
0.15). In one embodiment, the antibody binds to the extracellular
domain of Notch 4. In a particular embodiment, the antibody binds
to the polypeptide region LLCVSVVRPRGLLCGSFPE
(LeuLeuCysValSerValValArgProArgGlyLeuLeuCysGlySerPheProGlu) (SEQ
ill NO: 1). However, any anti-Notch 4 antibody that inhibits Notch
activation can be used to impair tumor survival.
[0075] Inhibitors of Notch signaling (such as Numb and Numb-like;
or antibodies or small molecules that block Notch activation) can
be used in the methods of the invention to inhibit solid tumor stem
cells. In this manner, the Notch pathway is modified to kill or
inhibit the proliferation of solid tumor stem cells.
[0076] By contrast, it had previously been found that stimulation
of Notch using soluble Delta (Han et al., Blood 95(5): 161625
(2000)), a Notch ligand, promoted growth and survival of tumor
cells in vitro. Thus, it had previously been found that stimulation
of the Notch pathway promotes growth and survival of the cancer
cells.
[0077] The invention differs from the manipulation of
non-terminally differentiated cells using the Notch pathway
provided in U.S. Pat. No. 5,780,300. U.S. Pat. No. 5,780,300
addresses the modification of normal cells, not cancer cells. That
patent is directed to methods for the expansion of non-terminally
differentiated cells (normal precursor cells) using agonists of
Notch function, by inhibiting the differentiation of the cells
without inhibiting proliferation (mitotic activity) such that an
expanded population of non-terminally differentiated cells is
obtained.
[0078] These expanded cells can be used in cell replacement
therapy, a use that is incompatible with the goal of killing or
inhibiting the proliferation of solid tumor stem cells by modifying
Notch signaling in this invention.
[0079] Therapeutic Aspects of the Invention.
[0080] A corollary to the solid tumor stem cell model of the
invention is that, to effectively treat cancer and achieve higher
cure rates, anti-cancer therapies must be directed against solid
tumor stem cells. Since current therapies are directed against the
bulk population, they may be ineffective at eradicating solid tumor
stem cells. The limitations of current cancer therapies derive from
their inability to effectively kill solid tumor stem cells. The
identification of solid tumor stem cells permits the specific
targeting of therapeutic agents to this cell population, resulting
in more effective cancer treatments. This concept would
fundamentally change our approach to cancer treatment.
[0081] Advances in modern biotechnology have facilitated the
identification of new therapeutic targets for cancer treatment.
Advances in genomics have made it possible to sequence and identify
the 10,000 to 30,000 genes that are expressed in individual cell
types. The human genome has been sequenced. This has resulted in
the identification of new proteins involved in a myriad of
biological processes such as proliferation, cell death and
immortalization, providing targets for drug intervention. Although
genomics provides a powerful means for identifying drug targets in
cancer cells, these targets are only valid if the targets are
present within the tumorigenic cell population. To be effective,
genomics must be focused on individual populations within the
heterogeneous cells that compose a tumor that are responsible for
tumorigenic growth. In solid tumors, these are the solid tumor stem
cells. Additionally, genomics has not yet been used to identify
genes expressed in purified cell populations derived from cancerous
tissues.
[0082] One of the major problems in identifying new cancer
therapeutic agents is determining which of the myriad of genes
identified in large scale sequencing projects are the most
clinically important drug targets. This is made especially
difficult since solid tumors consist of a mixture of a many types
of normal cells and a heterogeneous population of tumor cells. One
way to reduce the complexity is to make cDNA after microdissection
of solid tumors to enrich for tumor cells (see, below). This
technique is based on the assumption that the pathologist
dissecting out the tumor cells can predict which cells are
tumorigenic based upon appearance. However, cells can be
morphologically similar and yet remain functionally heterogeneous.
Moreover, cells obtained by microdissection are not viable and
therefore the functional properties of such cells cannot be tested
or verified.
[0083] Instead, by the methods of the invention, one can use flow
cytometry (such as FACS) and the xenograft animal model of the
invention to enrich for specific cell populations. This technique
has the advantage of being able to simultaneously isolate
phenotypically pure populations of viable normal and tumor cells
for molecular analysis. Thus, flow cytometry allows us to test the
functions of the cell populations and use them in biological assays
in addition to studying their gene expression profiles.
Furthermore, such cells can also be characterized in biological
assays. For example, mesenchymal (stromal) cells can be analyzed
for production of growth factors, matrix proteins and proteases,
endothelial cells can be analyzed for production of specific
factors involved in solid tumor growth support (such as
neo-vascularization), and different subsets of tumor cells from a
solid tumor can be isolated and analyzed for tumorigenicity, drug
resistance and metastatic potential.
[0084] "Enriched", as in an enriched population of cells, can be
defined based upon the increased number of cells having a
particular marker in a fractionated set of cells as compared with
the number of cells having the marker in the unfractionated set of
cells. However, the term "enriched" can be preferably defined by
tumorigenic function as the minimum number of cells that form
tumors at limit dilution frequency in test mice. Thus, if 500 tumor
stem cells form tumors in 63% of test animals, but 5000
unfractionated tumor cells are required to form tumors in 63% of
test animals, then the solid tumor stem cell population is 10-fold
enriched for tumorigenic activity (see, EXAMPLES). The solid tumor
stem cell model (FIG. 1A) provides the linkage between these two
definitions of (phenotypic and functional) enrichment.
[0085] FACS methods using CD44 alone can enrich solid tumor stem
cells at least 2-fold (see, EXAMPLE 1 and 3). FACS methods using
B38.1 and CD24 can enrich for solid tumor stem cells 5-6 fold (see,
EXAMPLE 3). Enrichment using additional markers can enrich 10-fold
or more and can be used to isolate solid tumor stem cells.
[0086] "Isolated" refers to a cell that is removed from its natural
environment (such as in a solid tumor) and that is isolated or
separated, and is at least about 75% free, and most preferably
about 90% free, from other cells with which it is naturally
present, but which lack the marker based on which the cells were
isolated.
[0087] Purification (enrichment or isolation) of subsets of cancer
cells from a solid tumor allows one of skill in the art of oncology
to distinguish between classic models of cancers and the solid
tumor stem cell model (FIG. 1). If indeed a minority of solid tumor
cells has stem cell properties, then to efficiently identify the
genes necessary for tumor proliferation and drug resistance, the
genomics must be focused on the stem cell population. If however,
the genomics is targeted to the bulk population rather than the
solid tumor stem cells, then the most promising drug targets are
obscured or lost in a sea of other genes expressed by the other
cells within a tumor that do not have the capacity for extensive
proliferation.
[0088] In some of the EXAMPLES, we focused on the tumorigenic cells
from breast cancer. Focusing on the individual populations of cells
within a solid tumor provides a clearer understanding of how to
focus new cancer treatments and identify novel targets for drug
discovery. In addition, purifying solid tumor stem (such as breast
cancer tumorigenic) cells provides a material for screening for
drug sensitivity and identifying markers that predict
tumorigenicity or metastatic potential.
[0089] In Vivo Proliferation of Solid Tumor Stem Cells.
[0090] The in vivo proliferation of solid tumor stem cells can be
accomplished by injection of solid tumor stem cells into animals,
preferably mammals, more preferably in rodents such as mice (due to
the predictable methods that have been developed in the art for
injection into laboratory rodents), and most preferably into
immunocompromised mice, such as SCID mice, Beige/SCID mice or
NOD/SCID mice (see, EXAMPLES). NOD/SCID mice are injected with the
varying number of cells and observed for tumor formation. The
injection can be by any method known in the art, following the
enrichment of the injected population of cells for solid tumor stem
cells.
[0091] In one particular embodiment, to establish human breast
cancer tumors in the NOD/SCID mouse model, eight week old female
NOD-SCID mice were anesthetized by an intraperitoneal injection of
0.2 ml Ketamine/Xylazine (300 mg Ketamine combined with 20 mg
Xylazine in a 4 ml volume. Then, 0.02 ml of the solution was
diluted in HBSS is used per 20 g mouse. Mice were then treated with
VP-16 (etoposide) via an intraperitoneal injection (30 mg etoposide
per 1 kg, diluted in serum-free HBSS for a final injection volume
of 0.2 ml). At the same time, estrogen pellets were placed
subcutaneously on the back of the necks of the mice using a trocar.
The mice were then warmed and placed back in to the cages after
they awoke. All tumor injections/implantations were done 3-5 days
after this procedure.
[0092] For the implantation of fresh specimens, samples of human
breast tumors were received within an hour after the surgeries.
These tumors were cut up with scissors into small pieces, and the
pieces were then minced with a blade to yield 2.times.2 mm-size
pieces. Mincing was done in sterile RPMI 1640 medium supplemented
with 20% Fetal Bovine Serum under sterile conditions on ice. The
tumor pieces were then washed with serum free HBSS right before
implantation. A 2-mm incision was then made in the mid abdomen
area, and using a trocar, one to two small tumor pieces were
implanted onto the upper right and upper left mammary fat pats
(right below the second nipple on both sides). A 6-0 suture was
wrapped twice around the MFP-Nipple allowing it to hold the
implanted pieces in place. Sutures were removed 5 days later.
Nexaban was used to seal the incision and mice were weekly
monitored from tumor growth.
[0093] For the injection of the pleural effusions or dissociated
solid tumor cells, cells were received shortly after surgery and
washed with HBSS serum-free. Cells were then suspended in serum
free-RPMI/Matrigel mixture (1:1 volume) and then injected into the
upper right and left mammary pads using an 18.0 needle. To do this,
the desired number of cells were suspended in 0.2 ml and injected.
The site of the needle injection was sealed with Nexaban to prevent
any cell leakage.
[0094] For the injection of digested tumor cells, tumors from a
patient (solid tumors) or grown in mice (by the methods of the
invention) were cut up into small pieces and then minced completely
using sterile blades. The resulting pieces were then mixed with
ultra-pure Collagenase III in HBSS solution (200-250 U
collagenase/ml) and allowed to incubate at 37 C for 3-4 hr,
pipetting with a 10 ml pipette is done every 15-20 minutes. At the
end of the incubation, cells were filtered through a 45-micron
nylon mesh and washed with RPMI-20% FBS, then washed with HBSS
twice. Cells to be injected were then suspended in HBSS/Matrigel
mix (1:1 volume) and injected into the mammalian fat pad or
subcutaneously as described above. Nexaban can be used to seal the
injection site.
[0095] For analysis of the xenotransplant tumor, a solid tumor is
removed from the mice and made into a single cell suspension. Cells
are stained and analyzed by flow cytometry (FACS) using methods
known to those skilled in the art (Morrison & Weissman,
Immunity 1(8): 661-73 (1994)). The phenotype of tumorigenic cells
is CD44.sup.+CD24.sup.-/lo in all tumors, and
B38.1.sup.+CD44.sup.+CD24.sup.-/lo in most tumors. We then do
limiting dilution analysis of cells isolated by FACS based upon
expression of these markers. Next, we further purify the breast
cancer stem cell. Cells are stained with 7AAD (which stains dead
cells), anti H2K-PE (which stains mouse cells), and combinations of
antibodies against various markers that have heterogeneous
expression patterns by the cancer cells including anti-B38.1,
-annexin V, -Notch 4, -CD9, -CD24, -MUC1, -CD49F, -CD62P,
-P-glycoprotein, -Notch 1, -520C9, -260F9 and -317G5. FACS is used
to isolate viable human cells that either do or do not express one
of the differentially expressed antigens. A combination of markers
allows the greatest enrichment of tumorigenic cells. For the
limiting dilution assays, one hundred, one thousand, ten thousand
and one hundred thousand cells of each population are analyzed in
vivo.
[0096] SCID mice, NOD/SCID mice or Beige/SCID mice are injected
with the varying number of cells and observed for tumors. Any
tumors that form are removed for pathologic examination and FACS
analysis. The tests are repeated (for example, about ten times) to
confirm the results. The phenotypes of the tumorigenic cells are
thus determined.
[0097] Other general techniques for formulation and injection of
cells may be found in Remington's Pharmaceutical Sciences, 20th ed.
(Mack Publishing Co., Easton, Pa.). Suitable routes may include
parenteral delivery, including intramuscular, subcutaneous (see,
above), intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections, just to name a few. For injection, the
agents of the invention may be formulated in aqueous solutions,
preferably in physiologically compatible buffers such as Hanks's
solution, Ringer's solution, or physiological saline buffer. For
such transmucosal administration, penetrants appropriate to the
barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0098] By the use of populations of cells enriched for solid tumor
stem cells, the invention is an improvement over the methods of
Mueller & Reisfeld, Cancer Metastasis Rev. 10: 193-200 (1991)
(who used the SCID mouse, which allows disseminated growths for a
number of human tumors, particularly hematologic disorders and
malignant melanoma) and Sakakibara et al., Cancer J. Sci. Am. 2:
291-300 (1996) (who studied the growth and metastatic potential of
surgical specimens of breast carcinomas engrafted into the large
abdominal (gonadal) fat pad of severe combined immunodeficient
(SCID) mice). Sakakibara et al. observed that placement of human
breast tumors within the gonadal fat pad could result in tumors
that grew either rapidly, slowly, or not at all. Of 48 tumors
studied, 12 (25%), including one of the three lymph node-derived
tumors, grew rapidly enough within some or all of the implanted
mice (i.e., the tumors reached a diameter of 2-3 em within 2-6
months) to allow repeated passage.
[0099] By contrast, the injection of solid tumor stem cells can
consistently result in the successful establishment of tumors, more
than 75% of the time, preferably more than 80% of the time, more
preferably more than 85%, more than 90%, or more than 95% of the
time. We have achieved 100% successful establishment of tumors from
the five tumors tested, as well as from three pleural effusions
(see, EXAMPLES). Moreover, the invention provides for the
advantageous` establishment of solid tumors (particularly tumors
from breast tumor stem cells) in mammary fat pads, an area not
accessible for establishment using the methods of Sakakibara et
al., Cancer J. 2: 291-300 (1996).
[0100] In Vitro Proliferation of Solid Tumor Stem Cells.
[0101] Cells can be obtained from solid tumor tissue by
dissociation of individual cells. Tissue from a particular tumor is
removed using a sterile procedure, and the cells are dissociated
using any method known in the art (see, Sambrook et al., Molecular
Cloning: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring
Harbor, N.Y., 1989); Current Protocols in Molecular Biology,
Ausubel et al., eds., (Wiley Interscience, New York, 1993), and
Molecular Biology LabFax, Brown, ed. (Academic Press, 1991)),
including treatment with enzymes such as trypsin, collagenase and
the like, or by using physical methods of dissociation such as with
a blunt instrument. Methods of dissociation are optimized by
testing different concentrations of enzymes and for different
periods of time, to maximize cell viability, retention of cell
surface markers, and the ability to survive in culture (Worthington
Enzyme Manual, Von Worthington, ed. (Worthington Biochemical
Corporation, 2000). Dissociated cells are centrifuged at low speed,
between 200 and 2000 rpm, usually about 1000 rpm (210 g), and then
resuspended in culture medium. For guidance to methods for cell
culture, see Spector et al., Cells: A Laboratory Manual (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1998).
[0102] The dissociated tumor cells can be placed into any known
culture medium capable of supporting cell growth, including HEM,
DMEM, RPMI, F-12, and the like, containing supplements which are
required for cellular metabolism such as glutamine and other amino
acids, vitamins, minerals and useful proteins such as transferrin
and the like. Medium may also contain antibiotics to prevent
contamination with yeast, bacteria and fungi such as penicillin,
streptomycin, gentamicin and the like. In some cases, the medium
may contain serum derived from bovine, equine, chicken and the
like. However, a preferred embodiment for proliferation of solid
tumor stem cells is to use a defined, low-serum culture medium. A
preferred culture medium for solid tumor stem cells is a defined
culture medium comprising a mixture of Ham's F12, 2% fetal calf
serum, and a defined hormone and salt mixture, either insulin,
transferrin, and selenium or B27 supplement. Brewer et al., J.
Neuroscience Res. 35: 567 (1993).
[0103] The culture medium can be a chemically defined medium that
is supplemented with fetal bovine serum or chick embryo extract
(CEE) as a source of mitogens and survival factors to allow the
growth of tumor stem cells in culture. Other serum-free culture
medium containing one or more predetermined growth factors
effective for inducing stem cell proliferation, such as N2
supplement or B27 supplement, known to those of skill in the art
can be used to isolate and propagate solid tumor stem cells from
other bird and mammalian species, such as human. See, U.S. Pat.
Nos. 5,750,376, 5,851,832, and 5,753,506; Atlas et al., Handbook of
Microbiological Media (CRC Press, Boca, Raton, La., 1993);
Freshney, Cutler on Animal Cells, A Manual of Basic Technique, 3d
Edition (Wiley-Liss, New York, 1994), all incorporated herein by
reference.
[0104] The culture medium for the proliferation of solid tumor stem
cells thus supports the growth of solid tumor stem cells and the
proliferated progeny. The "proliferated progeny" are
undifferentiated tumor cells, including solid tumor stem cells,
since solid tumor stem cells have a capability for extensive
proliferation in culture.
[0105] Conditions for culturing should be close to physiological
conditions. The pH of the culture medium should be close to
physiological pH, preferably between pH 6-8, more preferably
between about pH 7 to 7.8, with pH 7.4 being most preferred.
Physiological temperatures range between about 30.degree. C. to
40.degree. C. Cells are preferably cultured at temperatures between
about 32.degree. C. to about 38.degree. C., and more preferably
between about 35.degree. C. to about 37.degree. C. Similarly, cells
may be cultured in levels of O.sub.2 that are comparatively reduced
relative to O.sub.2 concentrations in air, such that the O.sub.2
concentration is comparable to physiological levels (1-6%), rather
than 20% O.sub.2 in air.
[0106] A particular patient's solid tumor stem cells, once they
have been proliferated in vitro, can be analyzed and screened.
Solid tumor stem cell proliferated in vitro can also be genetically
modified using techniques known in the art (see, below; see also,
Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1989); Current
Protocols in Molecular Biology, Ausubel et al., eds., (Wiley
Interscience, New York, 1993)). The in vitro genetic modification
may be more desirable in certain circumstances than in vivo genetic
modification techniques when more control over the infection with
the genetic material is required.
[0107] Solid tumor stem cells and stem cell progeny can be
cryopreserved until they are needed by any method known in the art.
The cells can be suspended in an isotonic solution, preferably a
cell culture medium, containing a particular cryopreservant. Such
cryopreservants include dimethyl sulfoxide (DMSO), glycerol and the
like. These cryopreservants are used at a concentration of 5-15%,
preferably 8-10%. Cells are frozen gradually to a temperature of
-10.degree. C. to --ISO.degree. C., preferably -20.degree. C. to
-100.degree. C., and more preferably --ISO.degree. C.
[0108] Additional guidance for the in vitro culture of solid tumor
stem cells is provided in EXAMPLE 9 and FIG. 9.
[0109] Genetic Modification of Solid Tumor Stem Cells and Solid
Tumor Stem Cell Progeny.
[0110] In the undifferentiated state, the solid tumor stem cells
rapidly divide and are therefore excellent targets for genetic
modification. The term "genetic modification" as used herein refers
to the stable or transient alteration of the genotype of a
precursor cell by intentional introduction of exogenous DNA. DNA
may be synthetic, or naturally derived, and may contain genes,
portions of genes, or other useful DNA sequences. The term "genetic
modification" as used herein is not meant to include naturally
occurring alterations such as that which occurs through natural
viral activity, natural genetic recombination, or the like. General
methods for the genetic modification of eukaryotic cells are known
in the art. See, Sambrook et al., Molecular Cloning: A Laboratory
Manual (Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989);
Current Protocols in Molecular Biology, Ausubel et al., eds.,
(Wiley Interscience, New York, 1993).
[0111] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use with solid tumor stem
cells in vivo, in vitro, and ex vivo. Vectors may be introduced
into hematopoietic stem cells taken from the patient and clonally
propagated. By the method of the invention, such methods are
extended to solid tumor stem cells.
[0112] "Transformation" or "genetically modified" as defined
herein, describes a process by which exogenous DNA enters and
changes a recipient cell. Transformation may occur under natural or
artificial conditions according to various methods well known in
the art, and may rely on any known method for the insertion of
foreign nucleic acid sequences into a prokaryotic or eukaryotic
host cell. The method for transformation is selected based on the
type of host cell being transformed and may include, but is not
limited to, viral infection, electroporation, heat shock,
lipofection, and particle bombardment. The term "transformed" cells
includes stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome, as well as transiently
transformed cells which express the inserted DNA or RNA for limited
periods of time.
[0113] Genetic manipulation of primary tumor cells has been
described previously by Patel et al., Human Gene Therapy 5: 577-584
(1994). Genetic modification of a cell may be accomplished using
one or more techniques well known in the gene therapy field.
Mulligan R C, Human Gene Therapy 5: 543-563 (1993). Viral
transduction methods may comprise the use of a recombinant DNA or
an RNA virus comprising a nucleic acid sequence that drives or
inhibits expression of a protein to infect a target cell. A
suitable DNA virus for use in the present invention includes but is
not limited to an adenovirus (Ad), adeno-associated virus (AAV),
herpes virus, vaccinia virus or a polio virus. A suitable RNA virus
for use in the present invention includes but is not limited to a
retrovirus or Sindbis virus. Several such DNA and RNA viruses exist
that may be suitable for use in the present invention.
[0114] Adenoviral vectors have proven especially useful for gene
transfer into eukaryotic cells for vaccine development (Graham F L
& Prevec L, In Vaccines: New Approaches to Immunological
Problems, Ellis R V ed., 363-390 (Butterworth-Heinemann, Boston,
1992).
[0115] Specific guidance for the genetic modification of solid
tumor stem cells is provides in EXAMPLE 13 and in FIGS. 15-18.
