U.S. patent application number 12/375443 was filed with the patent office on 2010-04-29 for methods of inhibiting tumor cell aggressiveness using the microenvironment of human embryonic stem cells.
This patent application is currently assigned to CHILDREN'S MEMORIAL HOSPITAL. Invention is credited to Mary Jessica Hendrix, Lynne-Maire Postovit, Elisabeth Ann Seftor, Richard Edward Barnet Seftor.
Application Number | 20100105610 12/375443 |
Document ID | / |
Family ID | 38802382 |
Filed Date | 2010-04-29 |
United States Patent
Application |
20100105610 |
Kind Code |
A1 |
Hendrix; Mary Jessica ; et
al. |
April 29, 2010 |
Methods of Inhibiting Tumor Cell Aggressiveness Using the
Microenvironment of Human Embryonic Stem Cells
Abstract
The invention provides compositions comprising one or more
isolated factors from a microenvironment of human embryonic stem
cells (hESCs), including, but not limited to, Lefty and inhibitors
of Nodal. The invention also provides methods of utilizing factors
derived from human embryonic stem cells (hESC) and their
microenvironment to treat and prevent tumor formation and
progression and to inhibit tumor cell aggressiveness. The invention
further provides methods of inhibiting tumor cell growth and/or
treating aggressive tumors in a mammal comprising administering to
the mammal, having at least one tumor cell present in its body, an
effective amount of an inhibitor of Nodal activity.
Inventors: |
Hendrix; Mary Jessica;
(Evanston, IL) ; Postovit; Lynne-Maire; (London,
CA) ; Seftor; Richard Edward Barnet; (Chicago,
IL) ; Seftor; Elisabeth Ann; (Chicago, IL) |
Correspondence
Address: |
K&L Gates LLP
P.O. Box 1135
CHICAGO
IL
60690
US
|
Assignee: |
CHILDREN'S MEMORIAL
HOSPITAL
Chicago
IL
|
Family ID: |
38802382 |
Appl. No.: |
12/375443 |
Filed: |
July 26, 2007 |
PCT Filed: |
July 26, 2007 |
PCT NO: |
PCT/US07/74529 |
371 Date: |
September 15, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60820740 |
Jul 28, 2006 |
|
|
|
60941343 |
Jun 1, 2007 |
|
|
|
Current U.S.
Class: |
514/1.1 ;
435/6.16; 435/7.1; 514/44A; 530/350 |
Current CPC
Class: |
G01N 33/574 20130101;
A61P 35/00 20180101; G01N 33/57415 20130101; A61P 15/00 20180101;
G01N 33/5011 20130101; C12N 15/1136 20130101; A61K 38/00 20130101;
C12N 2310/11 20130101; G01N 33/5743 20130101; G01N 33/57492
20130101; G01N 33/53 20130101; C12N 2310/14 20130101; C07K 14/495
20130101; C12N 2310/3233 20130101; A61K 35/54 20130101 |
Class at
Publication: |
514/12 ; 530/350;
514/44.A; 435/7.1; 435/6 |
International
Class: |
A61K 38/17 20060101
A61K038/17; C07K 14/00 20060101 C07K014/00; A61K 31/7088 20060101
A61K031/7088; G01N 33/53 20060101 G01N033/53; C12Q 1/68 20060101
C12Q001/68; A61P 35/00 20060101 A61P035/00 |
Claims
1. A composition comprising one or more isolated factors from a
microenvironment of human embryonic stem cells.
2. The composition of claim 1 wherein the isolated factor or
factors inhibit Nodal.
3. The composition of claim 1 wherein at least one factor is
Lefty.
4. An isolated Lefty protein produced by conditioning a matrix with
human embryonic stem cells.
5. A protein comprising glycosylated Lefty or a fragment or
derivative thereof.
6. The protein of claim 5 wherein the glycosylated Lefty is
isolated from the microenvironment of human embryonic stem
cells.
7. A composition comprising the protein of claim 5.
8. A method of inhibiting tumor cell growth in a mammal comprising
administering to the mammal the composition of claim 7 at a
physiologically acceptable dosage.
9. The method of claim 8 wherein the dosage is between 0.1 and 200
ng/mL.
10. A method of using one or more factors from a microenvironment
of human embryonic stem cells to inhibit tumor cell
aggressiveness.
11. The method of claim 10, wherein at least one of the factors is
an inhibitor of Nodal activity.
12. The method of claim 11, wherein the inhibitor of Nodal activity
is Lefty.
13. The method of claim 11, wherein the inhibitor of Nodal activity
is glycosylated Lefty.
14. The method of claim 11 wherein the inhibitor increases
apoptosis and decreases cell proliferation.
15. A method of inhibiting tumor cell growth and/or treating
aggressive tumors in a mammal comprising administering to the
mammal, having at least one tumor cell present in its body, an
effective amount of an inhibitor of Nodal activity.
16. The method of claim 15, wherein the inhibitor blocks binding of
Nodal to a receptor.
17. The method of claim 15, wherein the inhibitor is one or more
isolated factors from a microenvironment of human embryonic stem
cells.
18. The method of claim 15, wherein the inhibitor is glycosylated
Lefty.
19. The method of claim 16, wherein the inhibitor is an
antibody.
20. The method of claim 15, wherein the inhibitor blocks expression
of Nodal protein.
21. The method of claim 15, wherein the inhibitor is an
activin-like kinase receptor inhibitor.
22. The method of claim 21, wherein the activin-like kinase
receptor inhibitor is an ALK 4/5/7 inhibitor.
23. The method of claim 22 wherein the ALK 4/5/7 inhibitor is
SB431542.
24. The method of claim 15 wherein the inhibitor is a Cripto
inhibitor.
25. The method of claim 20, wherein the inhibitor is an antisense
oligonucleotide.
26. The method of claim 25, wherein the antisense oligonucleotide
is an anti-Nodal Morpholino.
27. The method of claim 15, wherein the inhibitor is Lefty.
28. The method of claim 15, wherein the inhibitor is recombinant
Lefty.
29. The method of claim 15, wherein the inhibitor is glycosylated
Lefty.
30. The method of claim 20, wherein the inhibitor is a Notch
inhibitor.
31. The method of claim 30, wherein the Notch inhibitor is a Notch
siRNA.
32. The method of claim 30, wherein the inhibitor is a Notch4
inhibitor.
33. The method of claim 32, wherein the inhibitor is a Notch4
siRNA.
34. The method of claim 15, wherein the inhibitor is derived from a
microenvironment of human embryonic stem cells.
35. A method of inhibiting tumor cell growth in a mammal comprising
administering to the mammal, having at least one tumor cell present
in its body, an effective amount of a preconditioned
microenvironment, which has been in contact with human embryonic
stem cells.
36. A method of detecting aggressive tumor cells comprising the
steps of: a. obtaining a sample from a patient; b. assaying the
sample for the presence of Nodal and Lefty; and c. detecting
aggressive tumor cells if Nodal is present and Lefty is absent in
the sample.
37. The method of claim 36, wherein assaying for the presence of
Nodal and the absence of Lefty comprises a nucleic acid based
assay.
38. The method of claim 36, wherein assaying for the presence of
Nodal and the absence of Lefty comprises a protein based assay.
39. A method of identifying a compound for treating aggressive
tumors, comprising: a. providing a plurality of cells that express
Nodal; b. assaying the cells for Nodal activity in the presence and
absence of a candidate compound; and c. identifying the compound as
a compound for treating aggressive tumors if the Nodal activity is
less in the presence of the candidate compound than in the absence
of the candidate compound.