[0116] "Non-viral" delivery techniques that have been used or
proposed for gene therapy include DNA-ligand complexes,
adenovirus-ligand-DNA complexes, direct injection of DNA, CaP04
precipitation, gene gun techniques, electroporation, and
lipofection. Mulligan R C, Science 260: 926-932 (1993). Any of
these methods are widely available to one skilled in the art and
would be suitable for use in the present invention. Other suitable
methods are available to one skilled in the art, and it is to be
understood that the present invention may be accomplished using any
of the available methods of transfection. Lipofection may be
accomplished by encapsulating an isolated DNA molecule within a
liposomal particle and contacting the liposomal particle with the
cell membrane of the target cell. Liposomes are self-assembling,
colloidal particles in which a lipid bilayer, composed of
amphiphilic molecules such as phosphatidyl serine or phosphatidyl
choline, encapsulates a portion of the surrounding media such that
the lipid bilayer surrounds a hydrophilic interior. Unilammellar or
multilammellar liposomes can be constructed such that the interior
contains a desired chemical, drug, or, as in the instant invention,
an isolated DNA molecule. Delivery by transfection, by liposome
injections, or by polycationic amino polymers may be achieved using
methods which are well known in the art (see, e.g., Goldman, C. K.
et al. Nature Biotechnology 15:462-466 (1997)).
[0117] Two types of modified solid tumor stem cells of particular
interest are deletion mutants and over-expression mutants. Deletion
mutants are wild-type cells that have been modified genetically so
that a single gene, usually a protein-coding gene, is substantially
deleted. Deletion mutants also include mutants in which a gene has
been disrupted so that usually no detectable mRNA or bioactive
protein is expressed from the gene, even though some portion of the
genetic material may be present. In addition, in some embodiments,
mutants with a deletion or mutation that removes or inactivates one
activity of a protein (often corresponding to a protein domain)
that has two or more activities, are used and are encompassed in
the term "deletion mutants." Over-expression mutants are wild-type
cells that are modified genetically so that at least one gene, most
often only one, in the modified solid tumor stem cell is expressed
at a higher level as compared to a cell in which the gene is not
modified.
[0118] Genetically modified solid tumor stem cells can be subjected
to tissue culture protocols known in the art (see, U.S. Pat. Nos.
5,750,376 and 5,851,832, Spector et al., Cells: A Laboratory Manual
(Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1998)). Tumor
stem cells can be genetically modified in culture to promote
differentiation, cell death, or immunogenicity. For example, tumor
stem cells can be modified to enhance expression of products that
direct an immune response against the patient's solid tumor.
Alternatively, the solid tumor stem cells can be subjected to
various proliferation protocols in vitro prior to genetic
modification. The protocol used depends upon the type of
genetically modified solid tumor stem cell or solid tumor stem cell
progeny desired. Once the cells have been subjected to the
differentiation protocol, they are again assayed for expression of
the desired protein. Cells having the desired phenotype can be
isolated and implanted into recipients in need of the protein or
biologically active molecule that is expressed by the genetically
modified cell. Such molecules can enhance tumor regression or
inhibit tumor spread.
[0119] In Vitro Models of Solid Tumor Development, In Vivo Models,
and Methods for Screening Effects of Drugs on Solid Tumor Stem
Cells.
[0120] Solid tumor stem cells and solid tumor stem cell progeny
cultured in vitro (see, EXAMPLE 9) or in vivo (in the xenograft
model of the invention) can be used for the screening of potential
therapeutic compositions. These compositions for the treatment of
solid tumors can be applied to these cells in culture at varying
dosages, and the response of these cells monitored for various time
periods. Physical characteristics of these cells can be analyzed by
observing cells by microscopy. The induction of expression of new
or increased levels of proteins such as enzymes, receptors and
other cell surface molecules can be analyzed with any technique
known in the art see, Clarke et al.) Proc. Natl. Acad. Sci. USA 92:
11024-11028 (1995)) which can identify the alteration of the level
of such molecules. These techniques include immunohistochemistry,
using antibodies (see, EXAMPLES) against such molecules, or
biochemical analysis. Such biochemical analysis includes protein
assays, enzymatic assays, receptor binding assays, enzyme-linked
immunosorbant assays (ELISA), electrophoretic analysis, analysis
with high performance liquid chromatography (HPLC), Western blots,
and radioimmune assays (RIA). Nucleic acid analysis such as
Northern blots can be used to examine the levels of mRNA coding for
these molecules or PCR (see, EXAMPLE 14).
[0121] Alternatively, such cells treated with these pharmaceutical
compositions can be transplanted into an animal (such as in the
xenograft model of the invention), and their survival, ability to
form tumors, and biochemical and immunological characteristics
examined.
[0122] The solid stem cells and solid stem cell progeny of the
invention can be used in methods of determining the effect of
biological agents on solid tumor cells. The term "biological agent"
or "test compound" refers to any agent (including a virus, protein,
peptide, amino acid, lipid, carbohydrate, nucleic acid, nucleotide,
drug, antibody, prodrug or other substance) that may have an effect
on tumor cells whether such effect is harmful, beneficial, or
otherwise.
[0123] To determine the effect of a potential test compound on
solid tumor stem cells, a culture of precursor cells derived from
tumor stem cells can be obtained from tissue of a subject, such as
a patient with a tumor or other cancerous disease, such as an
epithelial cancer or breast cancer. Once the solid tumor stem cells
or other desired populations of cells are obtained from the
subject, the cells are cultured in vitro. The choice of culture
depends upon the particular test compound being tested and the
diagnostic effects that the laboratory personnel want to
achieve.
[0124] The ability of various biological agents to increase,
decrease, or modify in some other way the number and nature of the
solid tumor stem cells and solid tumor stem cell progeny can be
screened. For example, it is possible to screen for test compounds
that decrease the proliferative ability of the solid tumor stem
cells, which would be useful for identifying anti-cancer
therapeutic agents. In these assays, the relevant cells are
cultured in the presence of the test compounds of interest and
assayed for the degree of proliferation or cell death that
occurs.
[0125] The effects of a test compound or combination of test
compounds on the extensive proliferation of solid tumor stem cells
and their progeny can be determined. It is possible to screen
non-tumorigenic solid tumor cells that have already been induced to
lose the ability to extensively proliferate before the screening.
It is also possible to determine the effects of the test compounds
on the proliferation process by applying them to solid tumor stem
cells. Generally, the test compound is solubilized and added to the
culture medium or to the mouse in the in vivo assay at varying
concentrations to determine the effect of the test compounds or
agent at each dose. The culture medium may be replenished with the
test compound or biological agent every couple of days in amounts
so as to keep the concentration of the agent somewhat constant.
Similarly, the test compound can be re-administered to the mouse at
different intervals to assess the effect of the compound over
time.
[0126] Changes in proliferation are observed by an increase or
decrease in the number of cells that form or an increase or
decrease in the size of the foci in vitro, or tumor size in vivo
(which is a reflection of the rate of proliferation and the rate of
cell death--determined by the numbers of cells per foci or tumor
size in the mouse). The effect of the test compound on tumor stem
cells are measured by determining the number of tumor stem cells
that persist in culture or in the tumors in vivo after treatment
with the test compound. In addition to determining the number of
tumor stem cells, the effects of the test compound on tumor stem
cell cell-cycle status, and marker expression are also determined
by flow-cytometry.
[0127] The test compounds or biological agents added to the culture
medium or injected into the mouse can be at a final concentration
in the range of about 10 pg/ml to 1 .mu.g/ml, preferably about 1
ng/ml (or 1 ng/cc of blood) to 100 ng/ml (or 100 ng/cc of
blood).
[0128] The effects of the test compounds or biological agents are
identified on the basis of significant difference relative to
control cultures with respect to criteria such as the ratios of
expressed phenotypes, cell viability, proliferation rate, number of
tumor stem cells, tumor stem cell activity upon transplantation in
vivo, tumor stem cell activity upon transplantation in culture,
cell cycle distribution of tumor cells, and alterations in gene
expression.
[0129] Therapeutic Compositions and Methods.
[0130] A pharmaceutical composition containing a Notch ligand, an
anti-Notch antibody, or other therapeutic agent that acts as an
agonist or antagonist of proteins in the Notch signal
transduction/response pathway can be administered by any effective
method. For example, a physiologically appropriate solution
containing an effective concentration of anti-Notch therapeutic
agent can be administered topically, intraocularly, parenterally,
orally, intranasally, intravenously, intramuscularly,
subcutaneously or by any other effective means. In particular, the
anti-Notch therapeutic agent may be directly injected into a target
cancer or tumor tissue by a needle in amounts effective to treat
the tumor cells of the target tissue. Alternatively, a cancer or
tumor present in a body cavity such as in the eye, gastrointestinal
tract, genitourinary tract (e.g., the urinary bladder), pulmonary
and bronchial system and the like can receive a physiologically
appropriate composition (e.g., a solution such as a saline or
phosphate buffer, a suspension, or an emulsion, which is sterile)
containing an effective concentration of anti-Notch therapeutic
agent via direct injection with a needle or via a catheter or other
delivery tube placed into the cancer or tumor afflicted hollow
organ. Any effective imaging device such as X-ray, sonogram, or
fiber-optic visualization system may be used to locate the target
tissue and guide the needle or catheter tube. In another
alternative, a physiologically appropriate solution containing an
effective concentration of anti-Notch therapeutic agent can be
administered systemically into the blood circulation to treat a
cancer or tumor that cannot be directly reached or anatomically
isolated.
[0131] All such manipulations have in common the goal of placing
the anti-Notch therapeutic agent in sufficient contact with the
target tumor to permit the anti-Notch therapeutic agent to contact,
transduce or transfect the tumor cells (depending on the nature of
the agent). In one embodiment, solid tumors present in the
epithelial linings of hollow organs may be treated by infusing the
vector suspension into a hollow fluid filled organ, or by spraying
or misting into a hollow air filled organ. Thus, the tumor cells
(such as a solid tumor stem cells) may be present in or among the
epithelial tissue in the lining of pulmonary bronchial tree, the
lining of the gastrointestinal tract, the lining of the female
reproductive tract, genitourinary tract, bladder, the gall bladder
and any other organ tissue accessible to contact with the
anti-Notch therapeutic agent. In another embodiment, the solid
tumor may be located in or on the lining of the central nervous
system, such as, for example, the spinal cord, spinal roots or
brain, so that anti-Notch therapeutic agent infused in the
cerebrospinal fluid contacts and transduces the cells of the solid
tumor in that space. (Accordingly, the anti-Notch therapeutic agent
can be modified to cross the blood brain barrier using method known
in the art). One skilled in the art of oncology can appreciate that
the anti-Notch therapeutic agent can be administered to the solid
tumor by direct injection of the vector suspension into the tumor
so that anti-Notch therapeutic agent contacts and affects the tumor
cells inside the tumor.
[0132] One skilled in the art of oncology can understand that the
vector is administered in a composition comprising the vector
together with a carrier or vehicle suitable for maintaining the
transduction or transfection efficiency of the chosen vector and
promoting a safe infusion. Such a carrier may be a pH balanced
physiological buffer, such as a phosphate, citrate or bicarbonate
buffers a saline solution, a slow release composition and any other
substance useful for safely and effectively placing the anti-Notch
therapeutic agent in contact with solid tumor stem cells to be
treated.
[0133] In treating a cancer patient who has a solid tumor, a
therapeutically effective amount of an anti-Notch therapeutic agent
is administered. A therapeutically effective dose refers to that
amount of the compound sufficient to result in amelioration of
symptoms or a prolongation of survival in a patient. Toxicity and
therapeutic efficacy of such compounds can be determined by
standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for determining the LD.sub.50 (the dose lethal to
50% of the population) and the ED.sub.50 (the dose therapeutically
effective in 50% of the population). The dose ratio between toxic
and therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds that exhibit
large therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography (HPLC).
[0134] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition (see e.g. Fingl et al., In The Pharmacological Basis of
Therapeutics, Ch. 1, pg. 1 (1975)). The attending physician would
know how to and when to terminate, interrupt, or adjust
administration due to toxicity, or to organ dysfunctions.
Conversely, the attending physician would also know to adjust
treatment to higher levels if the clinical response were not
adequate (precluding toxicity). The magnitude of an administrated
dose in the management of the clinical disorder of interest can
vary with the severity of the condition to be treated and the route
of administration. See, Merck Index An Encyclopedia of Chemicals,
Drugs and Biologicals, 12 Edition (CRC Press 1996); Physicians'
Desk Reference 55th Edition (2000)). The severity of the condition
may, for example, be evaluated, in part, by appropriate prognostic
evaluation methods. Further, the dose and perhaps dose frequency,
also vary according to the age, body weight, and response of the
individual patient. A program comparable to that discussed above
may be used in veterinary medicine.
[0135] Depending on the specific conditions being treated, agents
may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
Remington's Pharmaceutical Sciences, 20th ed. (Mack Publishing Co.,
Easton, Pa.). Suitable routes may include oral, rectal,
transdermal, vaginal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections, just to name a few.
[0136] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. For such transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0137] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically acceptable
carriers comprising excipients and auxiliaries, which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. The preparations formulated for oral
administration may be in the form of tablets, capsules, or
solutions. The pharmaceutical compositions of the present invention
may be manufactured in a manner that is itself known, e.g., by
means of conventional mixing, dissolving, granulating, levigating,
emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include
aqueous solutions of the active compounds in water-soluble form.
Additionally, suspensions of the active compounds may be prepared
as appropriate oily injection suspensions. Suitable lipophilic
solvents or vehicles include fatty oils such as sesame oil, or
synthetic fatty acid esters, such as ethyl oleate or triglycerides,
or liposomes. Aqueous injection suspensions may contain substances
that increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol, or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents that
increase the solubility of the compounds to allow for the
preparation of highly concentrated solutions.
[0138] Polynucleotides and Polypeptides Obtained from Solid Tumor
Stem Cells.
[0139] "Polynucleotide" refers to chain of nucleotides, which can
be a nucleic acid, nucleic acid sequence, oligonucleotide,
nucleotide or any fragment thereof. It may be DNA or RNA of genomic
DNA. mRNA, cDNA, or synthetic origin, double-stranded or
single-stranded and combined with carbohydrate, lipids, protein or
other materials to perform a particular activity or form a useful
composition. "Oligonucleotide" is substantially equivalent to the
terms amplimer, primer, oligomer, element, and probe. The term
"probe" refers to a polynucleotide sequence capable of hybridizing
with a target sequence to form a polynucleotide probe/target
complex. A "target polynucleotide" refers to a chain of nucleotides
to which a polynucleotide probe can hybridize by base pairing. In
some instances, the sequences will be complementary (no mismatches)
when aligned. In other instances, there may be up to a 10%
mismatch.
[0140] DNA or RNA can be isolated from the sample according to any
of a number of methods well known to those of skill in the art. For
example, methods of purification of nucleic acids are described in
Tijssen, Laboratory Techniques in Biochemistry and Molecular
Biology: Hybridization With Nucleic Acid Probes, Part 1. Theory and
Nucleic Acid Preparation (Elsevier. New York N.Y. 1993). "Sample"
is used in its broadest sense. A sample containing polynucleotides
or polypeptides can be a bodily fluid; an extract from a cell,
chromosome, organelle or membrane isolated from a cell; genomic
DNA, RNA or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; and the like.
[0141] Total RNA can be isolated using the TRIZOL reagent (Life
Technologies, Gaithersburg Md. USA), and mRNA is isolated using
oligo d(T) column chromatography or glass beads. Alternatively,
when target polynucleotides are derived from an mRNA, the target
polynucleotides can be a cDNA reverse transcribed from an mRNA, an
RNA transcribed from that cDNA, a DNA amplified from that cDNA, an
RNA transcribed from the amplified DNA and the like. When the
target polynucleotide is derived from DNA, the target
polynucleotide can be DNA amplified from DNA or RNA reverse
transcribed from DNA.
[0142] Several technologies produce pools of restriction fragments
of limited complexity for electrophoretic analysis, such as methods
combining double restriction enzyme digestion with phasing primers
(see, e.g., European patent application EP 0 534 858 A1), or
methods selecting restriction fragments with sites closest to a
defined mRNA end (see, e.g. Prashar et al., Proc. Natl. Acad. Sci.
USA 93: 659-663 (1996)). Other methods statistically sample cDNA
pools, such as by sequencing sufficient bases (in each of, multiple
cDNAs to identify each cDNA or by sequencing short tags which are
generated at known positions relative to a defined mRNA end (see,
e.g., Velculescu, Science 270: 484-487 (1995)).
[0143] Methods of modifying RNA abundances and activities currently
fall within three classes, ribozymes, antisense species (PCT patent
application WO 88/09810), and RNA aptamers (Good et al., Gene
Therapy 4: 45-54 (1997)). Ribozymes are RNAs which are capable of
catalyzing RNA cleavage reactions. (PCT patent application WO
90/11364). Ribozyme methods involve exposing a cell to, inducing
expression in a cell, etc. of such small RNA ribozyme molecules.
(Grassi & Marini, Annals of Medicine 28: 499-510 (1996);
Gibson, Cancer and Metastasis Reviews 15: 287-299 (1996)).
[0144] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to the "sense" strand of a specific nucleic acid
sequence. Antisense molecules may be produced by any method
including synthesis or transcription. Once introduced into a cell,
the complementary nucleotides combine with natural sequences
produced by the cell to form duplexes and to block either
transcription or translation. The designation "negative" can refer
to the antisense strand, and the designation "positive" can refer
to the sense strand.
[0145] Oligonucleotides may be synthesized by standard methods
known in the art, e.g. by use of an automated DNA synthesizer (such
as are commercially available from Biosearch, Applied Biosystems,
etc.).
[0146] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. See, e.g., Dieffenbach C W
& Dveksler G S, PCR Primer, a Laboratory Manuall-5 (Cold Spring
Harbor Press, Plainview, N.Y., 1995). Amplification can be
polymerase chain reaction (PCR), ligase chain reaction (LCR),
nucleic acid sequence-based amplification (NASBA), or T7 based RNA
amplification.
[0147] "Polypeptide" refers to an amino acid, amino acid sequence,
oligopeptide, peptide, or protein or portions thereof whether
naturally occurring or synthetic.
[0148] Methods for direct measurement of protein activity are well
known to those of skill in the art. Such methods include, e.g.,
methods which depend on having an antibody ligand for the protein,
such as Western blotting, see, e.g., Burnette, A. Anal. Biochem.
112: 195-203 (1981). Such methods also include enzymatic activity
assays, which are available for most well-studied protein drug
targets. Detection of proteins can be accomplished by antibodies
(see, EXAMPLES). The term "antigenic determinant," as used herein,
refers to that fragment of a molecule (i.e., an epitope) that makes
contact with a particular antibody. When a protein or a fragment of
a protein is used to immunize a host animal, numerous regions of
the protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0149] Proteins isolated from an enriched population of solid tumor
stem cells or isolated solid tumor cells can be separated by
two-dimensional gel electrophoresis systems. Two-dimensional gel
electrophoresis is well-known in the art and typically involves
iso-electric focusing along a first dimension followed by SDS-PAGE
electrophoresis along a second dimension. See, e.g., Hames et al,
Gel Electrophoresis of Proteins: A Practical Approach (IRL Press,
New York 1990); Lander, Science 274:536-539 (1996). The resulting
electrophoretograms can be analyzed by numerous techniques,
including mass spectrometric techniques, western blotting and
immunoblot analysis using polyclonal and monoclonal antibodies, and
internal and N-terminal micro-sequencing. Using these techniques,
it is possible to identify a substantial fraction of all the
proteins produced under given physiological conditions, including
in cells (e.g., in solid tumor stem cells) exposed to a drug, or in
cells modified by, e.g., deletion or over-expression of a specific
gene.
[0150] cDNA Libraries.
[0151] The purified solid tumor stem cells, solid tumor stem cell
progeny, non-tumorigenic cells, and unfractionated tumor cells can
be used to make arrays or cDNA libraries using methods known in the
art (see, Sambrook et al., Molecular Cloning: A Laboratory Manual
(Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989); Current
Protocols in Molecular Biology, Ausubel et al., eds., (Wiley
Interscience, New York, 1993)) to identify potential novel drug
targets.
[0152] Molecular biology comprises a wide variety of techniques for
the analysis of nucleic acids and proteins, many of which form the
basis of clinical diagnostic assays. These techniques include
nucleic acid hybridization analysis, restriction enzyme analysis,
genetic sequence analysis, and separation and purification of
nucleic acids and proteins (Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989). Many molecular biology techniques
involve carrying out numerous operations on a large number of
samples. For guidance to genomics and other molecular biological
methods useful in the invention, see Birren et al., Genome
Analysis: A Laboratory Manual Series, Volume 1, Analyzing DNA (Cold
Spring Harbor Press, Cold Spring Harbor, N.Y., 1997); Birren et
al., Genome Analysis: A Laboratory Manual Series, Volume 2,
Detecting Genes (Cold Spring Harbor Press, Cold Spring Harbor,
N.Y., 1998); Birren et al., Genome Analysis: A Laboratory Manual
Series, Volume 4, Mapping Genomes (Cold Spring Harbor Press, Cold
Spring Harbor, N.Y., 1999).
[0153] Nucleic acid hybridization analysis generally involves the
detection of a very small numbers of specific target nucleic acids
(DNA or RNA) with probes among a large amount of non-target nucleic
acids. Multiple sample nucleic acid hybridization analysis has been
conducted on a variety of filter and solid support formats. The
"dot blot" hybridization, involves the non-covalent attachment of
target DNAs to a filter, which are subsequently hybridized with a
labeled probes. The "dot blot" hybridization has been further
developed for multiple analysis (European Patent application EP 0
228 075) and for the detection of overlapping clones and the
construction of genomic maps (U.S. Pat. No. 5,219,726). Another
format, the so-called "sandwich" hybridization, involves attaching
oligonucleotide probes covalently to a solid support and using them
to capture and detect multiple nucleic acid targets (U.S. Pat. No.
4,751,177; PCT International patent application WO 90/01564).
Multiplex versions of these formats are called "reverse dot
blots".
[0154] Methods are known in the art for amplifying signal using
sensitive reporter groups (enzyme, fluorophores, radioisotopes,
etc.) and associated detection systems (fluorometers, luminometers,
photon counters, scintillation counters, etc.). These methods can
be combined with amplification methods, such as the polymerase
chain reaction (PCR) for the amplification of target nucleic acid
sequences. See, Innis et al., PCR Protocols: A Guide to Methods and
Applications, (Academic Press, 1990).
[0155] Microarrays.