40. A method for monitoring the effectiveness of a pharmaceutical
composition as an agent for treating aggressive tumors in a patient
comprising the steps of: a. obtaining a sample from a patient; b.
assaying the sample for the presence of Nodal; c. administering an
amount a pharmaceutical composition to the patient; d. repeating
steps (b) and (c) on subsequently-collected samples from the
patient; and e. comparing the amount of Nodal detected in the
sample from step (a) with the amount of Nodal detected in the
samples from step (d), wherein the effectiveness of the
pharmaceutical composition is monitored by detecting changes in the
amount of Nodal in the subsequently-collected samples compared with
the sample taken in step (a).
41. A method for detecting the presence of aggressive tumor cells
comprising the steps of: a. obtaining a sample of tumor cells from
a patient; b. conducting a sequence based methylation analysis of
the Nodal CpG island in the tumor cells; c. comparing the degree of
methylation in the CpG island of Nodal in the tumor cells to that
of non-aggressive or non-tumor cells; d. correlating
hypermethylation of Nodal with the presence of aggressive tumor
cells.
42. A method for detecting the presence cells having a
dedifferentiated, multipotent plastic phenotype in a mammal
comprising the steps of: a. obtaining a sample from a mammal; b.
assaying the sample for the presence of Nodal; c. correlating the
presence of Nodal with the presence cells having a
dedifferentiated, multipotent plastic phenotype.
43. The method of claim 42 wherein the sample is a bodily
fluid.
44. The method of claim 43 wherein the bodily fluid is serum.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application Nos. 60/820,740, filed on Jul. 28, 2006, and
60/941,343, filed on Jun. 1, 2007, the disclosures of which are
incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The invention relates to methods of using compounds produced
by embryonic stem cells to treat and/or prevent the growth and/or
dissemination of aggressive tumor cells in a patient. More
specifically, the invention relates to the administration to the
patient of inhibitors of Nodal activity, including, but not limited
to, those that are exclusively produced by human embryonic stem
cells. The invention also relates to methods for detecting
aggressive tumors in a patient comprising detecting the presence of
Nodal in the patient's cells.
BACKGROUND
[0003] Aggressive tumor cells share a number of characteristics
with embryonic progenitors. During vertebrate development,
multipotent precursor cells are gradually specified to particular
fates through the autocrine or paracrine delivery of signaling
molecules, and during cancer progression, malignant cells similarly
release and receive cues that promote tumor growth and metastasis.
Aggressive tumor cells, particularly melanoma cells, display stem
cell-like plasticity as demonstrated by their molecular signature
that signifies a dedifferentiated, multipotent plastic phenotype
(i.e. one that is capable of responding to microenvironmental
factors as well as influencing other cells via epigenetic
mechanisms) (Bittner et al., 2000, Nature 406:536-540; Hendrix et
al., 2003, Nat. Rev. Cancer 3:411-421). Furthermore, aggressive
melanoma cells are capable of vasculogenic mimicry, i.e. they are
able to form vasculogenic-like networks while simultaneously
expressing genes associated with an endotheilial cell type. (Seftor
et. al., 2002, Crit. Rev. Oncology Hematol. 44:17-27; Maniotis et.
al., Am. J. Pathol. 155:739-752).
[0004] Previous studies capitalized on the similarities between
cancer and stem cells by examining the ability of embryonic
microenvironments to modulate tumor cell behavior (Pierce et al.,
1982, Cancer Res. 42:1082-1087; Gerschenson et al., 1986, Proc.
Natl. Acad. Sci. U.S.A 83:7307-7310; Lee et al., 2005, Dev. Dyn.
233:1560-1570; Mintz et al., 1975, Proc. Natl. Acad. Sci. U.S.A
72:3585-3589). For example, Pierce and colleagues reported that
neural stage mouse embryos regulate neuroblastoma cells, and that
embryonic skin inhibits melanoma growth ((Pierce et al., 1982,
Cancer Res. 42:1082-1087; Gerschenson et al., 1986, Proc. Natl.
Acad. Sci. U.S.A 83:7307-7310). Although studies have focused on
the role of embryonic signals in the regulation of tumor cells, few
have utilized embryonic models as a tool to discover molecular
mechanisms by which cancer cells modulate their microenvironment
and the resulting reciprocal interactions.
[0005] One of the major factors contributing to the plasticity of
stem cells is Nodal. Nodal is a highly conserved morphogen
belonging to the transforming growth factor beta (TGF.beta.) super
family (Schier et al., 2003, Annu. Rev. Cell Dev. Biol.
19:589-621). By acting as an organizing signal before gastrulation,
Nodal initiates embryonic axis formation, and previous studies
demonstrated that the ectopic expression of Nodal induces
mesendodermal fates in ectopic positions (Whitman, 2001, Dev. Cell
1:605-617; Schier, 2003, Annu. Rev. Cell Dev. Biol. 19:589-621;
Iannaccone et al., 1992, Dev. Dyn. 194:198-208; Smith, 1995, Curr.
Opin. Cell Biol. 7:856-861; Zhou et al., 1993, Nature 361:543-547;
Rebagliati et al., 1998, Proc. Natl. Acad. Sci. U.S.A 95:9932-9937;
Toyama et al., 1995, Development 121:383-391).
[0006] Activation of Nodal includes binding to the co-receptor
Cripto and subsequent phosphorylation of the type I and type II
activin-like kinase receptors (ALK). In turn, SMAD2 and SMAD3 are
activated (Lee et. al., 2006, Nature Medicine 12:882-884).
Furthermore, human embryonic stem cells express Nodal and secrete
endogenous inhibitors of Nodal such as Lefty A/B (Besser, D., 2004,
J. Biol. Chem. 279:45076-45084). Lefty A and Lefty B, human
homologs to murine Lefty 2 and Lefty 1, respectively, are separated
by approximately 50 kb on chromosome 1q42 and are 96% identical to
each other (Kosaki et. al., 1999, Am. J. Hum. Genet. 64:712-21).
Lefty A and Lefty B are members of the TGF.beta. superfamily, and
are considered one of the powerful inhibitors of Nodal.
SUMMARY OF THE INVENTION
[0007] The invention provides compositions comprising one or more
isolated factors from a microenvironment of human embryonic stem
cells (hESCs), including, but not limited to, Lefty and inhibitors
of Nodal. The invention further provides an isolated Lefty protein
produced by conditioning a matrix with human embryonic stem cells.
The invention further provides a protein comprising glycosylated
Lefty, including glycosylated Lefty isolated from the
microenvironment of human embryonic stem cells, compositions
thereof, and methods of inhibiting tumor cell growth in a mammal
comprising administering to the mammal such compositions.
[0008] The invention also provides methods of utilizing factors
derived from human embryonic stem cells (hESC) and their
microenvironment to treat and prevent tumor formation and
progression and to inhibit tumor cell aggressiveness. The invention
further provides methods of inhibiting tumor cell growth and/or
treating aggressive tumors in a mammal comprising administering to
the mammal, having at least one tumor cell present in its body, an
effective amount of an inhibitor of Nodal activity, including, but
not limited to, hESC-derived Lefty and synthetic derivatives as
discussed herein, glycosylated Lefty, recombinant Lefty, anti-Nodal
antibodies, inhibitors of one or more of activin receptor-like
proteins ALK 4, ALK 5, and/or ALK7, inhibitors of Cripto,
anti-Nodal antisense moieties such as anti-Nodal Morpholinos, and
Notch inhibitors including, but not limited to, Notch 4 inhibitors
such as Notch 4 siRNA.