[0156] Mimicking the in situ hybridization in some aspects, new
techniques are being developed for carrying out multiple sample
nucleic acid hybridization analysis on micro-formatted multiplex or
matrix devices (e.g., DNA chips) (see, Barinaga, Science 253: 1489
(1991); Bains, Bio/Technology 10: 757-758 (1992)). Guidance for the
use of microarrays is provided by Wang, E et al., Nature
Biotechnology 18; 457-459 (2000); Diehn Met al., Nature Genetics
25: 58-62 (2000).
[0157] Microarrays are known in the art and consist of a surface to
which probes that correspond in sequence to gene products (e.g.,
cDNAs, oligonucleotides, mRNAs, cRNAs polypeptides, and fragments
thereof), can be specifically hybridized or bound at a known
position. In one embodiment, the microarray is an array (i.e., a
matrix) in which each position represents a discrete binding site
for a product encoded by a gene (e.g., a protein or RNA), and in
which binding sites are present for products of most or almost all
of the genes in the organism's genome.
[0158] The polynucleotides, polypeptides, or analogues are attached
to a solid support or substrate, which may be made from glass,
plastic (e.g., polypropylene, nylon), polyacrylamide,
nitrocellulose, or other materials. "Substrate" refers to any
suitable rigid or semi-rigid support to which polynucleotides or
polypeptides are bound and includes membranes, filters, chips,
slides, wafers, fibers, magnetic or nonmagnetic beads, gels,
capillaries or other tubing, plates, polymers, and microparticles
with a variety of surface forms including wells, trenches, pins,
channels and pores. The polynucleotides can be immobilized on a
substrate by any method known in the art. Preferably, the
substrates are optically transparent.
[0159] A variety of methods are currently available for making
arrays of biological macromolecules, such as arrays of nucleic acid
molecules or proteins. One method for making ordered arrays of DNA
on a porous membrane is a "dot blot" or "slot-blot" method. A more
efficient technique employed for making ordered arrays of fragments
uses an array of pins dipped into the wells, e.g., the 96 wells of
a microtiter plate, for transferring an array of samples to a
substrate, such as a porous membrane. An alternate method of
creating ordered arrays of nucleic acid sequences is described by
U.S. Pat. No. 5,143,854 (to Pirrung) and also by Fodor et al.,
Science 251:767-773 (1991). The method involves synthesizing
different nucleic acid sequences at different discrete regions of a
support. Khrapko et al., DNA Sequence 1:375-388 (1991) describes a
method of making an oligonucleotide matrix by spotting DNA onto a
thin layer of polyacrylamide, manually with a micropipette. U.S.
Pat. No. 5,807,522 (to Brown et al., incorporated by reference)
describes methods for fabricating microarrays of biological samples
by dispensing a known volume of a reagent at each selected array
position, by tapping a capillary dispenser on the support under
conditions effective to draw a defined volume of liquid onto the
support.
[0160] Spotters can use pin, ink-jet, and other technologies to
deposit samples onto the support material. Several of the more
common methods utilize metal pins, which can be either solid or
split. When the pins are dipped into wells that contain the
compounds of interest, each picks up a small amount of the
material. The pin is then brought into contact with the solid
support and a nanoliter volume is dispensed at the desired
location. In split pins (otherwise known as quills) a slot cut into
the head of the pin functions as a reservoir for the compound being
spotted. Quill pins are most often used with glass slides, while
solid pins are typically used for spotting membranes. Amersham
Pharmacia Biotech, GeneMachines, and other companies offer spotting
robots.
[0161] Ink-jet technology provides another method of spotting
microarrays. Adapted from the printer industry and redesigned for
use in biotechnological applications, this uses piezoelectric
crystal oscillators and an electrode guidance system to deposit the
compound in a precise location on the slide or membrane. Companies
such as Cartesian Technologies and ProtoGene Laboratories use this
technology.
[0162] A method for attaching the nucleic acids to a surface is by
printing on glass plates, as is described generally by PCT
publication WO 95/35505; DeRisi et al., Nature Genetics 14:457-460
(1996); Shalon et al., Genome Res. 6:639-645 (1996); and Schena et
al., Proc. Natl. Acad. Sci. USA 93: 10614-10619 (1995). Another
method for making microarrays is by making high-density
oligonucleotide arrays. Techniques are known for producing arrays
containing thousands of oligonucleotides complementary to defined
sequences, at defined locations on a surface using
photolithographic techniques for synthesis in situ (see, Fodor et
al., Science 251:767-773 (1991); Pease et al., Proc. Natl. Acad.
Sci. USA 91:5022-5026 (1994); Lockhart et al., Nature Biotech
14:1675 (1996); U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270,
each of which is incorporated by reference).
[0163] U.S. Pat. No. 6,110,426 (to Shalon et al.) a method and
apparatus for fabricating microarrays of biological samples for
large scale screening assays, such as arrays of DNA samples to be
used in DNA hybridization assays for genetic research and
diagnostic applications. U.S. Pat. No. 6,221,674 (to Sluka et al.)
discloses a process is described for applying spatially defined
reagent areas to a solid phase which is characterized in that a
liquid containing an adsorptive binding reagent is contacted with
spatially defined areas of a solid phase which comprises an
essentially continuous metal or metal oxide surface for an adequate
time period to enable the formation of adsorptive bonds between the
binding reagent and the solid phase. A process is described in PCT
application WO 92/1 0092 which can be used to generate a plurality
of different structures on a glass support by means of
photoreactive compounds and irradiation using masks. A process is
described in U.S. Pat. No. 4,877,745 in which differently
functionalized spots can be applied to plastic supports by means of
ink-jet.
[0164] Among the vendors of microarrays and microarray technology
useage are Affymetrix, Inc. (USA), NimbleGen Systems, Inc.
(Madison, Wis., USA), and Incyte Genomics (USA) (producing
microarrays for core facilities in large industrial and academic
departments); Agilent Technologies (USA) and Graffinity
Pharmaceutical Design, GmbH (Germany) (which provide specific
services such as printing and fingerprinting arrays designed and
used by individual researchers); and CLONTECH Laboratories (Becton
Dickinson Bioscience) and BioRobotics, Ltd. (Great Britain) (which
provide the basic tools necessary for individual researchers to
carry out the entire process of producing microarrays, including
printing). See, Gwynne P & Heebner G, "DNA Chips and
Microarrays" Science (2001).
[0165] In contrast to plastic surfaces, metal and metal oxide
surfaces have the advantage that they can be coated with an exactly
defined matrix layer by self-assembly techniques. A self-assembled
monolayer (SAM) is formed for example when organic alkylthiols are
absorbed onto a gold surface, the spontaneous organization of such
a densely packed monolayer being based on strong specific
interactions between the support material and the adsorbent. Nuzzo
et al., J. Am. Chem. Soc. 105: 4481 (1983). In this manner it is
possible to apply an exactly defined monolayer of a binding matrix
to the surface of metals such as e.g. gold or silver. Furthermore
the specific binding capability of self-assembled solid phases can
be further optimized by dilution of the specific solid phase
reactants as described in EP-A-0 515 615.
[0166] The coating of metal surfaces with microstructures based on
self-assembled monolayers is also known and can be used to attach
components isolated from solid tumor stem cells. Whitesides et al.,
Langmuir 10 (1994) 1498-1511 describe a process in which reagents
are stamped onto a noble metal surface by means of a special
microstructured silicone stamp. This enables microstructured
monolayers to be generated with zones that are spatially separated
from one another. Microstructures of self-assembled monolayers on
noble metal surfaces can be formed by irradiation through masks of
substrates whose whole area is covered with thiols and subsequent
washing. Hemminger et al., Langmuir 10: 626-628 (1994). Spatially
separate zones are also formed in this process which are all
identically functionalized. A further possibility of producing
reagent spots is firstly to apply gold spots to a support that are
already spatially separated from one another which are then
subsequently coated with reagents.
[0167] The binding of analytes to a functionalized solid phase
matrix according to the invention can for example be detected by
confocal scanner fluorescence microscopy or by plasmon resonance
spectroscopy. Ruthenhausler B et al., Nature, 332:615-617
(1988).
[0168] U.S. Pat. No. 6,228,659 describes an apparatus for producing
a plurality of arrays of reagent regions is disclosed. A dispensing
assembly in the apparatus has a plurality of heads which are spaced
for depositing reagents at selected positions in different array
areas in a substrate.
[0169] Transcript arrays can be employed for analyzing the
transcriptional state in a cell, and especially for measuring the
transcriptional states of cells exposed to graded levels of a
therapy of interest such as graded levels of a drug of interest or
to graded levels of a disease state of interest. In one embodiment,
transcript arrays are produced by hybridizing detectably labeled
polynucleotides representing the mRNA transcripts present in a cell
(e.g., fluorescently labeled cDNA synthesized from total cell mRNA)
to a microarray. In alternative embodiments, the cDNA or RNA probe
can be synthesized in the absence of detectable label and may be
labeled subsequently, e.g., by incorporating biotinylated dNTPs or
rNTP, or some similar means (e.g., photo-cross-linking a psoralen
derivative of biotin to RNAs), followed by addition of labeled
streptavidin (e.g., phycoerythrin-conjugated streptavidin) or the
equivalent. The label for the probe may be selected from the group
consisting of biotin, fluorescent, radioactive, and enzymatic
labels. When fluorescently-labeled probes are used, many suitable
fluorophores are known, including fluorescein, lissamine,
phycoerythrin, rhodamine (perkin Elmer Cetus), Cy2, Cy3, Cy3.5,
Cy5, Cy5.5, Cy7, Fluor X (Amersham) and others (see, e.g., Kricka,
Nonisotopic DNA Probe Techniques (Academic Press San Diego, Calif.,
1992). It will be appreciated that pairs of fluorophores are chosen
that have distinct emission spectra so that they can be easily
distinguished. In another embodiment, a label other than a
fluorescent label is used. For example, a radioactive label, or a
pair of radioactive labels with distinct emission spectra, can be
used (see Zhao et al., Gene 156:207 (1995); Pietu et al., Genome
Res. 6:492 (1996); see also, EXAMPLE 21). For example, .sup.32P can
be used.
[0170] These methods of attaching transcripts usually attach
specific polynucleotide sequences to very small specific areas of a
solid support, such as micro-wells of a DNA chip. These
hybridization formats are micro-scale versions of the conventional
"reverse dot blot" and "sandwich" hybridization systems. The
micro-formatted hybridization can be used to carry out "sequencing
by hybridization" (see, Barinaga, Science 253: 1489 (1991); Bains,
Bio/Technology 10: 757-758 (1992). Sequencing by hybridization
makes use of all possible n-nucleotide oligomers (n-mers) to
identify n-mers in an unknown DNA sample, which are subsequently
aligned by algorithm analysis to produce the DNA sequence (see,
U.S. Pat. No. 5,202,231; see also, United Kingdom patent
application GB 8810400 (1988); Southern et al., Genomics 13: 1008
(1992); Fodor et al., Nature 364: 555-556 (1993); Fodor et al.,
Science 251: 767-773 (1991); U.S. Pat. No. 5,143,854.
[0171] Probes can be synthesized, in whole or in part, on the
surface of a substrate using a chemical coupling procedure and a
piezoelectric printing apparatus, such as that described in PCT
publication WO 95/251116 (Baldeschweiler et al.). Alternatively,
the probe can be synthesized on a substrate surface using a
self-addressable electronic device that controls when reagents are
added (U.S. Pat. No. 5,605,662).
[0172] Furthermore, the probes do not have to be directly bound to
the substrate, but rather can be bound to the substrate through a
linker group. The linker groups are typically about 6 to 50 atoms
long to provide exposure to the attached polynucleotide probe.
Preferred linker groups include ethylene glycol oligomers,
diamines, diacids and the like. Reactive groups on the substrate
surface react with one of the terminal portions of the linker to
bind the linker to the substrate. The other terminal portion of the
linker is then functionalized for binding the polynucleotide
probe.
[0173] Devices and computer systems for forming and using arrays of
materials on a chip or substrate are known. For example, PCT
International patent applications WO 92/10588 and WO 95/11995, both
incorporated herein by reference, describe techniques for
sequencing or sequence checking nucleic acids and other materials.
Arrays for performing these operations can be formed in arrays
according to the methods of, for example, the pioneering techniques
disclosed in U.S. Pat. Nos. 5,445,934, 5,384,261 and 5,571,639,
each incorporated herein by reference. Improved methods of forming
high-density arrays of peptides, polynucleotides, and other polymer
sequences in a short period of time have been devised using
combinatorial solid phase synthesis. Very Large Scale Immobilized
Polymer Synthesis (VLSIPS) technology has greatly advanced
combinatorial solid phase polymer synthesis and paved the way to
clinical application of deoxyribonucleic acid (DNA) array chips
such as those sold under the name GENECHIP.TM. Kozal et al., Nature
Medicine 2: 753-759 (1996). VLSIPS technology is disclosed in U.S.
Pat. No. 5,143,854, PCT International patent applications WO
90/15070, WO 92/110092, and WO 95/11995; and Fodor et al., Science
251: 767-777 (1991).
[0174] Nucleic acid hybridization and wash conditions are chosen so
that the probe "specifically binds" or "specifically hybridizes" to
a specific array site, i.e., the probe hybridizes, duplexes or
binds to a sequence array site with a complementary nucleic acid
sequence but does not hybridize to a site with a non-complementary
nucleic acid sequence. As used herein, one polynucleotide sequence
is considered complementary to another when, if the shorter of the
polynucleotides is less than or equal to 25 bases, there are no
mismatches using standard base-pairing rules or, if the shorter of
the polynucleotides is longer than 25 bases, there is no more than
a 5% mismatch. Preferably, the polynucleotides are perfectly
complementary (no mismatches). It can easily be demonstrated that
specific hybridization conditions result in specific hybridization
by carrying out a hybridization assay including negative controls.
Optimal hybridization conditions will depend on the length (e.g.,
oligomer versus polynucleotide greater than 200 bases) and type
(e.g., RNA, DNA, PNA) of labeled probe and immobilized
polynucleotide or oligonucleotide. General parameters for specific
(i.e., stringent) hybridization conditions for nucleic acids are
described in Ausubel et al., Current Protocols in Molecular Biology
(Greene Publishing and Wiley-Interscience, New York 1987). Useful
hybridization conditions are also provided in, e.g., Tijessen,
Hybridization With Nucleic Acid Probes, (Elsevier Science
Publishers B.V., 1993) and Kricka, Nonisotopic DNA Probe
Techniques, (Academic Press, San Diego, Calif., 1992).
[0175] When cDNA complementary to the RNA of a cell is made and
hybridized to a microarray under suitable hybridization conditions,
the level of hybridization to the site in the array corresponding
to any particular gene will reflect the prevalence in the cell of
RNA transcribed from that gene. For example, when detectably
labeled (e.g., with a fluorophore) cDNA complementary to the total
cellular mRNA is hybridized to a microarray, the site on the array
corresponding to a gene (i.e., capable of specifically binding the
product of the gene) that is not transcribed in the cell will have
little or no signal (e.g., fluorescent signal), and a gene for
which the encoded mRNA is prevalent will have a relatively strong
signal.
[0176] U.S. Pat. No. 6,183,968 (to Bandman et al;) discloses
polynucleotide probes that can be used as hybridizable array
elements in a microarray, each of the polynucleotide probes having
at least a portion of a gene which encodes a protein associated
with cell proliferation or a receptor.
[0177] When fluorescently labeled probes are used, the fluorescence
emissions at each site of a transcript array can be, preferably,
detected by scanning confocal laser microscopy. In one embodiment,
a separate scan, using the appropriate excitation line, is carried
out for each of the two fluorophores used. Alternatively, a laser
can be used that allows simultaneous specimen illumination at
wavelengths specific to the two fluorophores and emissions from the
two fluorophores can be analyzed simultaneously (see Shalon et al.,
Genome Research 6:639-645 (1996)). Signals are recorded and,
preferably, analyzed by computer, using commercially available
methods. The abundance sort program of the invention described in
U.S. Pat. No. 5,840,484 can be used to tabulate and sort by
frequency the mRNA transcripts corresponding to each gene
identified. Since some of the polynucleotide sequences are
identified solely based on expression levels, it is not essential
to know a priori the function of a particular gene in solid tumor
stem cells.
[0178] Transcript image comparisons can be obtained by methods well
known to those skilled in the art. Transcript levels and images can
be obtained and compared, for example, by a differential gene
expression assay based On a quantitative hybridization of arrayed
DNA clones (Nguyen et al. Genomics 29:207-216 (1995), based on the
serial analysis of gene expression (SAGE) technology (Velculescu et
al. Science 270:484-487 (1995)), based on the polymerase chain
reaction (Peng et al. Science 257:967-971 (1992), based on a
differential amplification protocol (U.S. Pat. No. 5,545,522), or
based on electronic analysis, such as comparative gene transcript
analysis (U.S. Pat. No. 5,840,484) or the GEMTOOLS gene expression
analysis program (Incyte Pharmaceuticals, Palo Alto, Calif., USA).
Preferably, comparisons between two or more transcript profiles are
performed electronically.
[0179] U.S. Pat. No. 6,215,894 discloses a system for scanning
biochip arrays includes a unique image array identifier recorded
for each array, and a computer-stored record corresponding to each
identifier and containing the parameters of the experiment in the
array identified by the identifier. The system further includes
means for accessing a protocol library to retrieve the scanning
protocols associated with the identified arrays and then scanning
the arrays in accordance with the respective protocols. The
resulting image maps generated by the scanners are stored in
locations corresponding to the associated identifiers.
[0180] Measurement of the translational state may be performed
according to several methods. For example, whole genome monitoring
of protein (i.e., the "proteome,") can be carried out by
constructing a microarray in which binding sites comprise
immobilized, preferably monoclonal, antibodies specific to a
plurality of protein species encoded by the cell genome.
[0181] Use of Microarrays.
[0182] The microarrays describe above can be employed in several
applications including solid tumor: cancer diagnostics, prognostics
and treatment regimens, drug discovery and development,
toxicological and carcinogenicity studies, forensics,
pharmacogenomics and the like.
[0183] In one embodiment, the microarray is used to monitor the
progression of disease. Physicians can assess and catalog the
differences in gene expression between healthy and cancerous
tissues by analyzing changes in patterns of gene expression
compared with solid tumor stem cells from the patient. Thus, cancer
can be diagnosed at earlier stages before the patient is
symptomatic. The invention can also be used to monitor the efficacy
of treatment. For some treatments with known side effects, the
microarray is employed to "fine tune" the treatment regimen. A
dosage is established that causes a change in genetic expression
patterns indicative of successful treatment. Expression patterns
associated with undesirable side effects are avoided. This approach
may be more sensitive and rapid than waiting for the patient to
show inadequate improvement, or to manifest side effects, before
altering the course of treatment.
[0184] Alternatively, animal models which mimic a disease, rather
than patients, can be used to characterize expression profiles
associated with a particular disease or condition. This gene
expression data may be useful in diagnosing and monitoring the
course of disease in a patient, in determining gene targets for
intervention, and in testing novel treatment regimens.
[0185] Also, researchers can use the microarray to rapidly screen
large numbers of candidate drug molecules, looking for ones that
produce an expression profile similar to those of known therapeutic
drugs, with the expectation that molecules with the same expression
profile will likely have similar therapeutic effects. Thus, the
invention provides the means to determine the molecular mode of
action of a drug.
[0186] U.S. Pat. Nos. 6,218,122, 6,165,709, and 6,146,830 (all to
Friend et al.) discloses methods for identifying targets of a drug
in a cell by comparing (i) the effects of the drug on a wild-type
cell, (1i) the effects on a wild-type cell of modifications to a
putative target of the drug, and (iii) the effects of the drug on a
wild-type cell which has had the putative target modified of the
drug. In various embodiments, the effects on the cell can be
determined by measuring gene expression, protein abundances,
protein activities, or a combination of such measurements. In
various embodiments, modifications to a putative target in the cell
can be made by modifications to the genes encoding the target,
modification to abundances of RNAs encoding the target,
modifications to abundances of target proteins, or modifications to
activities of the target proteins. The present invention provides
an improvement to these methods of drug discovery by providing the
tumorigenic solid tumor stem cells, for a more precise drug
discovery program.
[0187] An "expression profile" comprises measurement of a plurality
of cellular constituents that indicate aspects of the biological
state of a cell. Such measurements may include, e.g., RNA or
protein abundances or activity levels. Aspects of the biological
state of a cell of a subject, for example, the transcriptional
state, the translational state, or the activity state, are
measured. The collection of these measurements, optionally
graphically represented, is called the "diagnostic profile".
Aspects of the biological state of a cell which are similar to
those measured in the diagnostic profile, e.g., the transcriptional
state, are measured in an analogous subject or subjects in response
to a known correlated disease state or, if therapeutic efficacy is
being monitored, in response to a known, correlated effect of a
therapy. The collection of these measurements, optionally
graphically represented, is called herein the "response profile".
The response profiles are interpolated to predict response profiles
for all levels of protein activity within the range of protein
activity measured. In cases where therapeutic efficacy is to be
monitored, the response profile may be correlated to a beneficial
effect, an adverse effect, such as a toxic effect, or to both
beneficial and adverse effects.
[0188] As is commonly appreciated, protein activities result from
protein abundances; protein abundances result from translation of
mRNA (balanced against protein degradation); and mRNA abundances
result from transcription of DNA (balanced against mRNA
degradation). Therefore, genetic level modifications to a cellular
DNA constituent alters transcribed mRNA abundances, translated
protein abundances, and ultimately protein activities. RNA level
modifications similarly alter RNA abundance and protein abundances
and activities. Protein level modifications alter protein
abundances and activities. Finally, protein activity modifications
are the most targeted modification methods. As is commonly
appreciated, it is ultimately protein activities (and the
activities of catalytically active RNAs) that cause cellular
transformations and effects. Also, most drugs act by altering
protein activities.
[0189] In one embodiment, cDNAs from two different cells (one being
the solid tumor stem cells of the invention) are hybridized to the
binding sites of the microarray. In the case of therapeutic
efficacy (e.g., in response to drugs) one cell is exposed to a
therapy and another cell of the same type is not exposed to the
therapy. In the case of disease states one cell exhibits a
particular level of disease state and another cell of the same type
does not exhibit the disease state (or the level thereof). The cDNA
derived from each of the two cell types are differently labeled so
that they can be distinguished. In one embodiment, for example,
cDNA from a cell treated with a drug (or exposed to a pathway
perturbation) is synthesized using a fluorescein-labeled dNTP, and
cDNA from a second cell, not drug-exposed, is synthesized using a
rhodamine-labeled dNTP. When the two cDNAs are mixed and hybridized
to the microarray, the relative intensity of signal from each cDNA
set is determined for each site on the array, and any relative
difference in abundance of a particular mRNA detected. The use of a
two-color fluorescence labeling and detection scheme to define
alterations in gene expression has been described, e.g., in Shena
et al., Science 270:467-470 (1995). An advantage of using cDNA
labeled with two different fluorophores is that a direct and
internally controlled comparison of the mRNA levels corresponding
to each arrayed gene in two cell states can be made, and variations
due to minor differences in experimental conditions (e.g.,
hybridization conditions) will not affect subsequent analyses.
Additional guidance is provided in EXAMPLE 21.
[0190] U.S. Pat. No. 6,194,158 (to Kroes et al.) for a diagnostic
assay for cancer with a DNA chip of specific sequences for
measuring expression levels of certain sequences within a cancer
cell to determine whether expression is up-or down-regulated. The
DNA chip comprising nucleotide sequences capable of hybridizing to
one or more members of a panel of DNA sequences may be synthesized
using commonly available techniques. mRNA is isolated from a
normal, non-cancer cell and a cancer cell and hybridized to the DNA
chip comprising one of more of the sequences from the panel.
Hybridization is then detected by any of the available methods. In
such a manner, sequences that are either overexpressed or
underexpressed in a cancer cell as compared to a normal cell are.
In a similar manner, mRNA from a cancer cell that has been
contacted with a compound may be hybridized to sequences on the DNA
chip to determine whether that compound affects expression of a
particular sequence. The present invention provides an improvement
over this method, in that the "cancer cell" from which mRNA can be
isolated is the tumorigenic solid tumor stem cell of the
invention.
[0191] Gene expression profiles of purified stem cells could give
clues for the molecular mechanisms of stem cell behavior. Terskikh
A V et al., Proc Natl Acad Sci USA 98(14): 7934-7939 (2001)
analyzed hematopoietic stem cells (HSC)-enriched cells by
comparison with normal tissue and mouse neurospheres (a population
greatly enriched for neural progenitor cells) by comparison with
terminally differentiated neural cells, using cDNA microarray
techniques and in situ hybridization, thus identifying potential
regulatory gene candidates. The invention provides an improved
method of drug discovery over the methods of Terskikh, in that the
use of the solid tumor stem cells of the invention can provide a
distinct set of drug targets when compared with a patient's normal
tissue (such as from the area of the solid tumor) or compared with
the other populations of cells obtained from the solid tumor.
[0192] Several other methods for utilizing DNA chips are known,
including the methods described in U.S. Pat. Nos. 5,744,305;
5,733,729; 5,710,000; 5,631,734; 5,599,695; 5,593,839; 5,578,832;
5,556,752; 5,770,722; 5,770,456; 5,753,788; 5,688,648; 5,753,439;
5,744,306 (all of which are incorporated by reference in their
entirety). U.S. Pat. No. 5,807,522 (to Brown et al.) discloses a
method to monitor early changes in a cell that correlate with
levels of a disease state or therapy and which precede detectable
changes in actual protein function or activity.
[0193] Moreover, microarrays of genomic DNA from solid tumor stem
cells can be probed for single nucleotide polymorphisms (SNP), to
localize the sites of genetic mutations that cause cells to become
precancerous or tumorigenic. Guidance for such methods are
available from the commercial vendors described above and may be
found in general genetic method books, such as those described
herein.
[0194] Vaccines.
[0195] The solid tumor stem cells of the invention can be used to
raise anti-cancer cell antibodies. In one embodiment, the method
involves obtaining an enriched population of solid tumor stem cells
or isolated solid tumor stem cells; treating the population to
prevent cell replication (for example, by irradiation); and
administering the treated cell to a human or animal subject in an
amount effective for inducing an immune response to solid tumor
stem cells. For guidance as to an effective dose of cells to be
injected or orally administered; see, U.S. Pat. Nos. 6,218,166,
6,207,147, and 6,156,305, incorporated herein by reference. In
another embodiment, the method involves obtaining an enriched
population of solid tumor stem cells or isolated solid tumor stem
cells; mixing the tumor stem cells in an in vitro culture with
immune effector cells (according to immunological methods known in
the art) from a human subject or host animal in which the antibody
is to be raised; removing the immune effector cells from the
culture; and transplanting the immune effector cells into a host
animal in a dose that is effective to stimulate an immune response
in the animal.
[0196] Monoclonal antibodies to solid tumor stem cells may be
prepared using any technique which provides for the production of
antibody molecules by continuous cell lines in culture.
[0197] These include, but are not limited to, the hybridoma
technique, the human B-cell hybridoma technique, and the
EBV-hybridoma technique (see, e.g., Kozbor, D. et al., J. Immunol.
Methods 81:31-42 (1985); Cote R J et al. Proc. Natl. Acad. Sci.
80:2026-2030 (1983); and Cole S P et al. Mol. Cell. Bioi.
62:109-120 (1984>>.
[0198] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used (see,
e.g., Morrison S L et al. Proc. Natl. Acad. Sci. 81:6851-6855
(1984); Neuberger M S et al. Nature 312:604-608 (1984); and Takeda
S et al. Nature 314:452-454 (1985>>.
[0199] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art.
[0200] The antibody can also be a humanized antibody. The term
"humanized antibody," as used herein, refers to antibody molecules
in which the amino acid sequence in the non-antigen binding regions
has been altered so that the antibody more closely resembles a
human antibody, and still retains its original binding ability.
Antibodies are humanized so that they are less immunogenic and
therefore persist longer when administered therapeutically to a
patient.
[0201] Human antibodies can be generated using the XenoMouse.TM.
technology from Abgenix (Fremont, Calif., USA), which enables the
generation and selection of high affinity, fully human antibody
product candidates to essentially any disease target appropriate
for antibody therapy. See, U.S. Pat. Nos. 6,235,883, 6,207,418,
6,162,963, 6,150,584, 6,130,364, 6,114,598, 6,091,001, 6,075,181,
5,998,209, 5,985,615, 5,939,598, and 5,916,771, each incorporated
by reference; Yang X et al., Crit. Rev Oncol Hemato 38(1): 17-23
(2001); Chadd H E & Chamow S M. Curr Opin Biotechnol
12(2):188-94 (2001); Green L L, Journal of Immunological Methods
23111-23 (1999); Yang et al., Cancer Research 59(6): 1236-1243
(1999); and Jakobovits A, Advanced Drug Delivery Reviews 31: 33-42
(1998). Antibodies with fully human protein sequences are generated
using genetically engineered strains of mice in which mouse
antibody gene expression is suppressed and functionally replaced
with human antibody gene expression, while leaving intact the rest
of the mouse immune system.
[0202] Moreover, the generation of antibodies directed against
markers present in or on the solid tumor stem cells of the
invention can be used as a method of identifying targets for drug
development. The antibodies that are raised in an immune response
to the solid tumor stem cells can be used to identify antigenic
proteins on the solid tumor stem cells using methods known in the
art (Harlow, Using Antibodies: A Laboratory Manual (Cold Spring
Harbor Press, Cold Spring Harbor, N.Y., (1999)) and can further be
used to identify polynucleotides coding for such proteins (Sambrook
et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor
Press, Cold Spring Harbor, N.Y., 1989); Current Protocols in
Molecular Biology, Ausubel et al., eds., (Wiley Interscience, New
York, 1993). Once identified, the proteins and polynucleotides can
be compared with other proteins and polynucleotides previously
identified to be involved in cancer. In one embodiment, the
XenoMouse.TM. technology to produce fully human antibodies can be
used to generate antibodies directed against drug development
targets (see, Jeffrey Krasner, Boston Globe (Jul. 25, 2001) at F4).
The present invention provides an improvement to these
antibody-based methods of drug discovery by providing the
tumorigenic solid tumor stem cells, to which the immune response is
raised, for a more precise drug discovery program.
[0203] The Microarray Network.
[0204] In addition to the cDNA libraries and DNA array techniques
described above for systematically studying gene expression
patterns, one of skill in the molecular biological art can obtain
guidance for the use of microarray technology from the University
of Michigan Microarray Network (see also, EXAMPLE 9). Furthermore,
the University of Michigan has made a large commitment to
bioinformatics, by providing funding specifically for microarray
data management and analysis, as well as by founding a new
Bioinformatics Center for the University.
[0205] Alternatively, methods for raising an immune response can
take advantage of the "stem cell" qualities of the solid tumor stem
cell of the invention. Solid tumor stem cells, solid tumor stem
cell protein extracts, purified proteins from solid tumor stem
cells, or proteins derived from the expression of cDNAs from solid
tumor stem cells (see, above for genetic modification of solid
tumor stem cells) to induce an immune response in an animal. The
immune response can be directed against cancer cells, as shown by
standard immunological methods. For example, the solid tumor stem
cells (enriched populations of isolated cells) or proteins can be
contacted with o dendritic cells in culture, antigen presenting
cells in culture, or antigen presenting cells and T cells in
culture. Then antigen-stimulated cells are infused back into the
patient.
[0206] Alternatively, the solid tumor stem cells of the invention
can be genetically engineered to promote an immune response against
the tumor stem cells. For example, hematopoietic stem cells can be
engineered to contain a T-cell receptor targeting a tumor stem cell
antigen. See, U.S. Pat. Nos. 5,914,108 and 5,928,638 incorporated
by reference. Thus T cell receptors that recognize antigens
expressed by tumor stem cells can be identified, then cloned into
hematopoietic stem cells. The engineered hematopoietic stem cells
can then be transplanted into a patient and allowed to engraft,
giving rise to large numbers of T cells that express receptors
recognizing the tumor stem cells. By increasing the numbers of
tumor stem cell-specific T cells the anti-tumor immune response can
be potentiated.
[0207] Other means are also available for increasing the anti-tumor
immune response, including using the tumor stem cells as the basis
of a vaccine, using the tumor stem cells to stimulate antigen
presenting dendritic cells, and using the tumor stem cells as an
innoculum to generate anti-tumor antibodies. Tumor stem cells can
be used as a vaccine by killing a patient's tumor stem cells, such
as by irradiation, and readministering the killed stem cells back
into the patient in a physiological and immunologically acceptable
carrier, for the purpose of generating an immune response against
the tumor stem cells. See, U.S. Pat. No. 4,960,716, in which
antibodies were raised to membrane vesicle preparations of breast
carcinoma cell cells; U.S. Pat. No. 4,584,268, in which anti-human
mammary epithelial antibody was produced a membrane fraction of
delipidated human milk fat globules; both incorporated by
reference.
[0208] Dendritic cells from a patient can be cultured in vitro and
killed tumor stem cells from the same patient can be added to the
cultures to stimulate the dendritic cells. The activated dendritic
cells, presenting tumor stem cell antigens, can then be
re-administered to the patient to stimulate the patient's
anti-tumor response. Finally, tumor stem cells can be administered
to an animal such as a mouse, rat, hamster, goat, sheep, rabbit, or
donkey to generate antibodies against the tumor stem cells.
Preferably, monoclonal anti-tumor stem cell antibodies are made in
mouse, rat, or hamster. Monoclonal antibodies that are made in this
way can then be administered to patients, or first humanized (as
described above) and then administered to patients, to promote an
immune response against the tumor stem cells in the patient.
[0209] Furthermore, adenoviral vectors have proven especially
useful for gene transfer into eukaryotic cells for vaccine
development. Graham F L & Prevec L, In Vaccines: New Approaches
to Immunological Problems, Ellis R V ed., 363-390
(Butterworth-Heinemann, Boston, 1992).
[0210] Probe for Scanning Microarrays.
[0211] The complete sequencing of the human genome makes possible
the identification of the genes expressed by a particular
population of cells. Probes from enriched populations of tumor stem
cells can be made using methods known to the art (see, Wang et al.,
Nature Biotechnology 18: 457 (2000). Analysis of gene expression
patterns and protein expression patterns for tumor stem cells and
tumor cell progeny populations can be performed by comparing
results to known Gene Pattern Databases or to known Protein Pattern
Databases (for example, PROSITE, PRINTS: Protein Motif Fingerprint
Database, BLOCKS, PFAM, nOMa, PROnOM, or other databases). Searches
can be preformed, for example, using the BCM Search Launcher:
General Protein Sequence/Pattern Searches (Baylor College of
Medicine, Human Genome Sequencing Center, One Baylor Plaza,
Houston, Tex.). Commercially available programs for gene and
protein analysis are also available (such as CGC (from Genetics
Computer Group, Inc.) and DNA STRIDER).
[0212] The factors involved in the proliferation, differentiation,
or survival of tumor stem cells and tumor stem cell progeny, or
their responses to biological agents can be isolated either by
constructing cDNA for libraries or to probe microarrays from tumor
stem cells, or tumor stem cell, non-tumorigenic cancer cells, or
normal tumor cells using techniques known in the art. cDNA can also
be made from any of the different populations after exposure to
biological agents or drugs to determine the response to such
manipulations. The libraries from cells of one population are
compared with those of cells of different populations to determine
the sequence of gene expression during development and to reveal
the effects of various biological agents or to reveal new
biological agents that alter gene expression in cancer cells. When
the libraries are prepared from neoplastic tissue, genetic factors
may be identified that play a role in the cause of cancer cell
growth, for example, by comparing the libraries from the cancerous
tissue with those from normal tissue. This information can be used
in the design of anti-cancer therapies. Additionally, probes can be
identified for use in the diagnosis of various cancers or for use
in identifying cells at a particular stage in tumor
development.
[0213] Diagnostic and Prognostic Evaluation of Tumors.
[0214] A variety of methods can be employed for the diagnostic and
prognostic evaluation of tumor and metastasis, and for the
identification of subjects having a predisposition to such
conditions. Among the methods well known in the art are the use of
bone scans, X-ray imaging, MRI tests, CAT scans, and blood tests
for tumor associated antigens (see, American Cancer Society, Cancer
Facts and Figures 1999: Selected Cancers (1999); American Cancer
Society, Breast Cancer Guidelines and Statistics (1999); Kopans
Breast Imaging. rd Edition (JB Lippincott, Philadelphia, Pa.,
1998); Potter & Partin, NCCN Practice Guidelines for Early
Detection of Prostate Cancer 13(11A) Oncology (November 1999). For
additional methods of detection, see Franklin et al, Breast Cancer
Research & Treatment 41(1): 1-13 (1996); Kufe et al., Cancer
Research 43(2): 851-7 (1983). For bone scans, nuclear medicine
imaging can be used. Nuclear medicine may be used in addition to
mammography to help identify certain abnormalities. Nuclear
medicine is also a good tool for evaluating the metastasis of
cancer into the lymphatic system, other organs and skeletal system.
Tumor associated antigens include for example, BCA 225 (U.S. Pat.
No. 5,681,860); Bladder Tumor Associated Antigen (BTA stat test,
ARUP Laboratories, Salt Lake City, Utah); tumor-associated antigen
CAl25 (Wagner et al., Hybridoma 16(1): 33-40 (1997)); 22-1-1 Ag, YH
206, GA 733, CA 125, carcinoembryonic antigen, and sialyl Lex
(Sonoda et al., Cancer 77(8): 1501-1509 (1996). For breast cancer
prognosis, cancerous cells can be looked for in the patientls bone
marrow. See, for example, Bruan et al., New Engl. J. Med (Feb. 24,
2000>>.
[0215] Such methods may, for example, utilize reagents such as VEGF
nucleotide sequences and VEGF antibodies. Specifically, such
reagents may be used, for example, for: (1) the detection of the
presence or over-expression of VEGF mRNA relative to the
non-carcinogenic tissue state; (2) the detection of an
over-abundance of VEGF protein relative to the non-carcinogenic
tissue state; (3) the detection of hypoxic conditions in the tumor
mass; (4) the detection of the expression of VEGF tyrosine kinase
receptors and other angiogenic receptors in adjacent endothelial
tissues; and (5) the detection of the expression of oncogenes. The
methods may be performed, for example, by utilizing pre-packaged
diagnostic kits comprising at least one specific VEGF nucleotide
sequence or VEGF antibody reagent described herein, which may be
conveniently used, e.g., in clinical settings, to diagnose patients
at risk for tumor angiogenesis and metastasis.
[0216] Further, the expression of different oncogene alleles may be
assessed using these methods. The additional information obtained
regarding the expression of other markers provides guidance for the
design of appropriate therapies to inhibit angiogenesis or tumor
proliferation tailored to the molecular stage of the cancer in a
particular patient.
[0217] Drug Discovery.
[0218] The invention provides a method for identifying a test
compound for reducing solid tumors. The practice of the method can
be further determined using the guidance provided in the EXAMPLES
below. The steps of the method include assaying the response of
tumor cells to biological agent and determining the effects of the
biological agent on the tumor stem cell. In other words, the
invention provides improved methods of drug discovery by the use of
the solid tumor stem cells of the invention.
[0219] Proof of principle of the use of the invention for drug
discovery is provided in drug discovery in EXAMPLE 11 and FIG. 11,
where the epidermal growth factor (EGF) receptor (EGF-R) and
HER2/neu markers (known to be involved in cancers) were identified
on solid tumor stem cells. Accordingly, therapies directed against
the EGF-R (e.g., Yang X et al., Crit. Rev Oncol Bematol. 38(1):
17-23 (2001)) and HER2/neu markers (see, Breast Disease, Vol. II,
HER2: Basic Research, Prognosis and Therapy, Y. Yarden, ed. (IOS
Press, Amsterdam, 2000)) can be effectively targeted to solid tumor
stem cells.
[0220] The identification of biological pathways is an important
part of modern drug discovery process. Biological pathways in solid
tumor stem cells and other cell populations obtained from solid
tumors, particularly pathways involved in drug actions, i.e.,
pathways that originate at a drug target (e.g., proteins), can be
identified for use as shown by U.S. Pat. No. 5,965,352, which is
incorporated herein by reference.
[0221] In one set of methods, drugs are screened to determine the
binding of test compounds to receptors, in which the binding
activates a cell's biological pathway to cause expression of
reporter polypeptides. Frequently the reporter polypeptides are
coded for on recombinant polypeptides, in which the coding
polynucleotide is in operable linkage with a promoter.
[0222] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related polynucleotides. A
promoter is operably associated or operably linked with a coding
sequence if the promoter controls the translation of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in the same reading frame,
certain genetic elements, e.g., repressor genes, are not
contiguously linked to the sequence encoding the polypeptide but
still bind to operator sequences that control expression of the
polypeptide.
[0223] The detectable signal can be fluorescence, absorbence, or
luminescence, depending on the reporter polypeptide, which can be,
for example, luciferase (firefly luciferase, Vibrio fisceri
luciferase, or Xenorhabdus luminescens luciferase), green
fluorescent protein, green fluorescent protein variant,
chloramphenicol acetyltransferase, .beta.-glucuronidase,
.beta.-galactosidase, neomycin phosphotransferase, guanine xanthine
phosphoribosyltransferase, thymidine kinase, .beta.-lactamase,
alkaline phosphatase, invertase, amylase (for yeast based assays)
human growth hormone (for activity based assays). The fluorescent
detectable signal can be fluorescence resonance energy transfer
(FRET), bioluminescence resonance energy transfer (BRET),
time-resolved fluorescence (TRF) or fluorescence polarization (FP).
Where appropriate, the detectable signal is detected by a machine
such as a fluorometer, luminometer, fluorescence microplate reader,
dual-monochromator microplate spectrofluorometer,
spectrophotometer, confocal microscope (laser scanner), or a
charge-coupled device (CCD). The detectable signal is determined by
comparing the amount of signal produced when the reporter
polypeptide is expressed in the tumor stem cell with the signal
produced when the reporter polypeptide is not expressed in the
tumor stem cell.
[0224] Another technique for drug screening provides for high
throughput screening of compounds (see, e.g., PCT application WO
84/03564.) In this method, large numbers of different small test
compounds are synthesized on a solid substrate, such as plastic
pins or some other surface. The test compounds are reacted with
solid tumor stem cells, or portions thereof, and washed. Bound
solid tumor stem cells are then detected by methods well known in
the art, using commercially available machinery and methods, for
example, the Automated Assay Optimization (AAO) software platforms
(Beckman, USA) that interface with liquid handlers to enable direct
statistical analysis that optimizes the assays; modular systems
from CRS Robotics Corp. (Burlington, Ontario), liquid handling
systems, readers, and incubators, from various companies using
POLARA.TM. (CRS), an open architecture laboratory automation
software for a Ultra High Throughput Screening System; 3P
(Plug&Play Peripherals) technology, which is designed to allow
the user to reconfigure the automation platform by plugging in new
instruments (ROBOCON, Vienna, Austria); the Allegro.TM. system or
Staccato.TM. workstation (Zymark), which enables a wide range of
discovery applications, including HTS, ultra HTS, and high-speed
plate preparation; MICROLAB Vector software (Hamilton Co., Reno,
Nev., USA) for laboratory automation programming and integration;
and others.
[0225] For any of these machines and methods, the assays measure a
response the target cells (solid tumor stem cells or genetically
modified solid tumor stem cells) that provides detectable evidence
that the test compound may be efficacious. The detectable signal is
compared to control cells and the detectable signal identified by
subtraction analysis. The relative abundance of the differences
between the "targeted" and "untargeted" aliquots are simultaneously
compared using a "subtraction" analysis (differential analysis)
technique such as differential display, representational difference
analysis (RDA), GEM-Gene Expression Microarrays (U.S. Pat. No.
5,545,531), suppressive subtraction hybridization (SSH) and direct
sequencing (PCT patent application WO 96/17957). The subtraction
analysis can include the methods of differential display,
representational differential analysis (RDA), suppressive
subtraction hybridization (SSB), serial analysis of gene expression
(SAGE), gene expression microarray (GEM), nucleic acid chip
technology, or direct sequencing.