[0009] The invention also provides a method of inhibiting tumor
cell growth in a mammal comprising administering to the mammal,
having at least one tumor cell present in its body, an effective
amount of a preconditioned microenvironment, which has been in
contact with human embryonic stem cells.
[0010] The invention further provides methods for detecting
aggressive tumors (including but not restricted to melanoma and
breast carcinoma) in a patient comprising the steps of: obtaining a
sample from a patient; assaying the sample for the presence of
Nodal and the absence of a Nodal inhibitor (such as Lefty or
modified Lefty or Lefty derivatives); and detecting aggressive
tumor cells if Nodal is present and Lefty is absent in the sample.
The invention also provides methods of identifying compounds for
treating aggressive tumors comprising providing a plurality of
cells that express Nodal; assaying the cells in the presence and
absence of a candidate compound for activity of Nodal; and
identifying the compound as a compound for treating aggressive
tumors if the Nodal activity is less in the presence of the
candidate compound than in the absence of the candidate
compound.
[0011] In addition, the invention provides methods for monitoring
the effectiveness of a pharmaceutical composition as an agent for
treating aggressive tumors in a patient, for detecting the presence
of aggressive tumor cells, and for methods for detecting the
presence of cells having a dedifferentiated, multipotent plastic
phenotype in a mammal
[0012] Specific embodiments of the invention will become evident
from the following more detailed description of certain preferred
embodiments and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 shows an experimental methodology flow chart
demonstrating the utilization of human embryonic stem cell
microenvironments to inhibit tumor cell aggressiveness.
[0014] FIG. 2 shows that the microenvironment of human embryonic
stem cells induces melanoma cell spheroid formation. (A-G) Phase
contrast microscopy showing the confluent growth of C8161
amelanotic, human metastatic cutaneous melanoma cells on 3-D
Matrigel matrix (A), compared with the formation of colonies by H1
(B) and HSF-6 (C) human embryonic stem cells (hESCs) on 3-D
Matrigel matrix; following removal of the hESCs from their 3-D
matrix (leaving a denuded preconditioned matrix, CMTX, shown in
inset), the C8161 tumor cells seeded onto the H1 (D,E) and HSF-6
(F) hESC preconditioned matrices, CMTX (Matrigel), now form
spheroids (D-F) similar to hESC colonies. (Bar in "A" equals 200
.mu.m). In contrast, C8161 cells exposed to medium conditioned by
H1 cells are unable to form spheroids (G).
[0015] FIG. 3 shows the epigenetic changes in human metastatic
cutaneous melanoma cells exposed to the microenvironment of human
embryonic stem cells. (A) Western blot analysis of whole cell
lysates (with an equal amount of protein loaded per sample), for a
melanocyte marker, Melan-A, shows its absence in H1 hESCs on
Matrigel and C8161 tumor cells on Matrigel; and the induction of
Melan-A in C8161 cells exposed to the H1 hESCs preconditioned
matrix, CMTX (Matrigel), compared with Melan-A in control human
epidermal melanocytes (HEMn) on Matrigel (upper panel). (B)
Semi-quantitative RT-PCR analysis of Melan-A gene expression in
HEMn cultured on Matrigel compared to C8161 cells exposed to a HEMN
preconditioned matrix, CMTX (Matrigel), compared with C8161 cells
on Matrigel. The CMTX lane serves as a control demonstrating the
complete removal of the HEMn cells from the preconditioned matrix
prior to seeding the C8161 melanoma cells. GAPDH was used as a
loading control for RNA (lower panel). (C) Semi-quantitative RT-PCR
analysis demonstrating that a collagen I 3-D matrix preconditioned
by human melanocytes (HEMn18 or HEMn20 CMTX) does not change the
expression of the genes tested. This indicates that the benign
melanocyte microenvironment does not epigenetically influence
metastatic melanoma cells to change their plastic, molecular
phenotype.
[0016] FIG. 4 shows that the microenvironment of human embryonic
stem cells decreases melanoma cell invasion and tumorigenesis. (A)
Invasion of C8161 cells following culture on unconditioned Matrigel
(Control) or Matrigel preconditioned by either H1 or HSF-6 hESCs
was calculated as a percentage of cells able to invade through a
defined matrix (collagen IV, laminin, and gelatin)-coated membrane
during a 24 hour period using the MICS (Membrane Invasion Culture
System) assay. Bars represent the mean, normalized, invasion
indices.+-.standard deviations. The values indicated by an asterisk
(*) are significantly different from the invasion index of control
cells. (B) In vivo tumor formation in a mouse injected with C8161
cells pre-exposed for 3 days to either a control matrix (Matrigel)
or a matrix conditioned by hESCs (H9 CMTX) (n=21). Values represent
the median tumor volume (mm3).+-.interquartile range, and tumor
volumes were significantly different at the time points indicated
by an asterisk (*) (P<0.05).
[0017] FIG. 5 shows differential expression of Nodal, Lefty, and
Cripto in hESCs, aggressive tumor cells, and normal human cells.
(A) Western blot analyses of Nodal, Lefty and Cripto in: H1 and H9,
human embryonic stem cell (hESC) lines; C8161, human metastatic
melanoma cells; normal human melanocytes; MDA-MB-231, human
metastatic breast carcinoma cells; Hs 578 Bst normal human
myoepithelial cells; and HMEpC normal human mammary epithelial
cells. Actin is used as a loading control. (B) Real Time RT-PCR
analysis of Lefty-B mRNA expression in H9, human embryonic stem
cells (hESCs); C8161, human metastatic melanoma cells; normal human
melanocytes; MDA-MB-231, human metastatic breast carcinoma cells;
Hs 578 Bst normal human myoepithelial cells; HMEpC normal human
mammary epithelial cells; GM00473 and GM00957A human amniotic
fluid-derived stem cells; SC00125, human umbilical cord-derived
stem cells; human adult mesenchymal stem cells (MSC); and
HTR-8/SVneo, immortalized human cytotrophoblast cells. (C)
Immunofluorescence localization of Cripto in 100% of H9 hESCs and
in small subpopulations of C8161 melanoma and MDA-MB-231 breast
carcinoma cells. Bar equals 10 .mu.m. (D) Western blot analyses of
Nodal, Lefty and Cripto in: H9, hESCs; GM00473 and GM00957A human
amniotic fluid-derived stem cells; SC00125, human umbilical
cord-derived stem cells; human adult mesenchymal stem cells (MSC);
and HTR-8/SVneo, immortalized human cytotrophoblast cells. Actin is
used as a loading control.
[0018] FIG. 6 shows patterns of Nodal Expression in Primary and
Metastatic Melanoma Lesions. FIGS. 6 (A-F) show immunohistochemical
analysis of Nodal staining in (A) normal skin, (B-D) a primary
cutaneous melanoma and (E and F) cutaneous melanoma metastases.
Arrows delineate (A) normal melanocytes and (F) Nodal protein
localized to melanoma cell membranes. (C) and (D) represent radial
and vertical growth phases respectively. FIG. 6(A-D) are
representative of 5 patient samples and FIG. 6(E-F) are
representative of 10 patient samples. Isotype controls are pictured
in the insets, and bars equal 50 .mu.m.
[0019] FIG. 7 shows patterns of Nodal Expression in Breast Cancer
Carcinoma. Immunohistochemical analysis of Nodal staining in normal
breast tissue, ductal carcinoma in situ (DCIS), invasive ductal
carcinoma (IDC) and metastatic IDC. Bars equal 100 .mu.m.