[0226] The solid tumor stem cell of the invention is particularly
useful in the drug development process because solid tumor stem
cells provide a limited and enriched set of targets for drug
development. One of the most important steps in rational drug
design is the identification of a target, the molecule with which
the drug itself interacts. Frequently, the target will be a
receptor on or in a tumorigenic solid tumor stem cell.
[0227] Likewise, the genetically modified solid tumor stem cell of
the invention is particularly useful in the drug development. For
example, the genetically modified stem cell can contain
polynucleotide with a promoter operably linked to the
polynucleotide encoding a reporter polypeptide. The reporter
polypeptide is expressed in the tumor stem cell after a receptor of
the tumor stem cell is activated by binding to a test compound or
inactivated by binding to a test compound. Such a detectable signal
makes the genetically modified solid tumor stem cell appropriate
for use in high throughput screening (HTS).
[0228] The detectable signal can be a result of a positive
selection or a negative selection. The positive selection includes
manipulations that test the ability of cells to survive under
specific culture conditions, ability to express a specific factor,
changes in cell structure, or differential gene expression. The
selection can be based on the ability of the solid tumor stem cells
or genetically modified solid tumor stem cells to:
[0229] (a) Grow or survive under specific culture conditions, such
as in vitro cell culture.
[0230] (b) Express a specific factor that can be measured, the
measurement adaptable for a selection. This factor can be anything
that is accessible to measurement, including but not limited to,
secreted molecules, cell surface molecules, soluble and insoluble
molecules, binding activities, activities that induce activities on
other cells or induce other organic or inorganic chemical
reactions.
[0231] (c) Changes in cell structure, including morphological
changes that are measured by physical methods such as differential
sedimentation, differential light scattering, differential buoyant
density, differential cell volume selected by sieving.
[0232] (d) Differences in gene expression that can be directly
measured, including changes in cell surface markers, changes in
biochemical activities, any changes that would be re-selected in
changes in binding of fluorescent labeled probes that could be used
in conjunction with a Fluorescence Activated Cell Sorter (FACS) or
any property that can be used as a basis for a selection.
Genetically modified solid tumor stem cells containing
polynucleotides that express reporter polypeptides are particularly
useful here.
[0233] (e) Differences in gene expression that can be indirectly
measured, including changes in transcription factor activity that
are measured by a synthetic gene construct encoding a selective
marker (such as a drug resistance marker or a cell surface marker
that could be used in a FACS selection). This category would also
include changes in mRNA stability, mRNA localization, mRNA
translation control. All of these changes could be the basis of a
selection because a synthetic construct which is controlled by one
of these regulatory events could be constructed which would drive
the expression of an easily selected gene product.
[0234] Pharmacogenomics.
[0235] The invention provides an improved method of ascertaining
propensity for malignancy, monitoring the progress of chemotherapy
or other anticancer therapy, screening for re-occurrence of cancer,
or other similar detection of present or potential cancer, where
such method detects for the expression of at least one gene which
is over- or under-expressed in a potential cancer cell, as compared
with either a solid tumor stem cell isolated from the patient or a
collection of solid tumor stem cells. In one embodiment, the method
is the assaying of a biological sample (such as from the patient)
to be tested for a signal indicating the transcription of a
significant (by comparison with the solid tumor stem cell)
polynucleotide transcript. In addition, screening assays of
biological samples are contemplated, where such assays are
conducted during the course of chemotherapy alone, or after
surgical intervention to treat cancer, to monitor for the continued
presence or return of cancerous cells.
[0236] Other Embodiments of the Invention.
[0237] The invention provides an article of manufacture (a system
or a kit), comprising packaging material and a primary reagent
contained within said packaging material. The primary reagent is
solid tumor stem cell preparation as described above. The packaging
material includes a label that indicates that the primary reagent
can be used for identifying an agent for reducing solid tumors.
Also, the invention provides a kit for determining the activity
level of a particular polynucleotide or protein in a cell. Such
kits contain arrays or microarrays containing a solid phase, e.g.,
a surface, to which are bound, either directly or indirectly, solid
tumor stem cells (enriched populations of or isolated),
polynucleotides extracted from such solid tumor stem cells, or
proteins extracted from such solid tumor stem cells. The kit may
also contain probes that are hybridized or bound to the solid tumor
stem cell components at a known location of the solid phase. These
probes consist of nucleic acids of known, different sequence, with
each nucleic acid being capable of hybridizing to an RNA species or
to a cDNA species derived therefrom. In particular, the probes
contained in the kits of this invention are nucleic acids capable
of hybridizing specifically to nucleic acid sequences derived from
RNA species which are known to increase or decrease in response to
perturbations correlated to the particular diseases or therapies to
be monitored by the kit. The probes contained in the kits of this
invention preferably substantially exclude nucleic acids which
hybridize to RNA species that are not increased or decreased in
response to perturbations correlated to the particular levels of
disease states or therapeutic effects to be determined by the
kit.
[0238] The details of one or more embodiments of the invention have
been set forth in the accompanying description above. Although any
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are now described.
Other features, objects, and advantages of the invention will be
apparent from the description and from the claims.
[0239] In the specification and the appended claims, the singular
forms include plural referents. Unless defined otherwise in this
specification, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in
the art to which this invention belongs. All patents and
publications cited in this specification are incorporated by
reference.
[0240] The following EXAMPLES are presented in order to more fully
illustrate the preferred embodiments of the invention. These
EXAMPLES should in no way be construed as limiting the scope of the
invention, as defined by the appended claims.
Example 1
Isolation of Breast Cancer Stem Cells
[0241] The purpose of this EXAMPLE is to show structural and cell
functional characterization of breast cancer stem cells.
[0242] We have developed both a tissue culture and a mouse model to
identify the breast tumor clonogenic cell. In the mouse model,
NOD/SCID mice Lapidot et al., Nature 367(6464): 645-8 (1994>>
are treated with VP-16 (Etoposide) (available from commercial
sources, such as Moravek Biochemicals, Brea, Calif., USA), and
implanted with primary human breast cancer tissue (obtained from
mastectomy or lumpectomy specimens). Three of five primary tumors
formed tumors in this system.
[0243] The tumor cells isolated from malignant pleural effusions
obtained from two patients (see, Zhang et al., Invasion &
Metastasis 11(4): 204-15 (1991)) were suspended in Matrigel.TM.
(available from Becton Dickinson, Franklin Lakes, N.J., USA), then
were injected into mice. Tumors formed in the injected mice.
[0244] By this method, we can generate enough tumor cells for
analysis by FACS. We also can generate enough tumor cells to
perform biological assays to characterize the cells. (For
clonogenic assay for detecting rare tumor cells in hematopoietic
samples, see U.S. Pat. No. 5,674,694, incorporated by
reference).
[0245] Phenotypically distinct subsets of tumor cells can be
isolated by any suitable means known in the art, including FACS
using a fluorochrome conjugated marker-binding reagent. Any other
suitable method including attachment to and disattachment from
solid phase, is also within the scope of the invention. Procedures
for separation may include magnetic separation, using
antibody-coated magnetic beads, affinity chromatography and
"panning" with antibody attached to a solid matrix, e.g. plate, or
other convenient technique. Techniques providing accurate
separation include fluorescence activated cell sorters, which can
have varying degrees of sophistication, such as multiple color
channels, low angle and obtuse light scattering detecting channels,
impedance channels, etc. Dead cells may be eliminated by selection
with dyes that bind dead cells (such as propidium iodide (PI), or
7-AAD). Any technique may be employed that is not unduly
detrimental to the viability of the selected cells.
[0246] The marker-binding reagent can be directly or indirectly
conjugated to a magnetic reagent, such as a superparamagnetic
microparticle (microparticle). Direct conjugation to a magnetic
particle is achieved by use of various chemical linking groups, as
known in the art. Antibody can be coupled to the microparticles
through side chain amino or sufhydryl groups and heterofunctional
cross-linking reagents. A large number of heterofunctional
compounds are available for linking to entities. A preferred
linking group is 3-(2-pyridyldithio)propionic acid
N-hydroxysuccinimide ester (SPDP) or
4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid
N-hydroxysuccinimide ester (SMCC) with a reactive sulfhydryl group
on the antibody and a reactive amino group on the magnetic
particle. Alternatively, the marker-binding reagent is indirectly
coupled to the magnetic particles. The marker-binding reagent is
directly conjugated to a hapten, and hapten-specific, second stage
antibodies are conjugated to the particles. Suitable haptens
include digoxin, digoxigenin, FITC, dinitrophenyl, nitrophenyl,
avidin, biotin, etc. Methods for conjugation of the hapten to a
protein, i.e. are known in the art, and kits for such conjugations
are commercially available. Fluorochrome labeled antibodies are
useful for FACS separation, magnetic particles for immunomagnetic
selection, particularly high gradient magnetic selection (HUMS),
etc. Exemplary magnetic separation devices are described in PCT
patent applications WO 90/07380 and WO 96/09550, and European
patent 0 438 520.
[0247] We have extensively studied the tumors formed by one of the
primary tumors and one of the malignant pleural effusion cells. We
have identified low serum tissue culture conditions in which
primary breast cancer cells and cells isolated from a mouse
xenograft proliferates for at least 1-3 weeks in tissue culture.
Using the in vitro tissue culture model, we found that stimulation
of a specific receptor can affect the growth and survival of breast
cancer cells.
[0248] We have used the in vitro (tissue culture) and in vivo
(mouse xenograft) models of human breast cancer. A human tumor
growing in the mouse model was harvested and made into a single
cell suspension and analyzed by FACS. We found a heterogeneity of
expression of cell surface markers on tumor cells. Initially,
breast cancer cells isolated from a malignant pleural effusion were
separated into groups based upon CD44 expression. Cells were
analyzed for expression of markers 520C9 and CD44 (see, FIG. 2).
520C9 is known to recognize c-erbB-2 (HER-2/neu). Ring et al.,
Molecular Immunology 28:915 (1991). See also, U.S. Pat. No.
4,753,894, which discloses murine monoclonal antibodies that bind
selectively to human breast cancer cells. Four populations of cells
were identifiable. There was a small population of cells that
expressed both markers 520C9 and CD44, a population that expressed
either marker alone, as well as a population that expressed neither
marker. Cells were isolated with regard to CD44 expression (FIG.
2). CD44.sup.+ or CD44.sup.- cells were tested for their ability to
proliferate. Marker CD44.sup.+ tumor cells, but not marker
CD44.sup.- tumor cells, were able to form colonies in vitro and
tumors in vivo
(TABLE 1). Note that isolation of CD44.sup.+ cells results in at
least a 2.5-fold purification of the tumorigenic cells.
TABLE-US-00001 TABLE 1 ISOLATED POPULATIONS OF BREAST CANCER STEM
CELLS CD44.sup.+ CD44.sup.- Colonies in vitro + - Tumorigenicity in
mice + - Human breast cancer cells were collected using FACS.
Analysis of in vitro colonies was done in 2 separate wells using
5,000 cells of the respective phenotype. In vivo growth of sorted
cells was done by injecting mice with 2 .times. 10.sup.6 marker
CD44.sup.+ or CD44.sup.- cells. Mice were analyzed at week 3 in
experiment 1 and week 4 in experiment 2. The injections of marker
CD44.sup.+ cells, but not marker CD44.sup.- cells, resulted in
tumor formation and growth in vitro. The in vitro experiments have
been replicated using frozen cells isolated from the patient and
support the in vitro experiments. The in vivo experiments have been
replicated twice.
[0249] These results serve as a proof-of-principle of the stem cell
model of solid cancer and demonstrate the following:
[0250] (a) solid tumor cells are phenotypically and functionally
heterogeneous; some tumor cells are tumorigenic, while others have
limited proliferative potential and do not form tumors upon
transplantation;
[0251] (b) by separating cells by FACS, one can enrich for
tumorigenic cells; and
[0252] (c) by studying the tumorigenic fractions one can isolate
tumor stem cells and more carefully focus strategies for
identifying therapeutic targets.
[0253] This EXAMPLE shows that that the clonogenic breast cancer
tumor cell from two tumors express CD44. Other markers also allow
the further purification of the breast cancer stem cell. We have
analyzed the tumor cells for expression of several antigens. Some
antigens with heterogeneous expression patterns include MUCI,
Notch-4, annexin V, 317G5, CD9, CD24, 260F9.about.P-glycoprotein
and CD49F. 260F9 binds to a 55 kilodalton glycoprotein (mucin) B
cell surface antigen. Weiner L M et al., Cancer Res. 49:4062-4067
(1989); Gregg, E O. et al J. Immunol, 138:4502-4508 (1987). See
also, U.S. Pat. No. 4,753,894, which discloses murine monoclonal
antibodies that bind selectively to human breast cancer cells.
Combinations of these markers with CD44 permit increased enrichment
of the tumor stem cells beyond what was achieved with CD44
alone.
[0254] Remarkably, all of the CD44.sup.+ (tumorigenic) cells are
also B38.1.sup.+. See, Kufe et al., Cancer Research 43(2): 851-7
(1983) for description of B38.1 antibody. Annexin, Notch-4, and
CD24 expression is heterogeneous by the B38.1.sup.+ cells. We can
thus further purify the breast cancer stem cell from two tumors by
analyzing various subpopulations of B38.1.sup.+ or CD44.sup.+
cells. Indeed, we have isolated B38.1.sup.+CD24.sup.+, and
B38.1.sup.+CD24.sup.- cells obtained from a primary biopsy and
placed them in tissue culture. Only the B38.1.sup.+CD24.sup.- cells
formed colonies. In another tumor, we isolated the
260F9.sup.+CD24.sup.-, 260F9.sup.+CD24.sup.hi, and the
260F9.sup.+CD24.sup.-/lo populations of cells. Only the
CD24.sup.-/lo population of cells formed tumors (TABLE 2). Note
that there is a 5-6 fold enrichment of tumorigenic cells using
B38.1 or 260F9 and CD24.
TABLE-US-00002 TABLE 2 ANALYSIS OF TUMORIGENICITY OF CD24.sup.+ AND
CD24-/l0 CELLS Tumor formation CD24.sup.+ CD24.sup.-/lo Tumor T1 -
+ Tumor T2 - + NOD/SCID mice were injected with either
50,000-200,000 CD24.sup.+ or CD24.sup.-/lo cells and analyzed for
tumor formation two weeks later.
Example 2
Role for Notch in Breast Cell Proliferation
[0255] The purpose of this EXAMPLE is to provide preliminary
evidence that in at least two different tumors, Notch 4 is
expressed by a minority of the tumorigenic cells. Cells from tumor
T1 and tumor T2 from EXAMPLE 1 were analyzed for expression of
Notch 4. Cells were stained with an anti-Notch 4 polyclonal
antibody and analyzed by FACS. 5-15% of cells expressed detectable
levels of Notch4. Furthermore, different populations of
non-tumorigenic cells express different Notch ligands and members
of the Fringe family. To determine which Notch RNAs are expressed
by normal breast tissue and breast tumor tissue, we performed
RT-PCR using primers specific for each Notch mRNA. Interestingly,
Notch 1, Notch 3 and Notch 4, but not Notch 2, were expressed by
both normal breast cells and breast tumor cells. We prepared RNA
from 100,000 cells. RT-PCR of RNA from normal breast cells or
breast tumor cells was performed using primers specific for Notch
1, Notch 2, Notch 3, and Notch 4. A PCR product of the predicted
size was present for Notch 1, 3, and 4, showing the presence of
these markers in these cells. The signal was lost if the RNA was
pretreated with Rnase.
[0256] To determine the role of Notch in proliferation, a Notch
recombinant ligand or agonist peptides of the ligand were added to
the medium in cultures of normal mammary epithelial cells. Both
Notch agonists stimulated the survival/proliferation of single
cells: in the presence of the Notch agonists, 2-3 times more
colonies formed and they included a higher percentage of large,
mixed colonies (40% versus 20%). When single cells were plated at
clonal density, we obtained two kinds of colonies. There were large
colonies made up of hundreds to thousands of cells, presumably
arising from solid tumor stem cells, that were of a mixture of
myoepithelial cells and ductal epithelial cells. There were also
smaller colonies of cells that appeared to contain only a single
lineage and that probably represented the progeny of restricted
progenitors.
[0257] From a normal breast epithelial cell grown in vitro,
bilineage colonies (containing both myoepithelial and ductal
epithelial cells) were generated by single cells. Myoepithelial
cells were identified by staining with an anti-CALLA antibody.
Ductal epithelial cells were identified by staining with an
anti-ESA antibody. Organoids grown in Matrigel.TM. in the presence
or absence of the Notch agonist peptide branched and proliferated
more in the presence of the Notch agonist peptide. This agonist
peptide also inhibited differentiation, as indicated by the
inhibition of casein production. These results demonstrate that
these assays also provide a means for detecting multipotent
progenitors from normal breast epithelium. This assay may be used
for the purification of normal breast stem cells. These results
also demonstrate that Notch has a function in normal breast
development.
[0258] We then examined Notch 4 expression in breast cancer tumor
cells. High levels of Notch 4 were expressed on a minority of the
tumor cells. When the B38.1 population, which identifies the
tumorigenic population, was analyzed for Notch 4 expression, a
distinct minor population of cells that were B38.1Iow, Notch
4.sup.+became apparent.
[0259] We sorted Notch 4.sup.+ and Notch 4.sup.- cells and analyzed
them in vitro. Surprisingly, neither population grew in tissue
culture. There were two possible explanations. First, it was
possible that interaction between the two populations of cells was
required for cell growth. Next, the antibody may be either an
agonist or an antagonist of Notch 4 and may inhibit tumor cell
growth. To distinguish between these possibilities, tumor cells
were incubated with the anti-Notch 4 antibody, and then assayed in
vitro. Breast cancer cells were placed in tissue culture after
exposure to an anti-Notch 4 antibody. Cells were incubated on ice
for 1 hr. in HBSS containing no anti-Notch 4 antibody, anti-Notch 4
antibody, or anti-Notch 4 antibody that had been preincubated with
the peptide used to generate the antibody. Cells were also grown in
the presence of soluble Delta and without soluble Delta as a
control.
[0260] While control cells grew in tissue culture and formed
colonies, cells incubated with the Notch 4 antibody did not. When
the anti-Notch 4 antibody was incubated with the peptide used to
generate the antibody before addition to the cells, growth was
restored. To confirm a role for Notch for growth in vitro, cells
were incubated in serum free conditions in media that contained
soluble Delta, a Notch ligand, or a control culture without Delta.
The cultures with Delta formed many colonies, whereas only a few
small colonies formed without soluble Delta or in cultures using
cells exposed to the anti-Notch 4 antibody. When the antibody was
preincubated with the peptide, the antibody no longer blocked
proliferation. Thus, the Notch 4 pathway is critical for breast
cancer cell growth in vitro.
[0261] Tumor cells were exposed to the anti-Notch 4 antibody and
then the cells were injected into mice to determine whether the
antibody inhibits tumor formation. 35,000 tumor cells were
incubated with no antibody, the anti-Notch 4 antibody, or the
anti-Notch 4 antibody that had been preincubated with the peptide
used to generate the antibody. After two weeks, the diameter of
tumors formed by the cells exposed to the anti-Notch 4 antibody was
40% smaller than the control tumors. Thus the cells that were
exposed to the anti-Notch 4 antibody formed smaller tumors than
cells that were exposed to the anti-Notch 4 antibody that was
incubated with the peptide used to generate the antibody.
[0262] Expression of Notch, Notch Ligands, and Fringe Family
Members in Subpopulations of Breast Cancer Tumor Cells.
[0263] We examined the expression of members of the Notch receptor
family, Notch ligands and Notch signal modifying proteins in
populations of breast tumor cells. We initially focused on the
markers in the Notch 4.sup.+ and Notch 4.sup.- populations. One
hundred Notch 4.sup.+ or Notch 4.sup.- cells were isolated by FACS
(see, FIG. 4). Forty rounds of PCR were performed to detect the
indicated mRNA. The Notch 4.sup.+ population expressed Notch 1,
Notch 4, and Jagged (a Notch ligand). The Notch 4.sup.- population
expressed Notch 1 and Notch 3 as well as Jagged. Interestingly,
Notch 3 (which may inhibit signaling through other Notch receptors)
was not expressed by the Notch 4.sup.+ population.
[0264] Summary.
[0265] We have developed in vitro and in vivo assays for normal
human breast cells and human breast tumor stem cells. In two
different tumors arising in the NOD/SCID mouse model, the tumor
cells were heterogeneous with respect to the expression of several
cell surface markers. In both tumors, the phenotype of the
clonogenic tumor stem cell was B38.1.sup.+CD44.sup.+CD24.sup.-/lo.
The same population was found to be the clonogenic in the in vitro
assay. This B38.1.sup.+ population can be further subdivided using
several additional markers. In vitro and in vivo evidence strongly
implicates the Notch pathway, especially Notch 4, as playing a
central pathway in tumorigenesis.
Example 3
Mouse Xenograft Model
[0266] We have developed a xenograft model in which we have been
able to establish tumors from primary breast tumors via injection
of tumors in the mammary gland of severely immunodeficient mice.
Xenograft tumors have been established from mastectomy specimens of
all five patients that have been tested to date. We have also been
able to establish tumors from three malignant pleural effusions.
NOD/SC1D mice were treated with VP-16, and implanted with primary
human breast cancer tissue. Tumor cells isolated from three
malignant pleural effusions suspended in Matrigel.RTM. were
injected into mice and also formed tumors. This enabled us to
generate enough malignant tumor cells to facilitate analysis by
flow-cytometry and assay for the ability of different subsets of
cells to form tumors. We have extensively studied the tumors formed
by one of the primary tumors and one of the malignant pleural
effusion cells. Furthermore, in the three tumors that we have
attempted to do so, we have been able to make single-cell
suspensions and transfer the tumors. These improvements in the
xenograft assay have allowed us to do biological and molecular
tests to characterize the clonogenic breast cancer cell. In
addition, we have found tissue culture conditions in which primary
breast cancer cells and cells isolated from a mouse xenograft tumor
have proliferated for a short period of time (1-3 weeks) in tissue
culture.
[0267] A human tumor growing in the mouse model was harvested, made
into a single cell suspension, and analyzed by FACS. There was
heterogeneity of expression of cell surface markers by tumor cells.
Initially, breast cancer cells isolated from a malignant pleural
effusion were separated into groups based upon CD44 expression.