[0020] FIG. 8 shows the distribution of Nodal and Lefty on hESCs
cultured on Matrigel Immunofluorescence localization of Lefty and
Nodal in H9 hESCs cultured on Matrigel and Western blot analysis of
Lefty protein in matrix conditioned by hESCs (H9 CMTX). Top panels
represent reconstructed confocal images depicting the cross-section
of a hESC colony with its underlying matrix. Dashed line designates
the cell-matrix interface and the arrow points to the upper surface
of the hESC colony. Corresponding images on the right illustrate
Lefty and Nodal at the cell surface (arrow) and the cell-matrix
interface (dashed line). The large image is a 3-dimensional
confocal projection of hESC colonies stained with Lefty and Nodal
(inset). Bar equals 25 .mu.m.
[0021] FIG. 9 shows that the microenvironment of human embryonic
stem cells (hESCs) leads to the reduction of Nodal expression and
tumorgenicity in plastic metastatic melanoma and breast cancer
cells exposed to the embryonic preconditioned matrix. (A) Western
blot analyses demonstrating that the microenvironments of hESCs
reduced the expression of Nodal protein in multipotent melanoma
(C8161) and breast carcinoma (MDA-MB-231) cells. (B) Western blot
analyses of Nodal protein in human metastatic melanoma cells
(C8161) and human metastatic breast carcinoma cells (MDA-MB-231)
exposed for 3 days to either control (unconditioned) Matrigel or to
Matrigel conditioned by hESCs (H9 CMTX). Some cancer cells exposed
to H9 CMTX were subsequently recovered on control (unconditioned)
Matrigel for 2 or 7 days prior to Western blot analysis. Actin is
used as a loading control. (C) Real Time RT-PCR analysis of Nodal
mRNA in human metastatic melanoma cells (C8161) and human
metastatic breast carcinoma cells (MDA-MB-231) exposed for 3 days
to either control (unconditioned) Matrigel or to Matrigel
conditioned by hESCs (H9 CMTX). Some cancer cells exposed to H9
CMTX were subsequently recovered on control (unconditioned)
Matrigel for 2 or 7 days prior to analysis. (D)
Immunohistochemistry localization of Nodal in C8161 cells forming
tumors in nude mice at Day 19. Nodal staining is strongest in the
Matrigel control injected tumor cells vs. the diminished Nodal
staining in C8161 cells exposed to the H9 CMTX (correlated with a
lower tumor burden). (Bars equal 50 .mu.m.) (E) Relative colony
formation of C8161 and MDA-MB-231 cells cultured on soft agar for
14 days following 3 days of exposure to either control
(unconditioned) Matrigel or to H9 CMTX. Assays were conducted in
the presence or absence of rNodal (100 ng/mL). Bars represent mean
normalized colony formation.+-.standard deviation. The values
indicated by an asterisk (*) are significantly different from the
colony forming ability of control cells and the values indicted by
a double asterisk (**) are significantly different from the colony
forming ability of control cells and H9 CMTX treated cells (n=12,
P<0.05). (F) Western blot analyses of Nodal protein in human
metastatic melanoma cells (C8161) and human metastatic breast
carcinoma cells (MDA-MB-231) exposed for 3 days to either control
(unconditioned) Matrigel or to Matrigel conditioned by normal
melanocytes (melanocyte CMTX), normal myoepithelial cells (Hs 578
Bst CMTX), amniotic fluid-derived stem cells (GM00473/GM00957A
CMTX) or trophoblast cells (HTR-8/SVneo CMTX). Actin is used as a
loading control. (G) Real Time RT-PCR analysis of Nodal mRNA in
human metastatic melanoma cells (C8161) exposed for 3 days to
either control (unconditioned) Matrigel or to Matrigel conditioned
by amniotic fluid-derived stem cells (GM 00473/GM 00957A CMTX) or
trophoblast cells (HTR-8/SVneo CMTX). Gene levels were normalized
using 18s and bars represent mean gene expression normalized to H9
(A,B) or Matrigel (C,D) values.
[0022] FIG. 10 shows the role of hESC-derived Lefty in Nodal
down-regulation. (A) An abundance of the Nodal inhibitor Lefty
within the hESC conditioned matrices (CMTX). Lefty protein is
absent in the C8161 cells within its own conditioned matrix
(C8161+CMTX). (B) Immunofluorescence localization of
FITC-conjugated anti Lefty Morpholinos (MO.sup.LEFTY) in H9 hESC
colonies on Matrigel. (C) Western blot analysis of Lefty protein in
H9 hESCs treated with either vehicle (Control), MO.sup.Control (MO
Control), or MO.sup.Lefty (MO Lefty). Actin is used as a loading
control. (D) Real Time RT-PCR analysis of Oct-3/4 and Nanog
expression in H9 hESCs treated with either vehicle (H9),
MO.sup.CONTROL (MO Control), or MO.sup.LEFTY (MO Lefty). Gene
levels were normalized using 18s and bars represent mean gene
expression normalized to H9. (E) Real Time RT-PCR analysis of Nodal
mRNA expression in C8161 cells cultured for 3 days on control
(unconditioned) Matrigel, Matrigel conditioned by hESCs (H9 CMTX)
or Matrigel conditioned by hESCs in which Lefty protein expression
was knocked out with Lefty-specific Morpholinos (H9 CMTX MO Lefty).
(F) Western blot analysis of Nodal protein in human metastatic
melanoma (C8161) and breast carcinoma (MDA-MB-231) cells exposed
for 3 days to either control (unconditioned) Matrigel or to
Matrigel seeded with Lefty protein purified from hESCs (H9-derived
Lefty). MDA-MB-231 cells were allowed to recover on fresh Matrigel
for 2 days prior to analysis and Actin is used as a loading
control. (G) Relative colony formation of C8161 and MDA-MB-231
cells cultured on soft agar for 14 days following 3 days of
exposure to either control Matrigel or Matrigel seeded with Lefty
purified from hESCs (Lefty), in the presence or absence of rNodal
(100 ng/mL). Bars represent mean normalized colony
formation.+-.standard deviation. The values indicated by an
asterisk (*) are significantly different from the colony forming
ability of control cells (n=6, P<0.05).
[0023] FIG. 11 shows the down-regulation of Nodal by recombinant
Lefty. Western blot analysis of Nodal protein in C8161 cells
exposed for 48 hrs to varying concentrations (0-1000 ng/mL) of
rLefty-B showing that the addition of rLefty to C8161 cells reduces
Nodal expression
[0024] FIG. 12 shows that hESC derived Lefty is glycosylated.
Staining for glycoprotein and detection of Lefty-A and Lefty-B on a
Western blot containing recombinant Lefty (rLefty)-B, rLefty-A and
a lysate from H9 human embryonic stem cells (hESCs) plus matrix
conditioned by the H9 hESCs for 3 days (CMTX). After
SDS-polyacrylamide gel electrophoresis, the proteins were
transblotted and stained for glycoproteins, identified by green
bands (bottom). Lefty-A and -B were subsequently detected by
Western blot analysis (top). Arrows point to Lefty protein(s) on
the Western blot and to the identical locations on the image
showing the stained glycoproteins.
[0025] FIG. 13 shows that Nodal inhibition and the microenvironment
of hESCs abrogate tumorigenicity in vivo. (A) In vivo tumor
formation in a mouse injected with MDA-MB-231 cells pre-exposed for
3 days to either a control matrix (Matrigel) or a matrix
conditioned by hESCs (H9 CMTX) (n=10) (B) C81-61 cells, transfected
with either an empty vector or a Nodal expression construct (n=5),
and (C) MDA-MB-231 cells treated with either MO.sup.Control or
MO.sup.Nodal (n=10). Values represent the mean tumor volume
(mm.sup.3).+-.standard error (A) or standard deviation (B,C), and
tumor volumes were significantly different at the time points
indicated by an asterisk (*) (P<0.05). (D) The ratio of tumor
cell proliferation to apoptosis for C8161 and MDA-MB-231 derived
tumors, determined by immunohistochemical staining for Ki67 and
terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling
(TUNEL). Prior to injection into a mouse, C8161 and MDA-MB-231
cells were cultured for 3 days on control or hESC conditioned (H9
CMTX) matrices, or treated with MO.sup.Nodal to knock down Nodal
expression. Bars represent mean normalized values.+-.standard
deviation, and values indicated by an asterisk (*) are
significantly different from control values (P<0.05). (E)
Immunohistochemical analysis of Ki67 expression (red/brown) and
TUNEL staining in orthotopic melanoma (C8161) and breast carcinoma
(MDA-MB-231) tumors. Prior to injection into a mouse, cells were
treated with MO.sup.Nodal, exposed to H9 hESC CMTX, or left
untreated (control). Proliferation is indicated by Ki67 staining
and apoptotic nuclei were detected with confocal microscopy as red
staining localized to the nuclei of apoptotic C8161 or MDA-MB-231
cells. For the TUNEL analyses, cell nuclei are counterstained blue
with DAPI. Bar equals 25 .mu.m. (F) In vitro proliferation of C8161
and MDA-MB-231 cells treated with either MO.sup.Control or
MO.sup.Nodal. Values represent the mean cell count
(.times.1000).+-.standard deviation 4 days after the plating of
15,000 cells. An asterisk (*) indicates a significant difference
between control and MO.sup.Nodal treated cells (n=4,
P<0.05).
[0026] FIG. 14 shows Nodal inhibition abrogates melanoma
tumorigenicity. (A) Phase contrast microscopy of cells cultured for
7 days in a soft agar assay. The panels represent the
colony-forming ability of poorly aggressive C81-61 cells,
aggressive C8161 cells, C8161 cells treated with MO.sub.Nodal and
C8161 cells treated with MO.sub.Nodal and rescued with recombinant
Nodal (100 ng/mL). Bar equals 20 .mu.m. (B) In vivo tumor formation
in a mouse injected with C8161 cells treated with either
MO.sub.Control or MO.sub.Nodal. Values represent the median tumor
volume (mm.sup.3).+-.interquartile range, and the MO.sub.Control
and MO.sub.Nodal tumor volumes were significantly different at the
time points indicated by an asterisk (*) (n=5, p<0.05). (C)
Immunohistochemical analysis of Nodal staining in an orthotopic
tumor derived from C8161 cells treated with MO.sub.Nodal. The C8161
cells have begun to re-express Nodal by 17 days post-injection. Bar
equals 50 .mu.m. (MO.sub.Nodal=antisense morpholino to Nodal). (D)
Acquisition of tumorigenic potential by C81-61 poorly aggressive
melanoma cells transfected with Nodal cDNA, compared with mock
transfected C81-61 control: Values are reported as median tumor
volume after 38 days.+-.standard deviation (*p<0.05;
n=5/parameter).
[0027] FIG. 15 shows down-regulation of Nodal signaling results in
acquisition of a melanocyte-like phenotype and loss of the
dedifferentiated, plastic phenotype. (A) Western blot analyses of
Nodal, phosphorylated SMAD-2, total SMAD 2/3, and Actin in C8161
cells 48 hours after administration of either vehicle or an ALK
4/5/7 inhibitor (SB431542, 1 .mu.M, 10 .mu.M). (A,B) All Nodal
bands represent the pro-protein. (B) Western blot analyses of
Nodal, Tyrosinase and Actin in C8161 cells 24 hours after the
administration of either vehicle or different concentrations of ALK
inhibitor while (C) is a Western analyses for VE-Cadherin, Keratin
18 and Actin in C8161 cells cultured on 3-D collagen I matrices for
6 days in the presence of vehicle or different concentrations of
ALK inhibitor. (D) Reduction in invasive ability and (E) abrogation
of vasculogenic mimicry following down-regulation of Nodal with ALK
4/5/7 inhibitor.
[0028] FIG. 16 shows molecular cross-talk between Nodal and Notch.
(A) Knockdown of Nodal expression in C8161 cells by Notch siRNAs,
particularly Notch 4 siRNA. Western blot analysis of C8161 cells 72
hrs following the administration of siRNA. Real-time RT-PCR and
Western blot analyses confirmed the silencing of each Notch at this
time points. (B) Notch expression is relatively unaffected by
knockdown of Nodal in C8161 cells. Western blot analysis of Notch
1, 2 and 4 in C8161 cells treated with the Nodal inhibitor
SB431542.
[0029] FIG. 17 shows that exposure of aggressive C8161 human
melanoma cells to matrices conditioned by hESCs induces an increase
in the methylation of specific cytosine residues (CpG residues
6-17) in the first half of the CpG island in the Nodal promoter.
The graphs depict the methylation status of the first half of the
Nodal CpG island in aggressive C8161 human cutaneous melanoma cells
cultured on either Matrigel or on Matrigel conditioned by H9 hESCs.
Each circle represents a CpG dinucleotide in the CpG island. Black
and grey circles symbolize methylated and unmethylated residues
respectively. Each row represents an individual clone or allele.
Although culture in the presence of a hESC microenvironment (H9
CMTX) globally increases methylation by only 6.8%, the shaded area
experienced a 32% increase in methylation when cells were cultured
on H9 CMTX versus Matrigel alone.
DETAILED DESCRIPTION
[0030] In certain embodiments, the invention provides a composition
comprising one or more isolated factors from a microenvironment of
human embryonic stem cells (hESCs). As used herein, "one or more
isolated factors" refers to any one or any group of factors present
in a microenvironment of hESCs. The factors may be individually
isolated, or isolated in a manner that provides a group of factors
in combination. Alternatively, "one or more isolated factors" may
refer to any one or a group of factors present on a defined matrix.
As used herein, a "microenvironment" is an environment that
comprises a basement membrane or other defined matrix that is in
contact with embryonic stem cells, preferably human embryonic stem
cells, and that is influenced by the embryonic stem cells. A
"preconditioned" microenvironment is a microenvironment that has
been in contact with human embryonic stem cells under appropriate
conditions as described herein, and described for example, in
Postovit et al., 2006, Stem Cells 24:501-505 and illustrated in
FIG. 1 herein. FIG. 1 herein illustrates utilization of human
embryonic stem cell microenvironments to inhibit tumor cell
aggressiveness.
[0031] In one embodiment, the isolated factor(s) from a
microenvironment of hESCs inhibit Nodal. As described herein,
aggressive tumor cells express Nodal, and Nodal is essential for
plasticity, tumorgenicity and aggressiveness. Therefore, inhibiting
Nodal provides an excellent approach for treating and preventing
aggressive tumors. As used herein, the terms "aggressive tumor" and
"aggressive cancer," which include "aggressive melanoma" and
"aggressive breast carcinoma" refer to a malignant cell that has
neoplastic growth with or without metastatic involvement. In a
non-limiting example, an aggressive tumor may refer to a malignant
cell that has a transdifferentiated phenotype characterized by the
aberrant expression of genes normally restricted to other cell
lineages, concomitant with the loss of lineage-specific factors.