Cells were analyzed for expression of markers 520C9 and CD44. Three
populations of cells were identifiable. There was a small
population of cells that expressed both markers 520C9 and CD44, a
population that expressed either marker alone, as well as a
population that expressed neither marker. Cells were isolated with
regard to CD44 expression. CD44.sup.+ or CD44.sup.- cells were
tested for their ability to proliferate. Marker CD44.sup.+ tumor
cells, but not marker CD44.sup.- tumor cells, were able to form
colonies in vitro and tumors in vivo (TABLE 3). Note that isolation
of CD44.sup.+ cells results in a 2-fold enrichment of the
tumorigenic cells.
TABLE-US-00003 TABLE 3 HUMAN BREAST CANCER CELLS COLLECTED USING
FACS CD44 CD44 Colonies in vitro + - Tumorigenicity in mice + -
Analysis of in vitro colonies was done in 2 separate wells using
5,000 cells of the respective phenotype. In vivo growth of sorted
cells was done by injecting mice with 2 .times. 10.sup.6 marker
CD44.sup.+ or CD44.sup.- cells. Mice were analyzed at week 3 in
test 1 and week 4 in test 2. The injections of marker CD44.sup.+
cells, but not marker CD44.sup.- cells, resulted in tumor formation
and growth in vitro. The in vitro tests have been replicated using
frozen cells isolated from the patient and support the in vitro
tests. The in vivo tests have been replicated twice.
[0268] These results show that the clonogenic breast cancer tumor
cell expresses CD44. We have begun to search for other markers that
might allow us to further purify the breast cancer stem cell. To do
this, we have analyzed the tumor cells for expression of several
antigens.
[0269] Surprisingly, all of the CD44.sup.+ (tumorigenic) cells were
also B38.1.sup.+. Indeed, we have isolated B38.1.sup.+CD24.sup.+
cells and B38.1.sup.+CD24.sup.- cells obtained from a primary
biopsy and placed them in tissue culture. Only the
B38.1.sup.+CD24.sup.- cells formed colonies.
[0270] We next isolated cells from two of the tumors based upon
expression of marker CD24. In tumor T2, we isolated the CD24.sup.-,
the CD24.sup.lo and the CD24.sup.hi populations. In both cases,
only the CD24.sup.-/lo populations formed tumors (TABLE 4). Note
that there was a 5-6-fold enrichment of tumorigenic cells using
B38.1 and CD24.
TABLE-US-00004 TABLE 4 FRACTIONS OF HUMAN BREAST CANCER CELLS
ISOLATED BY FLOW-CYTOMETRY Mouse tumor formation CD24 CD24.sup.-/lo
Tumor T1 - + Tumor T2 - + Mice were injected with 50,000 CD24 or
CD24.sup.-/lo cells. The formation of tumors was determined 4 weeks
after injection.
[0271] Analysis of Tumors Arising from the CD24.sup.- Cell
Population.
[0272] By the solid tumor stem cell model, CD24.sup.- cells give
rise to tumors that contain both CD24.sup.+ and CD24.sup.- cells.
To test this hypothesis, secondary transplants were performed using
B38.1.sup.+CD24.sup.- cells.
[0273] The resultant tumors were removed and the cells were
re-analyzed with respect to B38.1 and CD24 expression. As predicted
by the stem cell model, cells obtained from a tumor arising from
transplanted B38.1.sup.+CD24.sup.- cells were heterogeneous with
respect to expression of both B38.1 and CD24. The marker expression
pattern of the cells isolated from the tumor initiated by the
B38.1.sup.+CD24.sup.- cells was similar to that of the original
tumor.
[0274] These results are a proof-of-principle of the solid tumor
stem cell model of solid cancer and demonstrate the following:
[0275] (1) tumor cells are phenotypically and functionally
heterogeneous;
[0276] (2) by separating cells by FACS, one can enrich for
tumorigenic cells; and
[0277] (3) by testing the tumorigenic fractions, one can isolate
tumor stem cells and more carefully focus strategies for
identifying therapeutic targets.
Example 4
Analysis of Primary Breast Tumor Cells in a Mouse Model
[0278] We have established tumors from eight patients in our mouse
model. We also characterize tumors established from three primary
tumors and two pleural effusions. Two are fast growing tumors and
three are slow growing tumors.
[0279] The phenotype of the tumorigenic cell is determined for each
different tumor. For analysis, a tumor is removed from the mice and
made into a single cell suspension. We first confirm the solid
tumor stem cell model of the invention and that the phenotype of
tumorigenic cells is indeed B38.1.sup.+CD44.sup.+CD24.sup.-/lo. In
all tumors, we do limiting dilution analysis of cells isolated by
FACS based upon expression of these markers.
[0280] Based on our preliminary data, the antibody cocktail that
leads to the greatest purification of the putative tumorigenic cell
follows: anti-38.1-APC, anti-CD44-FITC, and anti-CD24-PE,
anti-CD3-cytochrome, anti-CD2-cytochrome, anti-CD10-cytochrome,
anti-CD14-cytochrome, anti-CD16-cytochrome, anti-CD3I-cytochrome,
CD45-eytochrome, CD140b-cytochrome, anti-CD64-cytochrome,
anti-ESA-Phar-red and 7AAD (a viability marker). All antibodies
labeled with cytochrome are considered to be part of a lineage
cocktail (LINEAGE.sup.-). FACS is used to isolate the putative
breast cancer stem cell, which in xenograft tumor TI are the
CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population of cells, the
CD44.sup.+ B38.1.sup.+CD24.sup.-/loLINEAGE.sup.- population of
cells are more enriched, and the ESA.sup.+CD44.sup.+
B38.1.sup.+CD24.sup.-/loLINEAGE.sup.- population of cells are most
enriched for the breast cancer stem cell. The different populations
of malignant cells are tested in the in vivo and in vitro models to
confirm that the phenotype of the primary tumorigenic cell does not
change while growing in the mouse xenograft model As we
progressively enrich the tumorigenic cells, the number of cells and
time needed to form a tumor decreases.
Example 5
Role for Notch in Breast Cell Proliferation
[0281] The Notch protein is a receptor for the growth/survival
factors Delta and Jagged (Panin et al., Nature 387(6636): 908-912
(1997); Perron et al., Cellular & Molecular Life Sciences
57(2): 215-23 (2000); Shimizu et al., Journal of Biological
Chemistry 274(46): 32961-9 (1999)). In some normal stem cells,
Notch is known to play a role in proliferation, survival and
differentiation. Apelqvist et al., Nature 400(6747): 877-81 (1999);
Berry et al., Development 124(4): 925-36 (1997); Yasutomo et al.,
Nature 404(6777): 506-10 (2000); Morrison, Cell 101: 499-510
(2000). In certain situations, stimulation of Notch can promote
stem cell self-renewal while in other situations it can promote
differentiation. Delta activates all four Notch receptors.
[0282] We examined Notch 4 expression in the breast cancer tumor
cells. Notch 4 was expressed on the minority of the tumor cells.
When the B38.1 population, which identifies the tumorigenic
population, was analyzed for Notch 4 expression, a distinct minor
population of cells that were B38.110 and Notch 4.sup.+became
apparent.
[0283] We sorted Notch 4.sup.+and Notch 4.sup.- cells and analyzed
their ability to form colonies in vitro. Surprisingly, neither
population grew in tissue culture. There are two possible
explanations. First, it is possible that interaction between the
two populations of cells is required for cell growth. Alternately,
the antibody may be either an agonist or antagonist of Notch 4 and
inhibition or activation of the receptor may inhibit tumor cell
growth.
[0284] To distinguish between these possibilities, unseparated
tumor cells were incubated with the anti-Notch 4 antibody, and then
assayed for the ability to form colonies in vitro. While control
cells grew in tissue culture and formed colonies (FIG. 5A), cells
incubated with the Notch 4 antibody did not grow (FIG. 5B). When
the anti-Notch 4 antibody was pre-incubated with the peptide used
to generate the antibody (this peptide should theoretically block
binding of the antibody to the cells), the ability of the tumor
cells to form colonies was restored (FIG. 8C). To confirm that
Notch regulates colony formation by tumor cells, cells were
incubated in medium with or without soluble Delta (FIG. 5A and FIG.
5D, respectively). The cultures with Delta formed many colonies
(FIG. 5A), whereas only a few small colonies formed in medium
lacking soluble Delta or in cultures using cells exposed to the
anti-Notch 4 antibody (FIG. 5B, FIG. 5D, and FIG. 5E). Taken
together, these results show that the Notch 4 pathway regulates the
proliferation/survival of breast cancer cells in cells and that the
anti-Notch 4 antibody blocks activation in vitro.
[0285] Next, tumor cells were incubated with the anti-Notch 4
antibody and then the cells were injected into mice to determine
whether the antibody inhibits tumor formation. The cells that were
exposed to the anti-Notch 4 antibody formed smaller tumors than
cells that were exposed to the anti-Notch 4 antibody that was
incubated with the peptide used to generate the antibody before
addition to the cells. Approximately 350,000 tumor cells were
incubated with no antibody, the anti-Notch 4 antibody, or the
anti-Notch 4 antibody that had been incubated with the peptide used
to generate the antibody. After two weeks, the diameter of tumors
formed by the cells exposed to the anti-Notch 4 antibody was 40%
smaller than either of the control tumors. The simplest explanation
of the in vitro and in vivo assays is that Notch 4 activation
promotes proliferation/survival of stem cells and that the
anti-Notch 4 antibody blocks receptor activation.
Example 6
Further Characterization of Breast Cancer Stem Cells
[0286] Human breast cancer tumors have been established in NOD/SCID
mice from five tumors (mastectomy specimens of three primary breast
tumors and two malignant pleural effusions from metastatic breast
cancer). The five tumors were initially used to understand
neoplastic cell marker heterogeneity. Based upon these results,
experiments using cells isolated from primary tumors directly after
removal from a patient were done to confirm that the mouse
xenograft model accurately reflects human breast cancer.
[0287] CD44 expression and tumorigenicity. Cells obtained from the
metastatic breast cancer, designated T1, and one primary breast
tumor, designated T2, were chosen to expand in the mice to obtain
sufficient cells for analysis. To accomplish this,
10.sup.6-10.sup.7 pleural effusion cells from T1 (a frozen sample)
or a 1-2 mm.sup.3 piece of T2 (fresh biopsy sample) were grown in
the mouse mammary fat pad for 1 to 3 months. The resulting human
tumors were then harvested, made into a single cell suspension and
analyzed by flow-cytometry for expression of several different
antigens. Contaminating mouse cells were gated out of the analysis
by eliminating cells expressing mouse H-2K (major
histocompatibility complex class I) and dead cells were eliminated
using a viability dye.
[0288] As predicted by the solid tumor stem cell model, cells
displayed heterogeneous expression of a variety of cell
surface-markers including CD44 and B38.1. To determine whether
these markers could distinguish tumorigenic from non-tumorigenic
cells, CD44.sup.+ and CD44.sup.- cells (FIG. 6) were isolated from
these in vivo passaged n or T2 cells and NOD/SCID mice were
injected with CD44.sup.+ or CD44.sup.- cells.
[0289] Identification of other informative markers. Between 6 and
12 weeks after injecting, mice were examined for tumors by
observation and palpation, then all mice were necropsied to look
for growths at injection sites that might be too small to palpate.
All of the CD44.sup.+ injections gave rise to visible tumors, but
none of the CD44.sup.- injections formed detectable tumors (TABLE
5). Next, cells from first passage T1 and T2 were sorted based upon
the expression of B38.1 and injected into mice. Tumors appeared
from all injections of B38.1.sup.+ cells but no tumor formation was
detected from B38.1.sup.- cells (TABLE 5). Thus, tumorigenic cells
from both passaged tumors were B38.1.sup.+CD44.sup.+.
TABLE-US-00005 TABLE 5 Tumorigenicity of different populations of
tumor T1 and tumor T2 cells # tumors/# of injections injection
Cells 8 .times. 10.sup.5 5 .times. 10.sup.5 2 .times. 10.sup.5 T1
cells CD44.sup.- 0/2 0/2 -- CD44.sup.+ 2/2 2/2 -- B38.1.sup.- 0/2
0/2 -- B38.1.sup.+ 2/2 2/2 -- CD24.sup.+ -- -- 1/6 CD24.sup.- -- --
6/6 T2 cells CD44.sup.- 0/2 0/2 -- CD44.sup.+ 2/2 2/2 --
B38.1.sup.- 0/2 0/2 -- B38.1.sup.+ 2/2 2/2 -- CD24.sup.+ -- -- 1/6
CD24.sup.- -- -- 6/6 Cells were isolated by flow cytometry as
described in FIG. 2 based upon expression of the indicated marker
and assayed for the ability to form tumors after injection of 2-8
.times. 10.sup.5 cells into the mammary fat pad of NOD/SCID mice.
The number of tumors that formed/the number of injections that were
performed is indicated for each population of cells.
[0290] The tumors were also heterogeneous for CD24 expression. When
200,000 CD24.sup.+ or CD24.sup.-/lo cells were injected into
NOD/SCID mice, all mice injected with CD24.sup.+ cells grew tumors
(TABLE 5). Although no tumors could be detected by palpation in the
locations injected with CD24.sup.+ cells, two of twelve mice
injected with CD24.sup.+ cells did contain small growths at the
injection site that were detected only upon necropsy. The sites
injected with CD24.sup.-/lo cells, by contrast, all had tumors
greater than 1 cm in diameter that were readily apparent visually
and by palpation. Since it is extremely difficult to completely
eliminate CD24.sup.- cells from the CD24.sup.+ fraction by
flow-cytometry, the small growths likely represent contamination by
the 1-3% of CD24.sup.- cells that are typically present in the
sorted CD24.sup.+ population. Alternately, the small growths might
have arisen from CD24.sup.+ cancer cells that had reduced
proliferative capacity. At present we cannot distinguish between
these possibilities. Nonetheless, all of the cells that produced
palpable tumors in this xenograft model were
B38.1.sup.+CD44.sup.+CD24.sup.-/lo.
[0291] Several antigens associated with normal cell types, to which
we refer collectively as LINEAGE (CD2, CD3, CD10, CD14, CD16, CD19,
CD31, --CD45, --CD64 and --CD140b) markers, were found not to be
expressed by the cancer cells based on analyses of tumors that had
been passaged multiple times in mice. By eliminating LINEAGE.sup.+
cells, normal human leukocytes, endothelial cells, and fibroblasts
were eliminated, especially from unpassaged or first passage
tumors.
[0292] Flow-cytometric analysis of four of the tumors established
in NOD/SCID mice revealed that all of these tumors were
heterogeneous with respect to B38.1, CD44, and CD24 expression
(see, for example, FIG. 7A, FIG. 7B). However, each of the tumors
contained a distinct population of
B38.1.sup.+CD44.sup.+CD24.sup.-/lo cells. In four independent
tumors that were analyzed after one passage in NOD/SCID mice, the
frequency of B38.1.sup.+CD44.sup.+CD24.sup.-/lo cells was
9.5.+-.4.5% (mean.+-.standard deviation) of live human tumor cells.
Seven weeks after inoculation, the injection sites of
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells and
B38.1.sup.+CD44.sup.+CD24.sup.+ LINEAGE.sup.- cells were examined
by histology. The B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
injection sites contained tumors greater than 1 cm in diameter
while the B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- injection
sites contained no detectable tumors. Only normal mouse mammary
tissue was seen by histology at the sites of the
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- injections (FIG. 7K),
whereas the tumors formed by
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells contained
malignant cells as judged by morphology in hematoxylin and eosin
stained sections (FIG. 7L). Even when
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- injection sites were
examined after 11 weeks, no tumors were detected.
[0293] Enrichment of Tumorigenic Cells Using B38.1, CD44, CD24, and
LINEAGE Markers.
[0294] T1, T2, and T3, a third tumor that had been passaged once in
NOD/SCID mice, were tested to determine whether tumorigenic cells
could be enriched based upon expression of B38.1, CD44, CD24 and
LINEAGE markers. In all cases, the
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells were markedly
enriched for tumorigenic cells. When injecting unsorted T1 or T2
cells, 5.times.10.sup.4 cells consistently gave rise to a tumor,
but 10.sup.4 cells gave rise to tumors in only a minority of cases
(TABLE 6). In contrast when
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells were isolated
from T1 or T2 (FIG. 7), as few as 10.sup.3 cells from this
population were able to give rise to tumors in all cases (TABLE 6,
FIG. 7J and FIG. 7L). Cells that were
B38.1.sup.+CD44.sup.+LINEAGE.sup.- but CD24.sup.+ failed to form
tumors. These data indicate that the
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population is at
least 10-fold enriched for the ability to form tumors in NOD/SC1D
mice relative to unfractionated tumor cells. This population
accounted for 4-17% of the cells in T1 and T2 (5-23% of cancer
cells).
Example 7
Analysis of Primary Human Tumor Cells
[0295] To rule out the possibility that the tumorigenic activity in
the enriched population of EXAMPLE 7 arose as a result of
propagation in the xenograft model, we isolated unfrozen pleural
effusion breast cancer cells based upon expression of CD44 and
CD24. Approximately 11% of the neoplastic cells were
CD44.sup.+CD24.sup.-/loLINEAGE.sup.- and 75%
CD44.sup.+CD24.sup.+LINEAGE.sup.-. All three injections of 100,000
CD44.sup.+CD24.sup.-/loLINEAGE.sup.- but none of the
CD44.sup.+CD24.sup.+LINEAGE.sup.- cells, formed tumors (TABLE 6).
We next tested whether the B38.1.sup.+CD44.sup.+CD24.sup.-/lo
population from unpassaged breast tumor specimens (FIG. 7I) was
tumorigenic. Of four independent tumors that were analyzed after
being passaged in mice, there were only enough frozen unpassaged
cells available of T1 to permit flow-cytometry. In addition, frozen
unpassaged cells from a fifth patient (T5) were analyzed. Five
percent of unpassaged T1 cells and 35% of unpassaged T5 cells were
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-.
[0296] All 40,000 B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
cells or 40,000 B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- cells
that were isolated by flow-cytometry from unpassaged T1 were
injected into a single mouse. From the unpassaged T5 cells,
60,000-100,000 cells of the
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- (FIG. 7I) and
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- (FIG. 7F) populations
were isolated and injected into each of three NOD/SCID mice. The
CD24.sup.-/lo population formed tumors from the T1 cells and in 2
of 3 injections from T5 cells, but the CD24.sup.+ population never
formed tumors, demonstrating that the tumorigenic population
identified in the passaged tumors also existed in the unpassaged T1
and T5 samples. When tumors that arose from the unpassaged T1 cells
were analyzed, the frequency of
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells was similar
in both passaged and unpassaged T1 samples.
TABLE-US-00006 TABLE 6 Tumorigenic breast cancer cells are highly
enriched in the CD44.sup.+B38.sup.+CD24.sup.- population Number of
tumors/number of injections for each cell dose Cells/injection 5
.times. 10.sup.5 1 .times. 10.sup.5 5 .times. 10.sup.4 2 .times.
10.sup.4 1 .times. 10.sup.4 5 .times. 10.sup.3 1 .times. 10.sup.3 5
.times. 10.sup.2 2 .times. 10.sup.2 T1 cells (xenograft) Unsorted
4/4 4/4 6/6 -- 2/6 -- 0/6 -- -- B38.1.sup.+CD44.sup.+CD24.sup.+ --
-- -- 0/5 0/5 0/5 0/5 -- -- B38.1.sup.+CD44.sup.+CD24.sup.- -- --
-- 5/5 5/5 5/5 5/5 -- -- ESA.sup.+B38.sup.+CD24.sup.+ -- -- -- --
-- -- 8/8* 2/2 1/2 ESA.sup.+B38.sup.+CD24.sup.- -- -- -- -- -- --
0/8* 0/2 0/2 T2 cells (xenograft) Unsorted 4/4 4/4 4/4 -- 1/6 --
0/6 B38.1.sup.+CD44.sup.+CD24.sup.+ -- -- -- 0/5 0/5 0/5 0/5 -- --
B38.1.sup.+CD44.sup.+CD24.sup.- -- -- -- 5/5 5/5 5/5 5/5 -- -- T3
cells (xenograft) B38.1.sup.+CD44.sup.+CD24.sup.+ -- -- -- -- 0/2
-- -- -- -- B38.1.sup.+CD44.sup.+CD24.sup.- -- -- -- -- 2/2 -- --
-- -- T5 cells (primary cells) CD44.sup.+CD24.sup.+ 0/3
CD44.sup.+CD24.sup.- 3/3 T1 cells (primary cells)
B38.1.sup.+CD44.sup.+CD24.sup.+ 0/1 B38.1.sup.+CD44.sup.+CD24.sup.-
1/1 T6 cells (primary cells) B38.1.sup.+CD44.sup.+CD24.sup.+ 0/1
0/2 B38.1.sup.+CD44.sup.+CD24.sup.- 1/1 1/2 Cells were isolated
from first passage tumor T1, tumor T2, or tumor T3 cells.
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- and
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- cells were isolated by
flow-cytometry as described in FIG. 3. The indicated number of
cells of each phenotype was injected into the mammary fat pad of
NOD/SCID mice. The number of tumors that formed from injections of
each group of cells is indicated. Note that isolation of
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells results in
more than a ten-fold enrichment in tumorigenic cells and that no
tumors arose from injection of
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- cells. When cells were
sorted as ESA.sup.+ in addition to B38.1.sup.+CD24.sup.-,
injections of as few as 200 cells gave rise to a tumor, an
approximately 50-fold enrichment in tumorigenicity relative to
unsorted cells. * for these injections 2 .times. 10.sup.3 cells
were used instead of 1 .times. 10.sup.3.
Example 8
[0297] Further Characterization of the Cell Populations from a
Breast Cancer Tumor
[0298] In one tumor, tumorigenic cells were further enriched by
selecting the ESA.sup.+ subset of the
B38.1.sup.+CD44.sup.+CD24.sup.-/lo population. When
ESA.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells were isolated
from T1 (FIG. 8A), as few as 200 cells from this population were
able to give rise to a tumor while injection of 2,000
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- but ESA.sup.- cells
failed to form tumors in any case (TABLE 6). At least 10,000
unfractionated cells were required to form any tumors. Thus, the
ESA.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population was at
least 50 fold enriched for the ability to form tumors relative to
unsorted tumor cells. The
ESA.sup.+B38.1.sup.+CD24.sup.-/loLINEAGE.sup.- population accounted
for 2-4% of T1 cells (2.5-5% of cancer cells). The
ESA.sup.+B38.1.sup.+CD24.sup.-/loLINEAGE.sup.- and the
non-tumorigenic cells were examined by cytology and both
populations consisted of malignant cells that were virtually
indistinguishable in appearance.