For example, aggressive melanoma cells possess keratin-positive,
intermediate filaments indicative of epithelial cells, and they
aberrantly express genes, including VE-Cadherin, normally
associated with endothelial cells. Furthermore, the expression of
melanocyte specific markers, such as Tyrosinase, is dramatically
reduced, and sometimes absent, in aggressive melanoma cells.
Tyrosinase catalyses the conversion of tyrosine to the pigment
melanin, and is reduced by more than 35-fold in aggressive
melanomas as compared to their poorly aggressive counterparts.
Aggressive tumor cells also have the ability to express multiple
stem cell markers, suggestive of a multipotent, dedifferentiated
phenotype. These aggressive tumor cells are also highly
metastatic.
[0032] In one embodiment of the invention, the factor from a
microenvironment of hESCs is Lefty. As noted herein, Lefty,
including hESC-derived Lefty, is an inhibitor of Nodal. As used
herein, the terms "Lefty A/B" and "Lefty" are interchangeable and
refer to either Lefty A or Lefty B, or both Lefty A and Lefty B in
combination. In one embodiment, Lefty, isolated from a
microenvironment, may be substantially pure. In another embodiment,
Lefty may be present in combination with other hESC factors.
[0033] In another embodiment, the invention provides an isolated
Lefty protein produced by conditioning a matrix with human
embryonic stem cells. As used herein, "conditioning a matrix"
refers to preparing a preconditioned microenvironment as defined
herein. In certain embodiments, the matrix is conditioned with
hESCs from 0 to 10 days or any range in between, including, but not
limited to, from 0.5 to 10 days, from 2 to 8 days, from 3 to 6
days, from 3 to 5 days, from 3 to 4 days, or for 1, 2, 3, 4, or 5
days. Lefty may be isolated from the matrix by any method known to
one of skill in the art, including through use of anti-Lefty
antibodies.
[0034] In one embodiment, the invention provides a protein
comprising glycosylated Lefty. In this embodiment, Lefty may be
glycosylated to varying degrees, and may comprise one or more N-
and/or O-linked glycosylation sites, or a combination thereof. In
one embodiment, the glycosylated Lefty is characterized in that
more than 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the possible N- and/or
O-glycosylation sites are glycosylated. In another embodiment, the
glycosylated Lefty is characterized in that less than 100%, 95%,
90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%,
25%, 20%, 15%, 10%, or 5% of the possible N- and/or O-glycosylation
sites are glycosylated. In another embodiment, the glycosylated
Lefty is characterized in that the percentage of possible N- and/or
O-glycosylation sites that are glycosylated is based on a
combination of the "more than" and "less than" percentages recited
above. Thus, in one non-limiting example, the glycosylated Lefty is
characterized in that more than 30% and less than 70% of the
possible N- and/or O-glycosylation sites are glycosylated. In
another embodiment, 100% of the possible N- and/or O-glycosylation
sites are glycosylated.
[0035] In one embodiment, the glycosylated Lefty is glycosylated to
substantially the same extent as Lefty derived from hESCs.
[0036] Glycosylated Lefty may be prepared by any method, including
by recombinant methods (see, e.g. Sambrook et al., 2001, Molecular
Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.). In one embodiment,
glycosylated Lefty is prepared recombinantly in Chinese Hamster
Ovary (CHO) cells. Alternatively, glycosylated Lefty may be
prepared by chemical synthesis methods (such as solid phase peptide
synthesis) using techniques known in the art such as those set
forth by Merrifield et al., 1963, J. Am. Chem. Soc. 85:2149;
Houghten et al., 1985, Proc Natl Acad. Sci. USA 82:5132; and
Stewart and Young, Solid Phase Peptide Synthesis (Pierce Chemical
Co. 1984), or by a combination of synthetic and recombinant
techniques. Glycosylated Lefty may also be prepared by isolation
from hESCs, including by isolation from the microenvironment of
hESCs.
[0037] Included within the scope of the invention are fragments or
derivatives of Lefty or glycosylated Lefty. As used herein,
"fragment" means any portion of the full length Lefty sequence
having an activity of the full length protein, including, but not
limited to, the ability to inhibit Nodal. Included in the scope of
"fragments" are naturally occurring enzymatic cleavage products.
Included in the scope of the term "derivatives" are derivatives of
full length Lefty as well as fragments thereof. As used herein,
"derivative" or "derivatives" includes variations of Lefty having
one or more amino acid residues which have been added, deleted,
inserted or substituted, where the resulting polypeptide has an
activity of Lefty, including, but not limited to, the ability to
inhibit Nodal. As used herein, "derivatives" also includes chemical
derivatives of Lefty and variations thereof. It will be understood
to one of skill in the art that these variations may occur in any
combination.
[0038] Chemically modified derivatives of glycosylated Lefty may be
prepared by one skilled in the art, in view of the disclosures
described herein. Glycosylated Lefty derivatives are modified in a
manner that is different--either in the type or location of the
molecules naturally attached to the polypeptide. Derivatives may
include molecules formed by the deletion of one or more
naturally-attached chemical group, or they may be modified by the
covalent attachment of one or more polymers. For example, the
polymer selected is typically water-soluble so that the protein to
which it is attached does not precipitate in an aqueous
environment, such as a physiological environment. Included within
the scope of suitable polymers is a mixture of polymers.
Preferably, for therapeutic use of the end-product preparation, the
polymer will be pharmaceutically acceptable.
[0039] The polymers each may be of any molecular weight and may be
branched or unbranched. The polymers each typically have an average
molecular weight of between about 2 kDa to about 100 kDa (the term
"about" indicating that in preparations of a water-soluble polymer,
some molecules will weigh more, some less, than the stated
molecular weight). The average molecular weight of each polymer is
preferably between about 5 kDa and about 50 kDa, more preferably
between about 12 kDa and about 40 kDa and most preferably between
about 20 kDa and about 35 kDa.
[0040] Suitable water-soluble polymers or mixtures thereof include,
but are not limited to, N-linked or O-linked carbohydrates, sugars,
phosphates, polyethylene glycol (PEG) (including the forms of PEG
that have been used to derivatize proteins, including
mono-(C.sub.1-C.sub.10), alkoxy-, or aryloxy-polyethylene glycol),
monomethoxy-polyethylene glycol, dextran (such as low molecular
weight dextran of, for example, about 6 kD), cellulose, or other
carbohydrate based polymers, poly-(N-vinyl pyrrolidone)
polyethylene glycol, propylene glycol homopolymers, polypropylene
oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g.,
glycerol), and polyvinyl alcohol. Also encompassed by the present
invention are bifunctional crosslinking molecules which may be used
to prepare covalently attached glycosylated Lefty polypeptide
multimers.
[0041] In general, chemical derivatization may be performed under
any suitable condition used to react a protein with an activated
polymer molecule. The optimal reaction conditions will be
determined based on known parameters and the desired result. For
example, the larger the ratio of polymer molecules to protein, the
greater the percentage of attached polymer molecule. In one
embodiment, the glycosylated Lefty derivative may have a single
polymer molecule moiety at the amino-terminus. See, e.g., U.S. Pat.
No. 5,234,784.
[0042] The pegylation of a polypeptide may be specifically carried
out using any of the pegylation reactions known in the art. Such
reactions are described, for example, in the following references:
Francis et al., 1992, Focus on Growth Factors 3:4-10; European
Patent Nos. 0154316 and 0401384; and U.S. Pat. No. 4,179,337.