Example 9
Use of Cancer Stem Cells to Improve Tissue Culture Conditions for
Growth of Breast Cancer Cells
[0299] In tissue culture, it has long been known that only a
minority of primary tumor cells are capable of forming colonies.
According to the solid tumor stem cell model, while the
non-tumorigenic population of cells might retain some ability to
proliferate, the tumorigenic population of cells would have a
greater capacity to do so. Equal numbers of the tumorigenic
phenotype and the non-tumorigenic cells, isolated either directly
from a primary tumor or from tumor T1 and tumor T4 growing in mice,
were seeded at clonal density in tissue culture plates and colony
formation was measured. The primary patient cells were from an
aliquot of frozen primary cells from a tumor that easily forms a
breast cancer cell line and proliferates well in tissue
culture.
[0300] Cytology showed that each population contained cancer cells
of epithelial origin (FIG. 9).
[0301] Initially, colonies formed from not only the tumorigenic
cells but in some tumors also from the non-tumorigenic cells (FIG.
9). However, by day twelve, the colonies formed by the tumorigenic
cells had grown while the non-tumorigenic cells had died (FIG. 9).
By day 14, only B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- cells
formed numerous large colonies of cancer cells (FIG. 9C).
[0302] We have passed these cells in tissue culture over 3
passages, and the epithelial neoplastic cells continue to
proliferate. By contrast, the non-tumorigenic cells were capable of
forming only small colonies consisting of 2-4 cells that stopped
proliferating after a few days (FIG. 9C).
[0303] These results illustrate two important points. First, the
phenotype of the stem cells is not an artifact of the
immune-deficient mouse assay. The fact that the phenotypically
identical population of cells had increased proliferative ability
in both the mouse xenograft model and the in vitro assays shows
that this population is the tumorigenic population of cells found
in patients with breast cancer.
[0304] Second, at least some of the non-tumorigenic cells have the
capacity for limited proliferation. This shows that the markers
used to identify the non-tumorigenic population were not simply
selecting for cells that lacked viability.
[0305] These results of this EXAMPLE provides a solution to the
problem that the use of the in vitro clonogenic assay to predict
chemotherapy drug sensitivity frequently does not predict a
particular patient's response to therapy. In one of the tumors used
in this EXAMPLE, the non-tumorigenic population of cells formed
colonies almost as efficiently as the tumorigenic cells (the solid
tumor stem cells of the invention) on day 4. In using this tumor
for a drug-screening assay, if the non-tumorigenic and tumorigenic
cells that are clonogenic in vitro respond differently to a
particular test compound, then the assay, especially if scored
early, would not have reflected the ability of the test compound to
eliminate breast cancer stem cells in vivo.
Example 10
The Stem Cell Population Regenerates Both the Stem Cell and the
Non-Tumorigenic Populations of Cells
[0306] According to the deterministic solid tumor stem cell model
(FIG. 1B), B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
tumorigenic cells form tumors that contain additional
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- tumorigenic cells
as well as phenotypically heterogeneous populations of
non-tumorigenic cells. Classical models would suggest that tumors
that form from B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.-
cells consist solely of expanded numbers of
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells.
[0307] Tumors arising from 1,000
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells were
dissociated and analyzed by flow-cytometry with respect to B38.1,
CD44 and CD24 expression (FIG. 10). As expected from the
deterministic solid tumor stem cell mode, cells obtained from a
tumor arising from transplanted
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells were
heterogeneous with respect to expression of all markers (FIG. 10C
and FIG. 10F). In both cases, the marker expression pattern of the
cells isolated from the tumor initiated by the
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells was similar
to that of the original tumors (compare FIG. 10A and FIG. 10B with
FIG. 10C and FIG. 10F) none of the
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- population, gave rise
to new tumors.
[0308] We tested whether the tumors formed by
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells contained
tumorigenic B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells
in addition to other non-tumorigenic cells.
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells or
B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- cells were isolated
from second passage T1 tumors that had been initiated by injections
of 1,000 B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells
seven weeks earlier. 1,000 cells were used per injection into each
of four animals in an attempt to establish third passage tumors. As
with the original T1 and T2 tumors, all four of the injections of
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population, but
none of the B38.1.sup.+CD44.sup.+CD24.sup.+LINEAGE.sup.- population
gave rise to new tumors.
[0309] This EXAMPLE demonstrates that the
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population gives
rise to both additional tumorigenic
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells and
phenotypically distinct non-tumorigenic cells.
Example 11
Expression of Potential Therapeutic Targets by the Different
Populations of Neoplastic Cells
[0310] In the deterministic solid tumor stem cell model (FIG. 1B),
expression of genes by the tumorigenic and non-tumorigenic
populations of tumor cells may differ. Accordingly, the inability
of current cancer treatments to significantly improve outcome is
due to the tendency of therapeutics to target non-tumorigenic cells
but not the rare tumorigenic solid tumor stem cells. If only the
non-tumorigenic populations are killed by particular therapies,
then tumors may shrink, but the remaining tumorigenic solid tumor
stem cells can drive regrowth of the tumor.
[0311] We examined first passage T1 cells for expression of either
the EGF receptor (EGF-R) or HER2/neu. The EGF-R and HER2/neu are
potential therapeutic targets that have been implicated in breast
cancer cell proliferation. Tumorigenic Ti cells stained with lower
levels of anti-EGF-R antibody than non-tumorigenic cells, and EGF-R
expression could not be detected at the single cell level in
tumorigenic cells (FIG. 11).
[0312] To test whether cells that did not express detectable levels
of the EGF-R were tumorigenic, 1,000-2,000 EGFR.sup.-B38.1.sup.+
CD24.sup.-LINEAGE.sup.- cells were injected into NOD/SCID mice.
Tumors formed in four out of four cases, continuing that the
EGF-R.sup.- cells are tumorigenic. By contrast, we could not detect
a substantial difference in HER.sup.2/neu expression between
tumorigenic and non-tumorigenic T1 cells (FIG. 11B and FIG.
11E).
[0313] As expected from the solid tumor stem cell model,
1,000-2,000 HER2/neu.sup.+B38.1.sup.+CD24.sup.-LINEAGE.sup.- cells
gave rise to tumors in four out of four cases. This EXAMPLE shows
that there can be differences in the expression of therapeutic
targets between the tumorigenic and non-tumorigenic
populations.
[0314] These experiments serve as a proof-of-principle of our stem
cell model of solid cancer and demonstrate the following:
[0315] (a) tumor cells are phenotypically and functionally
heterogeneous;
[0316] (b) by separating cells by FACS one can enrich for
tumorigenic cells; and
[0317] (c) by studying the tumorigenic fractions, one can isolate
solid tumor stem cells to more carefully focus molecular and
biological assays.
Example 12
Further Evidence for a Role for Notch in Breast Cell
Proliferation
[0318] To determine whether Notch was a solid tumor stem cell
marker, we sorted Notch 4.sup.+ and Notch 4.sup.- cells and
analyzed their ability to form colonies in vitro. Surprisingly,
neither population grew in tissue culture.
[0319] There are two possible explanations for these results.
First, interaction between the two populations of cells may be
required for cell growth. Alternately, the antibody may be either
an agonist or antagonist of Notch 4 and inhibition or activation of
the receptor may inhibit tumor cell growth. To distinguish between
these possibilities, unseparated tumor cells were incubated with
the anti-Notch 4 antibody, and then assayed for the ability to form
colonies in vitro in tissue culture medium containing soluble
Delta, a Notch ligand. Delta was essential for colony formation
from cells isolated from this tumor. While control cells grew in
tissue culture and formed colonies (FIG. 12A), cells incubated with
the Notch 4 antibody did not grow (FIG. 12B). When the anti-Notch 4
antibody was pre-incubated with the peptide used to generate the
antibody (this peptide should theoretically block binding of the
antibody to the cells), the ability of the tumor cells to form
colonies was restored (FIG. 12C). An anti-Notch 2 antibody did not
affect colony formation.
[0320] To confirm that Notch regulates colony formation by tumor
cells, cells were incubated in medium with or without soluble Delta
(FIG. 12A and FIG. 12D, respectively). The cultures with Delta
formed many colonies (FIG. 12A), whereas only a few small colonies
formed in medium lacking soluble Delta or in cultures using cells
exposed to the anti-Notch 4 antibody (FIG. 12B, FIG. 12D, and FIG.
12E). Taken together, these data suggest that the Notch 4 pathway
regulates the proliferation/survival of breast cancer cells in
cells and that the anti-Notch 4 antibody blocks activation in
vitro.
[0321] We also tested whether the anti-Notch 4 antibody inhibits
breast cancer tumor cell growth in vivo. To do this, 20,000, 10,000
or 5,000 tumorigenic cells were incubated with the anti-Notch 4
antibody or the anti-Notch 4 antibody and the blocking peptide. The
antibody delayed the appearance of the tumors by one week when
either 20,000 or 10,000 tumorigenic cells were injected, and by 13
days when 5,000 cells were injected. This suggests that the
antibody may block tumor formation in vivo.
[0322] We then tested four breast cancer cell lines for expression
of Notch 4 and growth inhibition by the anti-Notch 4 antibody. We
found that the MCF-7 and the SKBR-3 breast cancer cell lines
expressed Notch 4 and that the anti-Notch 4 antibody inhibited the
growth of these cells. RT-PCR showed that both expressed Notch 4.
To determine whether the anti-Notch 4 antibody inhibits growth,
5,000 MCF-7 cells were incubated with the anti-Notch 4 antibody,
the anti-Notch 4 antibody and the peptide used to generate the
antibody, or an irrelevant control antibody. The anti-Notch 4
antibody, inhibited colony formation of both cell lines by more
than ten-fold.
[0323] This EXAMPLE shows that Notch 4 activation promotes
proliferation/survival of solid tumor stem cells and that the
anti-Notch 4 antibody blocks receptor activation.
Example 13
[0324] Targeting Breast Cancer Stem Cells with a Gene Therapy
Suicide Vector
[0325] The breast cancer stem cell comprises a minority of the
tumor cells in a breast tumor. It is an object of the invention
that gene therapy strategies target these cells (FIG. 13).
[0326] We targeted virus vectors to breast cancer stem cells using
markers expressed by stem cells. To do this, we stained different
cell lines with the B38.1 antibody and analyzed them by flow
cytometry. MCF7, SKBR3, T47D and BT474 breast cancer cell lines
were highly positive.
[0327] We had the anti-fiber and the B38.1 antibodies conjugated
with the Prolinx (Prolinx, Inc., Bothell, Wash., USA) method (FIG.
14, see Douglas J T et al., Nature Biotechnology. 14(11):1574-8
(1996)). When we mixed the modified anti-knob and anti-B38.1
antibodies together, they became cross-linked and generated the
bi-specific conjugate (FIG. 15). The anti-fiber antibody part of
the conjugate binds to the adenovirus, while the anti-B38.1 moiety
binds to the breast cancer stem cell. Incubation of the AdLacZ
virus with the anti-fiber alone blocks the infectivity of the virus
(FIG. 16). If we incubate the virus with the bi-specific conjugate,
the infectivity is restored only in the cells that express high
levels of the B38.1 antigen (FIG. 18). The re-targeting is
specific, because it can be inhibited by free B38.1 antibody (FIG.
18).
[0328] The conclusion is that the new bi-specific conjugate
modifies the infectivity of Adenovirus, blocking its natural
tropism and directing the infection to cells that express the
breast cancer stem cell surface marker.
Example 14
Downstream Targets in the Breast Cancer Stem Cells
[0329] The methods used in this EXAMPLE provide guidance for the
development of Notch-related and other anti-cancer therapies using
the cancer stem cells of the invention. We use array technology to
begin to understand the molecular pathways that might be regulated
by Notch-signaling induced by specific Notch ligands. Sequence
verified human cDNAs from Research Genetics, provided by the
University of Michigan Microarray Network, are arrayed by the
Cancer and Microarray Facility. Probes are prepared from
self-renewing breast cancer stem cells or cells from the various
populations of cells found in a tumor. Probes are hybridized to the
arrays and the hybridization patterns are read by the Cancer and
Microarray Facility. We then analyze the hybridization patterns to
identify genes that hybridize to probe from the breast cancer stem
cells stimulated with various Notch ligands and non-stimulated
cells. Such genes may represent those that are involved in the
regulation of breast cancer cell survival or self-renewal.
[0330] Preparation of Microarrays.
[0331] We have used the microarray technology to analyze gene
expression of hematopoietic stem cells. We now extend this work to
cancer stem cells.
[0332] The University of Michigan Microarray Network currently has
32,500 sequence verified human cDNAs from Research Genetics. A
"cancer" chip has been assembled in collaboration with the NCl.
This chip contains a comprehensive constellation of 1,200 genes
involved in proliferation and tumorigenesis. There is also an
"apoptosis chip" developed by the University of Michigan that
contains all genes known to be involved in programmed cell death.
Note that the HES genes, known to be downstream targets of Notch,
are included in the arrays.
[0333] Preparation of Probe from Breast Cancer Stem Cells.
[0334] Messenger RNA is isolated either from freshly purified
breast cancer stem cells or from breast cancer stem cells incubated
in the presence or absence of various Notch ligands. The RNA is
amplified if necessary, such as by PCR or linear RNA amplification
Wang et al., Nature Biotechnology. 18: 457-459 (April 2000). Probe
are prepared by reverse transcription from an oligo-dT primer, and
labeled by incorporating CY3 or CY5 conjugated nucleotides. Gene
expression profiles are examined using probe prepared from freshly
isolated, uncultured breast cancer cells, as well as from cultured
breast cancer cells, such as cells that have been exposed to the
appropriate Notch ligands, including Fringe family members, either
singly or in combination as determined by which ligands are
expressed by the different populations of tumor cells. To do these
assays, we expose the cells to a soluble form of Delta or Jagged
family members in which the transmembrane region has been deleted,
or one of the Fringes. The Fringes are secreted proteins. We make
recombinant protein of each Notch ligand of the Delta, Jagged and
Fringe families from insect cells or mammalian cells transfected
with a baculovirus or mammalian expression vector,
respectively.
[0335] Comparisons of gene expression patterns between control
breast cancer tumorigenic cells and tumorigenic cells exposed to
various Notch ligands are made. Probe from breast cancer stem cells
from each tumor is combined with probe labeled with a different
fluor made from cultured breast cancer stem cells exposed to
various Notch ligands to compare their hybridization patterns. To
do this, breast cancer stem cells are isolated by FACS. Cells are
seeded at single cell density to preclude Notch interactions
between cells. Cells are exposed to soluble forms of Delta (see,
EXAMPLE 6), Delta-like, Jagged 1, Jagged 2, or each of the Fringes.
Cells are exposed to each protein both alone and in combinations
suggested by the Notch-ligand expression pattern of individual cell
populations. The microarrays hybridized with probe from each test
condition are compared and analyzed to gain insights into molecular
pathways affected by Notch ligand interactions. For example, if a
particular population of cells expresses Delta and Manic Fringe,
then one group of breast cancer stem cells is exposed to Delta
alone, a second to Delta and Manic Fringe and a third to Manic
Fringe alone. cDNA is made from each population with CY5 or CY3
labeling, and used to probe a microarray chip. In addition, cDNA
from each population is used with cDNA made from cells cultured in
control medium and freshly isolated breast cancer cells to probe a
microarray chip. Each group is compared 5 times to assure that any
differences in expression profiles of the arrayed genes by each
test groups are real.
[0336] Preparation of Probe from Cells Treated with the Anti-Notch
4 Antibody.
[0337] An antibody against Notch 4 inhibits growth in vitro and
tumorigenesis in vivo. This effect could be explained if the
antibody acts as either a Notch-4 agonist or antagonist. Since
soluble Delta promotes cancer cell growth in vitro, the antibody
most likely is a Notch 4 antagonist. To confirm the mechanism by
which the anti-Notch 4 antibody inhibits tumor growth, probe is
made from cells incubated in the presence or absence of the
anti-Notch 4 or control irrelevant antibody and the various
combinations of the Notch ligands and used for microarray
expression analysis as described above. Another control group
includes cells incubated with the antibodies and no Notch ligand.
Each comparison is performed in at least six independent tests
employing independently prepared batches of probe. By comparing the
gene expression patterns of each group, we should be able to
determine how the anti-Notch 4 antibody affects Notch
signaling.
[0338] Making the cDNA Probe.
[0339] 1-2 .mu.g of mRNA is commonly used to synthesize probe for
screening gene expression profiles on microarrays (Wang et al.,
Nature Biotechnology. 18: 457-459 (April 2000)). Since we screen a
set of three slides (containing a total of 32,500 cDNAs) in each
test, 6 .mu.g of mRNA is required per assay (reverse transcription
of 6 .mu.g of mRNA should yield around 3 .mu.g of cDNA probe, or 1
.mu.g of probe per slide). Cancer cells tend to have a high RNA
content. In past assays, 10' cancercells yielded around 100 .mu.g
of total RNA, which in turn yielded around 3 .mu.g of poly A.sup.+
RNA. Thus in order to generate 6 .mu.g of mRNA, around
2.times.10.sup.7 cells would be required. As described in the
preliminary data, that number of flow-cytometrically purified
breast cancer stem cells can be isolated from approximately
five-ten 1 cm tumors.
[0340] The breast cancer stem cells represent approximately 5% of
the total number of cells within a tumor. It is not practical to
isolate more than 10.sup.6 freshly dissociated (uncultured) breast
cancer stem cells by flow-cytometry in one day. This would yield
less than 0.5 .mu.g of mRNA from one day of sorting. While breast
cancer stem can be combined from multiple days of sorting to pool
enough mRNA to prepare probe from freshly isolated cells, it may
not be practical to perform all assays in this manner. Some assays
require brief periods of tissue culture. Plating efficiency of the
sorted cells is approximately 10%. Thus it may be necessary to
enzymatically amplify the template prior to synthesizing probe.
This can be done either by PCR or by linear amplification of RNA
using T7 RNA polymerase. Our protocol employs 15-18 rounds of PCR
to amplify cDNA from small numbers of stem cells. This protocol was
used to construct a high quality hematopoietic stem cell (HSC) cDNA
library and to make probe from hematopoietic stem cells. To produce
probe from freshly isolated breast cancer stem cells, we test the
same approach. Alternately, a number of groups have reported
success in using linear RNA amplification to produce probe for
microarray hybridization. Thus we compare the two methods by
preparing probe both ways and examining the hybridization patterns
that result. cDNA is primed using an oligo-dT primer that contains
a T7 RNA polymerase binding site and synthesized by Superscript
reverse transcriptase (Gibco) and the Clontech 5's switch oligomer
that allows the tagging of the 5' end of the cDNA. Second strand
cDNA is synthesized using E. coli DNA polymerase. Then amplified
RNA (aRNA) is produced using T7 RNA polymerase or PCR. Which of the
two that amplification methods are used is determined by comparing
probe made with standard cDNA synthesis. After preparing aRNA, cDNA
is re-synthesized using random hexamers. This cDNA can then be used
for probe, or if necessary, additional rounds of amplification can
be performed. Both approaches are used to prepare probe from 40,000
MCF-7 cells (a human breast cancer stem cell line). This probe is
hybridized to human cDNA microarrays along with probe from
unamplified MCF-7 cells. The amplification approach that most
closely reproduces the hybridization pattern of the unamplified
probe is selected. Then amplification conditions are modified until
the amplified probe reproduces the hybridization pattern of the
unamplified probe as closely as possible.
[0341] Analysis of the Hybridization Pattern.
[0342] Hybridization patterns are analyzed in the Cancer and
Microarray Core facility using their laser scanning system. The use
of an integrated system for arraying, hybridizing, scanning, and
analyzing hybridization patterns in which all components are
provided by Genomics Solutions permits a seamless and efficient
analysis of hybridization patterns.
[0343] A transcript is differentially expressed if there is at
least a 3-fold difference in normalized hybridization levels
between probes. Hybridization signals from CY3 and CY5 labeled
probes within a single test are normalized to each other to correct
for potential differences in the effective concentration of each
probe and replicates of each test are done using the opposite fluor
for each group to correct for differences in the amounts or
labeling efficiencies of probes.
[0344] Verification of Differential Expression.
[0345] cDNAs that consistently hybridize to probe from groups of
cells but not to probe from the control groups of cells are further
characterized. The sequences of these cDNAs are obtained from the
Microarray Network. Two approaches are used to confirm the
differential expression of candidate genes between cell
populations. The first is to prepare in situ hybridization probes
against candidate genes, and then perform in situ hybridizations on
breast cancer stem cells cultured in medium with or without various
Notch ligands as described above. In situ hybridizations are then
performed on cultured cells. The advantage of this approach would
be that expression could be compared at the level of individual.
cells.
[0346] An alternate approach is to design nested PCR primers
against candidate genes, and to perform RT-PCR on multiple
1-10-cell aliquots of freshly purified breast cancer stem cells
(isolated as described above). By performing RT-PCR on small
numbers of cells it is possible to observe a difference in the
ability to amplify particular transcripts, even if the
"non-expressing" population contains rare expressing cells. We used
this approach to demonstrate the differential expression of RGS18
between different subpopulations of multipotent hematopoietic
progenitors.
[0347] Differential expression can be confirmed by Northern
analysis. Poly A.sup.+RNA from 1-2.times.10.sup.7 breast cancer
stem cells cultured with or without the Notch ligands, are
hybridized-to probes of the differentially expressed cDNAs.
Hybridization signals are quantitatively compared between these
samples.
[0348] We then confirm that genes are differentially expressed at
the protein level. In cases where immunocytochemical staining is
uninformative we also perform western blots on protein from the
different cells.
[0349] Certain molecular analyses are difficult using the primary
breast cancer cells that only proliferate for prolonged periods of
time in the xenograft model. These analyses can be done in cell
lines. One can use any of a large number of breast cancer cell
lines, including early passages of lines that we have studied in
the past. Clarke et al., Proc. Natl. Acad. Sci. USA. 92: 11024-28
(November 1995); Hernandez-Alcoceba et al., Human Gene Therapy.
11(11): (September 2000). These cell lines are plated at
single-cell density with and without various Notch ligands, as well
as the anti-Notch 4 or control antibodies as described in the
assays with the primary breast cancer cells. If clonogenicity is
affected by Notch signaling, then probe for the microarray analysis
is made using cDNA made from the cell line incubated in medium with
or without the various Notch ligands or anti-Notch 4 or control
antibodies. Since a virtually unlimited number of cells can be
analyzed, we can make probe that has not been amplified.
[0350] Finally, cell lines are useful for confirming whether the
anti-Notch 4 antibody is an agonist or antagonist. If a cell line
is identified that clonogenicity is enhanced by soluble Delta and
inhibited by the anti-Notch 4 antibody, then it is used in these
assays. The cells are stably transfected with a luciferase minigene
under the control of the Notch-inducible HET-1 promoter. Jarriault
et al., Molecular & Cellular Biology. 18(12): 7423.about.31
(December 1998). The cells are plated at single cell density to
prevent cell-cell Notch-Notch ligand interactions. They are treated
with the various combinations of Notch ligands and either the
anti-Notch 4 antibody or a control antibody. The cells are
harvested and a luciferase assay is done to determine how each
condition affects Notch signal transduction as reflected by trans
activation of the HET1 promoter.
[0351] A comprehensive functional analysis of candidate genes that
emerge from the microarray analysis can be performed. Full-length
cDNAs are isolated and cloned into a retroviral expression vector.
Breast cancer cell lines and breast cancer stem cells isolated from
the five xenograft tumors are infected in vitro and the effect of
the retroviral transgene on self-renewal and tumorigenicity is
assayed relative to clones infected with a control vector. The
transgene is expressed as a bicistranic message that contains
IRES-GFP. This allows identification of transduced cells via FACS
or fluorescent microscopy. The effect of the transgene on Notch
signaling is examined in vitro and in vivo. To do this, transduced
cells are tested for response to the various combinations of Notch
ligands found to affect colony formation in tissue culture and
tumorigenicity in mice.
[0352] The expression patterns of candidate genes are examined in
detail in vivo to determine how widely the genes are expressed
beyond the xenograft. In addition to performing more extensive in
situ hybridizations of tissue sections from slices of primary
breast cancer, we make antibodies against selected gene products
being studied. The Hybridoma Core facility at the University of
Michigan has extensive experience preparing monoclonal antibodies
using both peptides, and expressed recombinant proteins.
[0353] Ultimately, the functions of unknown genes are tested in
vivo, using gene targeting to make knockout mice. The University of
Michigan Transgenic Core has established murine ES cell technology,
they provide ES cells that `go germline at a high rate and assist
with the generation of homologous recombinant ES clones.
[0354] The ability of microarray analysis to simultaneously compare
the expression of many genes provides unparalleled power to screen
for changes in gene expression patterns. Combined with the ability
to purify stem cells and to regulate their self-renewal and
differentiation in vitro, microarray analyses can be applied with
great precision to screen for specific types of regulatory
genes.
Example 15
How Notch Signaling Affects Breast Cancer Tumorigenesis
[0355] To verify the importance of Notch in normal mammary growth
and development, we test the effects of Notch ligands or agonist
peptides on the growth and tumor formation of mammary tumor stem
cells.
[0356] Effect of Notch Activation on Tumor Formation In Vivo.
[0357] The effects of the various combinations of Notch ligands on
tumor formation are correlated with the microarray data obtained in
EXAMPLE 14. These assays are done using xenograft tumor cells
before and after enrichment of populations of malignant breast
cells, allowing us to differentiate Notch effects on both stem and
progenitor cells respectively. The effects of Notch ligands on
growth and differentiation of each population of cancer cells are
determined utilizing the in vivo assay.
[0358] To determine the effect of Notch stimulation on tumor growth
in vivo, one thousand, ten thousand, and one hundred thousand
breast cancer tumor cells are incubated with soluble Notch ligands,
alone or in combination, or control media and then injected (6
replicates of each) into NOD/SCID mice. The soluble forms of Delta,
Delta-like, Jagged 1, and Jagged 2 are used as well as members of
the Fringe family. The cells are incubated with each ligand
individually, as well as combinations of different ligands, for two
hours. The cells are suspended in Matrigel.RTM. with the Notch
ligands and then injected into the mice. The effects of each ligand
of Notch stimulation on both the number of cells needed to form a
tumor as well as the time needed to form a tumor are determined.
This allows us to determine whether Notch affects tumor growth in
vivo.
[0359] In previous EXAMPLES, the anti-Notch 4 antibody retarded
tumor formation after a single, brief incubation with cells. Next,
one thousand, ten thousand, and one hundred thousand tumorigenic
cells from each of the five xenograft tumors are incubated with
either a control antibody or the anti-Notch 4 antibody alone, or
with various combinations of Delta, Jagged and Fringe family
proteins. Each group is then mixed in Matrigel.RTM. containing the
same antibody or Notch ligands and injected into mice. The effects
of the anti-Notch 4 antibody on the number of cells and time needed
to form a tumor are measured. The results are correlated with the
microarray expression data, obtained in EXAMPLE 14.
[0360] Stimulation of Notch should drive the proliferation of the
normal breast stem cells. Like in normal stem cells, members of the
Fringe family should modulate Delta or Jagged signaling. Notch
should also play a role in the self-renewal of the breast cancer
stem cell.
Example 15
Human Subjects
[0361] The following human sources of material are obtained for use
in the methods of diagnosis of the invention: (1) primary tumors
from patients with breast cancer and (2) pleural fluid from
patients with metastatic breast cancer. A portion of the tumor or
the pleural fluid is obtained during routine treatment when the
patient's physician deems removal of the tumor or pleural fluid to
be clinically indicated.
[0362] For example, at the University of Michigan Hospital, there
is an IRB approved protocol to obtain the specimens. Patients are
asked to sign an informed consent where indicated. There are no
additional risks to patients.
Example 16
Vertebrate Animals
[0363] The University of Michigan complies with the Animal Welfare
Act as amended (7 U.S.C. .sctn.2131 et seq.), and other Federal
statutes and regulations relating to animals. We use the laboratory
mouse strain NOD/SC1D mice and other strains of immunocompromised
mice, such as Beige/SCID mice.
[0364] There should not be any discomfort from cancer cell growth,
because mice undergo euthanasia prior to development of illness. If
the mice do show evidence of discomfort from tumors (posture,
appetite, or behavior changes, weight loss), they can be
immediately sacrificed. Should the mice show evidence of more than
mild discomfort (posture, appetite, or behavior changes) they are
given analgesics such as oral morphine or codeine. Mice have bone
marrow cells inoculated into their retro-orbital vein while
anaesthetized with either ether or a similar general anesthetic
agent.
[0365] Mice are killed by the use of C0.sub.2, which is consistent
with the recommendations of the Panel on Euthanasia of the American
Veterinary Medical Association. This method was chosen because it
is safe and humane.
Example 17
Cell Cycle Analysis of Breast Cancer Stem Cells and Non-Tumorigenic
Breast Cancer Cells
[0366] We tested cells from a mouse xenograft tumor. The cell cycle
status of the breast cancer stem cell (FIG. 19A) and
non-tumorigenic populations of cells (FIG. 19B) in the tumor was
determined by flow cytometry. There was a similar distribution of
cells in the G1, S, and G2/M phases of the cell cycle in both
populations. Thus, both the tumorigenic and non-tumorigenic
populations exhibited similar cell-cycle distributions.
[0367] This EXAMPLE rules out the possibility that all cells that
expressed the solid tumor stem cell markers were at a particular
stage of the cell cycle in the tumor that was analyzed. By
selecting cells at a particular stage of the cell-cycle by
flow-cytometric sorting of Hoechst stained tumor stem cells it may
be possible to further enrich tumorigenic activity, just as it has
been possible to enrich hematopoietic stem cell activity by
selecting cells with low levels of Hoechst staining
Example 18
Rhodamine 123 Staining of Breast Cancer Stem Cells and
Non-Tumorigenic Breast Cancer Cells
[0368] The purpose of this EXAMPLE is to examine the activity of
the multi-drug resistance pump in breast cancer stem cells and the
non-tumorigenic cancer cells. We have stained the ESA.sup.+
CD44.sup.+CD24.sup.-/loLINEAGE.sup.- cells (breast cancer stem
cells) and the non-tumorigenic cells obtained from one of the
xenograft tumors with Rhodamine 123.
[0369] One of the major factors that determines the intensity of
Rhodamine 123 staining in a cell is the MDR pump activity that
eliminates this dye from the cells. Yumoto R. et al., Drug
Metabolism & Disposition 29(2): 145-51 (2001); Daoud R. et al.,
Biochemistry 39(50): 15344-52 (2000). We found that some of the
solid tumor stem cells from this tumor stained less intensely with
Rhodamine 123 than did the non-tumorigenic cancer cells (FIG.
20).
[0370] This EXAMPLE shows tumor cell heterogeneity and indicates
that MDR pump activity may be higher in a solid tumor cells.
Example 19
[0371] A B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- Population
of Cells Exists in Ovarian Cancer Tumors
[0372] We analyzed cancer cells obtained from both a tumor and
ascites fluid obtained from a cyto-reduction surgery for a patient
with ovarian cancer. Notably, B38.1 is known to be expressed by
ovarian cancer cells. Surprisingly, the flow cytometry analysis
revealed multiple cell populations, and there was a distinct
B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- population of cells
(FIG. 21).
[0373] This distinct cell population phenotypically resembles the
breast cancer stem cell and may represent an ovarian cancer stem
cell.
Example 20
[0374] A B38.1.sup.+CD44.sup.+CD24.sup.-/loLINEAGE.sup.- Population
of Cells Exists in Sarcoma Tumors/Comparison to Breast Cancer Stem
Cell Data
[0375] The B38.1 antigen had previously been described to be
expressed only in breast cancer and ovarian cancer. We established
two sarcomas by placing sarcoma cells in the flanks of NOD/SCID
mice that had been treated with VP16. One of the tumors was excised
and examined by flow cytometry. Surprisingly, the sarcoma cells
expressed the B38.1 antigen. Furthermore, there were three distinct
cell populations with respect to CD44.sup.+ high, low and negative
(FIG. 22).
[0376] Thus sarcoma cells include a CD44.sup.+ population that
phenotypically resembles breast cancer stem cells and that may
represent sarcoma stem cells.
TABLE-US-00007 TABLE 7 Tumorigenicity of Different Populations of
Tumor T1 and T2 Cells # tumors/# of injections injection Cells 8
.times. 10.sup.5 5 .times. 10.sup.5 2 .times. 10.sup.5 T1 cells
CD44.sup.- 0/2 0/2 -- CD44.sup.+ 2/2 2/2 -- B38.1.sup.- 0/2 0/2 --
B38.1.sup.+ 2/2 2/2 -- CD24.sup.+ -- -- 1/6 CD24.sup.- -- -- 6/6 T2
cells CD44.sup.- 0/2 0/2 -- CD44.sup.+ 2/2 2/2 -- B38.1.sup.- 0/2
0/2 -- B38.1.sup.+ 2/2 2/2 -- CD24.sup.+ -- -- 1/6 CD24.sup.- -- --
6/6 Cells were isolated by flow cytometry as described in FIG. 2
based upon expression of the indicated marker and assayed for the
ability to form tumors after injection of 2-8 .times. 10.sup.5
cells into the mammary fat pad of NOD/SCID mice. The number of
tumors that formed/the number of injections that were performed is
indicated for each population of cells.
Example 21
Preparation of Polynucleotide Probes
[0377] Single Round RT Labeling from Total RNA Source.
[0378] The polynucleotides isolated from isolated solid tumor stem
cells or enriched populations of solid tumor stem cells can be RNA
extracted from the cells or complementary DNA (cDNA) made from the
extracted RNA.
[0379] For some embodiments of the methods of the invention (such
as those involving hybridization), labeled cDNA is useful. In one
embodiment, we use the methods of single round RT labeling from
total RNA source.
[0380] The quality of the RNA coming into a labeling reaction will
have a marked effect on the quality of the hybridization. RNA
preparations that look good by the standard molecular biology
criteria can give poor results. Typical problems include disperse,
fine-grain noise over the entire hybridized surface and
non-specific binding of fluor to the zones of DNA immobilization on
the slide. These problems seem likely to have some roots in
contaminating carbohydrate, and as would be expected with
carbohydrate, the problems are exacerbated by ethanol
precipitations before and after labeling. Very impure preparations
frequently produce visible aggregates if precipitated after
labeling and ethanol precipitation, which are essentially resistant
to solubilization. It is well known that nucleic acids form strong
aggregates with carbohydrate when either dried together or when
co-precipitated. This interaction is the basis for nucleic acid
immobilization onto chromatography supports such as cellulose. To
minimize this sort of problem, we recommend preparative procedures
which use few or preferably no ethanol precipitations during RNA
preparation and labeling. We use at least the volumes of extractant
and washing solutions suggested for the number of cells being
processed. Appropriate or slightly excessive extraction/wash
volumes tend to minimize noise in a hybridization assay.
[0381] Methods that give satisfactory results are Triazol
extraction (BRL) and Rneasy (Quiagen).
[0382] Total RNA is prepared from tissue (from solid tumor or
xenograft tumor), tissue culture or enriched populations of solid
tumor stem cells obtained by flow cytometry. We resuspend the
prepared RNA in a volume that produces an RNA concentration of
>6 mg/ml in DEPC water. If the RNA is recovered from a matrix as
the final preparative step, and is still too dilute, we concentrate
as needed for labeling, using a MicroCon 30 (Amicon) determine the
concentration of the RNA. We test by reading a small sample (A260)
in 50 mM NaOH.
[0383] For the nucleotide mix, we use 10.times. low T dNTPs (using
100 mM dNTPs from Pharmacia (St Louis, Mo., USA) [27-2035-02])
TABLE-US-00008 TABLE 8 NUCLEOTIDE MIX mM final (1/10) Nucleotide
.mu.l concentration dGTP 25 0.5 dATP 25 0.5 dCTP 25 0.5 dTTP 10 0.2
water 415 total volume 500
[0384] Fluorescent Nucleotides are from Amersham Life Sciences or
Perkin Elmer Applied Biosystems Division. We use FluoroLink
Cy3-dUTP (#401-896) or FluoroLink Cy5-dUTP. The Cy.sub.3 and
Cy.sub.5 nucleotides come at concentration of 1 mM.
[0385] The R110 nucleotide comes at concentration of 0.1 mM, and
must be dried and resuspended at 0.1.times. the initial volume to
bring it to 1 mM. Currently, the factors of labeling efficiency,
fluorescent yield, spectral separation, and tendency toward
non-specific binding make the Cy3/Cy5 pair the most useful for the
detection system.
[0386] For the labeling reaction, the primer can be Pharmacia oligo
(dT) 12-18 (27-7858-01). Unlabeled nucleotide mixes are prepared
from Pharmacia 100 mM stocks. The reverse transriptase is BRL
SuperScript II (18064-014). The 5.times. buffer is the buffer
supplied with the polymerase.
[0387] For use in microarrays, normalization in a two fluor
labeling is achieved by reference to housekeeping genes distributed
through the microarray.
[0388] A cocktail of synthetic cDNAs produced using phage RNA
polymerases on cloned E. coli genes in the pSP64 poly(A) vector as
a mass standard and RNA quality standard. If possible, we also
prepare a total RNA solution containing 100 .mu.g of RNA in 17
.mu.l of DEPC water. Otherwise, we ethanol precipitate 100 mg of
total RNA to concentrate sample for labeling reaction. We take care
to remove all residual 70% ethanol wash either by air drying or
vacuum, as residual ethanol may impede the efficiency of
labeling.
[0389] We resuspend the pellet in DEPC water to give a final volume
of 17 ml. Any residual precipitate is removed by centrifugation.
The RNA is added to the reaction last.
[0390] Fluor Nucleotide (NTP) RT Labeling:
[0391] We add the following:
TABLE-US-00009 TABLE 9 REVERSE TRANSCRIPTASE LABELING Component
.mu.l 5X first strand buffer 8 Oligo (dT)12-18 (500 .mu.g/ml) 2 10X
low dT NTP mix 4 Fluor dUTP (1 mM) 4 0.1M DTT 4 RNAsin 1 syn mRNA
std (0.06 .mu.g) 0.5 100 mg total RNA 17.0 total 40
[0392] We vortex the sample after adding RNAsin. We minimize
bubbles and foaming during the vortex or quick spin.
[0393] Next, we hold at 65.degree. C. for 5 minutes, bring to
42.degree. C. (program R1). Then, add 2 .mu.l of SSII enzyme. Make
sure enzyme is well mixed into the reaction. Next, incubate
42.degree. C. for 25 minutes. Add 2 .mu.l of SSII enzyme. Make sure
enzyme is well mixed into the reaction. Then, incubate 42.degree.
C. for 35 minutes. Add 5 .mu.l of 500 mM EDTA. Be sure to stop the
reaction with EDTA before adding NaOH.
[0394] Add 10 ml of 1M NaOH. Incubate at 650 C for 60 minutes to
hydrolyze residual RNA. Cool to room temperature and add 25 .mu.l
of 1 M Tris-HCl (pH 7.5).
[0395] For probe cleanup and analysis, we transfer to a Microcon
30, concentrate to about 20 .mu.l (approx 3.5 min at 14,000 rpm in
Eppendorf 5415C). We wash by adding 200 .mu.l of TE (pH 7.5) and
concentrating to about 20 .mu.l (4-4.5 min @14,000 rpm). We recover
the product by inverting the concentrator over a clean collection
tube and spinning for 3 min @ 3000 rpm.
[0396] In some cases, the Cy5 probe may produce a gelatinous blue
precipitate which is recovered in the concentrated volume. The
presence of this material signals the presence of contaminants. The
more extreme the contamination, the greater the fraction of probe
which will be captured in this gel. Even if heat solubilized, this
material tends to produce uniform, non-specific binding to the DNA
targets.
[0397] When concentrating by centrifugal filtration, the times
required to achieve the desired final volume are variable. Overly
long spins can remove nearly all the water from the solution being
filtered. Then, when fluor tagged nucleic acids are concentrated
onto the filter in this fashion, they are very hard to remove.
Thus, we approach the desired volume by conservative approximations
of the required spin times.
[0398] We take a 2 .mu.l aliquot of Cy5 probe for analysis, leaving
17-18 .mu.l for hybridization, then run this probe on a 2% agarose
gel in TAB (gel size is 6 cm wide.times.8.5 cm long, 2 mm wide
teeth). For maximal sensitivity when running samples on a gel for
fluor analysis, we use loading buffer with minimal dye and do add
ethidium bromide to the gel or running buffer.
[0399] We scan the gel on a Molecular Dynamics Storm fluorescence
scanner (Settings -red fluorescence, 200 micron resolution, 1000
volts on PMT). A successful labeling produces a dense smear of
probe from 400 bp to >1000 bp, with little pile-up of low
molecular weight transcripts. Weak labeling and significant levels
of low molecular weight material indicate a poor labeling.
[0400] Hybridization.
[0401] The blocking species is poly(dA) from Pharmacia (27-7988-01)
(resuspended at 8 mg/ml); yeast tRNA from Sigma (R8759)
(resuspended at 4 mg/ml); and CoT1 DNA from Life Technologies Inc.
(concentrated 10 fold to 10 mg/ml).
[0402] The volume required for the hybridization is dependent on
the size of array used. For hybridizations that require small
volumes (20-25 .mu.l), the probe volumes after microcon
concentration can be too large. If so, then we add the blockers to
the Cy3 probe and precipitate the Cy3 probe.
[0403] We add 8 mg poly (dA); 4 mg tRNA; and 10 mg CoT1 DNA per 10
ml hybridization.
TABLE-US-00010 TABLE 10 HYBRIDIZATION PROBE MIX Cy3 labeled probe
-20 .mu.l poly dA (8 mg/ml) 1 .mu.l yeast tRNA (4 mg/ml) 1 .mu.l
CoT1 DNA (10 mg/ml) 1 .mu.l
[0404] We add 2 .mu.l of 3 M sodium acetate (pH 5.5), then add 60
.mu.l of ethanol, centrifuge, dry lightly, and resuspend in the
.about.17 .mu.l of Cy5 probe. If the array requires approximately
40 .mu.l of probe, then the Cy3 and Cy5 concentrates are pooled and
the blockers are added directly, so no precipitation is
required.
[0405] Then, we add 3 .mu.l of 20.times.SSC per 20 .mu.l of
hybridization mix volume. At this point, we optionally add 1 .mu.l
of 50.times.Denhardt's blocking solution per 20 .mu.l of
hybridization mix. With very clean probe, the Denhardt's does not
make any visible difference.
[0406] Then, we heat at 98.degree. C. for 2 minutes, cool to
45.degree. C. and add 0.2 .mu.l of 10% SDS per 20 of hybridization
mix volume, then apply to the array, and hybridize (16-24) hours at
65.degree. e in a sealed, humidified chamber.
[0407] Washing.
[0408] Residual unbound probe is removed from the slides by washing
2-5 minutes each at room temperature. The first wash is
0.5.times.SSC, 0.01% SDS. The second wash is 0.06.times.SSC. Air
drying of the slides after this step can leave a fluorescent haze
on the slide surface, so buffer is removed from the slides by a
brief spin at low G. We place the slides in a slide holder and spin
in a centrifuge equipped with a swinging carrier (horizontal) which
can hold the slide holder. Most centrifuges that are adaptable to
centrifuging microtiter plates can be used for this purpose
[0409] The foregoing description has been presented only for the
purposes of illustration and is not intended to limit the invention
to the precise form disclosed, but by the claims appended hereto.
Sequence CWU 1
1
1119PRTHomo sapiens 1Leu Leu Cys Val Ser Val Val Arg Pro Arg Gly
Leu Leu Cys Gly Ser 1 5 10 15 Phe Pro Glu
* * * * *