[0043] In another embodiment, glycosylated Lefty polypeptides may
be chemically coupled to biotin. The biotin/glycosylated Lefty
polypeptide molecules are then allowed to bind to avidin, resulting
in tetravalent avidin/biotin/glycosylated Lefty polypeptide
molecules. Glycosylated Lefty polypeptides may also be covalently
coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the
resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to
form decameric conjugates with a valency of 10.
[0044] Generally, conditions that may be alleviated or modulated by
the administration of the present glycosylated Lefty derivatives
include those described herein for glycosylated Lefty. However, the
glycosylated Lefty derivatives disclosed herein may have additional
activities, enhanced or reduced biological activity, or other
characteristics, such as increased or decreased half-life, as
compared to the non-derivatized molecules.
[0045] In a further embodiment, the present invention provides a
composition comprising a glycosylated Lefty. Compositions may be
formulated as known to one of skill in the art or as described
herein. In another embodiment, the present invention provides
methods of inhibiting tumor cell growth in a mammal comprising
administering to the mammal a composition comprising a glycosylated
Lefty at a physiologically acceptable dosage. Such a composition
may be administered in an effective or therapeutically effective
amount. As used herein, "effective amount" and "therapeutically
effective amount" are used interchangeably.
[0046] By mammal it is meant humans, companion animals such as cats
and dogs, primates such as monkeys and chimpanzees, and livestock
animals such as horses, cows, pigs, and sheep, or any patient in
need of, or that will benefit from, administration of any of the
methods or compounds or compositions of the invention. The term
"patient" as used herein includes human and animal subjects.
[0047] In one embodiment, the invention comprises methods of
inhibiting tumor cell growth in a mammal comprising administering
to the mammal a composition comprising a glycosylated Lefty at a
dosage between 0.01 and 500 ng/mL, between 0.01 and 200 ng/mL,
between 0.1 and 200 ng/mL, between 0.1 and 100 ng/mL, between 1 and
100 ng/mL, between 10 and 100 ng/mL, between 10 and 75 ng/mL,
between 20 and 75 ng/mL, between 20 and 50 ng/mL, between 25 and 50
ng/mL, or between 30 and 40 ng/mL. In another embodiment, the
invention comprises methods of inhibiting tumor cell growth in a
mammal comprising administering to the mammal a composition
comprising a glycosylated Lefty at a dosage of about 1, 5, 10, 20,
25, 30, 35, 40, 45, 50, 75, 100, 200, or 500 ng/mL. As used in this
context, "about" means within 0, 1, 2, or 3 ng/mL of the recited
concentration.
[0048] In certain embodiments, the invention provides methods of
using one or more factors from a microenvironment of human
embryonic stem cells to inhibit tumor cell aggressiveness. In one
embodiment, the factor(s) is an inhibitor of Nodal, including, but
not limited to Lefty and glycosylated Lefty.
[0049] In one embodiment of these methods, the factor(s) inhibiting
tumor cell aggressiveness do so by increasing apoptosis. As used
herein, "apoptosis" refers to the physiologic process of programmed
cell death which normally occurs during embryonic development and
during maintenance of tissue homeostasis. In a further embodiment,
the factor(s) inhibiting tumor cell aggressiveness do so by
decreasing cell proliferation. Cell proliferation is defined as the
increase in number of cells resulting from completion of the cell
cycle, as contrast to growth, which is the increase in the
individual cell mass. In a further embodiment, the factor(s)
inhibiting tumor cell aggressiveness do so by both increasing
apoptosis and by decreasing cell proliferation and/or by decreasing
the tumor cell proliferation-to-apoptosis ratio.
[0050] In another embodiment, the invention provides a method of
inhibiting tumor cell growth in a mammal comprising administering
to the mammal, having at least one tumor cell present in its body,
an effective amount of a preconditioned microenvironment, which has
been in contact with human embryonic stem cells.
[0051] In further embodiments of the invention, Nodal and/or Lefty
are used as biomarkers for aggressive tumor cell aggressiveness and
for prognostic, diagnostic and clinical diagnoses for aggressive
carcinoma including, but not limited to, melanoma and breast
cancer. In certain embodiments, the invention provides methods for
detecting aggressive tumors (including but not restricted to
melanoma or breast cancer) in a patient comprising the steps of:
obtaining a sample from a patient; assaying the sample for the
presence of Nodal and Lefty; and detecting aggressive tumors if
Nodal is present and Lefty is absent in the sample. As used in this
context, "a sample" includes, but is not limited to, tumor cells,
tissue samples, and bodily fluids as defined herein. In a
non-limiting example, the sample can be serum.
[0052] In certain embodiments, the presence of Nodal can be
detected by assaying for the Nodal gene or gene product. For
example, a nucleic acid based assay or a protein based assay can be
used to detect the presence of Nodal in a tumor sample. Exemplary
assays that can be used to detect Nodal include those described
herein. The presence of Lefty can be similarly detected. Those of
skill in the art readily recognize that other assays can be
designed following conventional methods as described, for example,
in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual,
3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y.
[0053] In another embodiment, compounds for treating aggressive
tumors may be identified by providing a plurality of cells that
express Nodal, assaying the cells for Nodal activity in the
presence and absence of a candidate compound, and identifying the
compound as a compound suitable for treating aggressive tumors if
the Nodal activity is less in the presence of the compound than in
the absence of the candidate compound. As used in this context,
"Nodal activity" refers Nodal expression and/or to any of the
activities recited herein, including maintaining tumor cell
plasticity, tumorgenicity and aggressiveness.
[0054] In another method of the invention, the effectiveness of a
pharmaceutical composition as an agent for treating aggressive
tumors in a patient may be monitored. The method comprises
obtaining a first sample from a patient; assaying the first sample
for the presence of Nodal; administering an amount a pharmaceutical
composition to the patient; assaying subsequently-collected
biological samples from the patient for the presence of Nodal; and
comparing the amount of Nodal detected in the first sample with the
amount of Nodal detected in the subsequent samples, wherein the
effectiveness of the pharmaceutical composition is monitored by
detecting changes in the amount of Nodal in the
subsequently-collected samples compared with the first sample. As
used in this context, "a sample" or "biological sample" includes,
but is not limited to, tumor cells, tissue samples, and bodily
fluids as defined herein. In a non-limiting example, the sample can
be serum.
[0055] In another method of the invention, the presence of
aggressive tumor cells in a mammal may be detected by obtaining a
sample of tumor cells from a patient; conducting a sequence based
methylation analysis of the Nodal CpG island in the tumor cells;
comparing the degree of methylation in the CpG island of Nodal in
the tumor cells to that of non-aggressive or non-tumor cells; and
correlating hypermethylation of Nodal with the presence of
aggressive tumor cells. The sequence based methylation analysis may
be based on the entirety of the CpG island or on a subsection
thereof. In a further method of the invention the presence of cells
having a dedifferentiated, multipotent plastic phenotype in a
mammal may be detected by obtaining a sample from a mammal;
assaying the sample for the presence of Nodal; and correlating the
presence of Nodal with the presence cells having a
dedifferentiated, multipotent plastic phenotype. The sample may be
a bodily fluid. Bodily fluids include, but are not limited to,
whole blood, blood plasma, blood serum, urine, semen, saliva, lymph
fluid, meningal fluid, amniotic fluid, glandular fluid, sputum and
cerebrospinal fluid. Bodily fluid also includes experimentally
separated fractions of all of the preceding and solutions or
mixtures containing homogenized solid material, such tissues and
biopsy samples. These methods may be used as a prognostic or
diagnostic assay for aggressive cancer or susceptibility to
aggressive cancer, including, but not limited to, melanoma and
breast cancer.
[0056] In other embodiments, the invention provides methods of
inhibiting tumor cell growth in a mammal comprising administering
to the mammal, having at least one tumor cell present in its body,
an effective amount of an inhibitor of Nodal activity.
[0057] The invention also provides methods of treating aggressive
tumors in a mammal comprising administering to the mammal, having
at least one tumor cell present in its body, an effective amount of
an inhibitor of Nodal activity. As used herein, the phrase
"treating aggressive tumors" refers to a method comprising
administering a Nodal inhibitor to a mammal in need thereof,
wherein the Nodal inhibitor prevents aggressive tumor cell growth,
and/or prevents aggressive tumor cell metastasis in the mammal.
[0058] In one embodiment, the invention provides methods of
inhibiting tumor cell growth and/or treating aggressive tumors
comprising contacting the tumor cell with a microenvironment that
comprises human embryonic stem cells or a microenvironment that has
been preconditioned by human embryonic stem cells ("CMTX"). In
certain embodiments, the basement membrane matrix can be
Matrigel.TM.. There is variability between lots of Matrigel
basement membrane matrix, which can impact the preparation of the
preconditioned media. More specifically, occasional lots of
Matrigel will not produce a preconditioned microenvironment that
has the tumor inhibiting properties of the invention. In such
situations, an alternate lot can be used. One of skill in the art
will understand that other matrices may be used.
[0059] As used herein, an "inhibitor" can be any chemical compound,
nucleic acid molecule, endogenous protein such as Lefty A/B,
peptide or polypeptide such as an antibody against Nodal that can
reduce Nodal activity or interfere with expression of a Nodal gene.
Included within the scope of the term "inhibitor" is any
combination of two or more such inhibitors administered
concurrently or separately and in any order. A Nodal inhibitor can
inhibit the activity of a Nodal protein either directly or
indirectly. Direct inhibition can be accomplished, for example, by
binding to a Nodal protein and thereby preventing the Nodal protein
from binding an intended target, such as a receptor. Indirect
inhibition can be accomplished, for example, by binding to a Nodal
protein's intended target, such as a receptor or binding partner,
thereby blocking or reducing activity of the Nodal protein.
Furthermore, a Nodal inhibitor can inhibit a Nodal gene by reducing
or inhibiting expression of the gene, inter alia by interfering
with gene expression (transcription, processing, translation,
post-translational modification), for example, by interfering with
the Nodal mRNA and blocking translation of the Nodal gene product
or by post-translational modification of the Nodal gene product, or
by causing changes in intracellular localization.
[0060] A Nodal inhibitor can also be an endogenously produced
protein, including but not restricted to, Lefty A/B derived from
the microenvironment of human embryonic stem cells. For example,
Lefty A/B is produced in human embryonic stem cells and is secreted
into the microenvironment surrounding the cells. Lefty A/B can be
isolated from the microenvironment. Alternatively, Lefty A/B can be
isolated from the human embryonic stem cells directly (i.e. before
it is secreted into the microenvironment). In another embodiment, a
Nodal inhibitor within the scope of the invention is recombinant
Lefty A/B (rLefty) that may be prepared by any conventional methods
known in the art. Lefty A/B may be glycosylated or
non-glycosylated. In certain embodiments, Nodal inhibitors in
accordance with the invention are glycosylated Lefty A/B produced
by hESCs. In other embodiments, glycosylated Lefty A/B may be
prepared by using CHO (Chinese Hamster Ovary) cells. In some
instances, glycosylated Lefty A/B may be a more potent inhibitor of
Nodal than its non-glycosylated or recombinant counterpart, and may
therefore be administered in therapeutic applications at a lower
dose.
[0061] In other embodiments, Nodal inhibitors are molecules which
interfere with Nodal signaling, such as activin-like kinase (ALK)
inhibitors. For example, Nodal propagates its signal by binding to
heterodimeric complexes between type I (ALK 4/5/7) and type II
(ActRIIB) activin-like kinase receptors. Assembly of the complex
causes phosphorylation and activation of ALK 4/5/7 by ActRIIB,
which is followed by ALK 4/5/7 mediated phosphyorylation of
Smad-2/3. Inhibitors of ALK 4, ALK 5, and/or ALK7 are included
within the scope of the invention; as described herein, ALK 4/5/7
inhibitors can abrogate Nodal expression. In one embodiment, the
ALK inhibitor is SB431542 (Sigma, St. Louis, Mo.).
[0062] In one embodiment, an inhibitor can be, for example, a small
molecule inhibitor, an antibody, a nucleic acid such as an
antisense oligonucleotide, a short interfering RNA (siRNA)
molecule, or a short hairpin RNA (shRNA) molecule. In addition,
such inhibitors can be specifically designed using the methods
described herein or using methods known in the art.
[0063] In certain embodiments, an antisense oligonucleotide is
complementary to at least a portion of a Nodal gene, so long as
hybridization of the antisense oligonucleotide inhibits Nodal
activity. The term "oligonucleotide" as used herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and/or non-naturally occurring oligonucleotide
linkages. Oligonucleotides are a polynucleotide subset generally
comprising no more than 200 nucleotides. In certain embodiments,
oligonucleotides are 10 to 60 nucleotides in length. In certain
embodiments, oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, or 30 to 40 bases in length.
Oligonucleotides are single stranded, e.g. for use in the
construction of a gene mutant using site directed mutagenesis
techniques.
[0064] The oligonucleotides of the invention may also comprise
nucleotide analogs that may be better suited as therapeutic or
experimental reagents. An example of an oligonucleotide analogue is
a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose)
phosphate backbone in the DNA (or RNA), is replaced with a
polyamide backbone which is similar to that found in peptides (P.
E. Nielsen, et al Science, 1991, 254, 1497). PNA analogues have
been shown to be resistant to degradation by enzymes and to have
extended lives in vivo and in vitro. PNAs also bind stronger to a
complimentary DNA sequence due to the lack of charge repulsion
between the PNA strand and the DNA strand. Other oligonucleotides
may contain nucleotides containing polymer backbones, cyclic
backbones, or acyclic backbones. For example, the nucleotides may
have morpholino backbone structures (U.S. Pat. No. 5,034,506).
Oligonucleotides may also contain groups such as reporter groups, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide. Oligonucleotides may also have
sugar mimetics.
[0065] The antisense nucleic acid molecules may be constructed
using chemical synthesis and enzymatic ligation reactions using
procedures known in the art. The antisense nucleic acid molecules
of the invention or a fragment thereof, may be chemically
synthesized using naturally occurring nucleotides or variously
modified nucleotides designed to increase the biological stability
of the molecules or to increase the physical stability of the
duplex formed with mRNA or the native gene e.g. phosphorothioate
derivatives and acridine substituted nucleotides. The antisense
sequences may be produced biologically using an expression vector
introduced into cells in the form of a recombinant plasmid,
phagemid or attenuated virus in which antisense sequences are
produced under the control of a high efficiency regulatory region,
the activity of which may be determined by the cell type into which
the vector is introduced.
[0066] In one embodiment, the Nodal inhibitors of the invention are
anti-Nodal Morpholinos.
[0067] In one embodiment, certain inhibitors provided by the
invention are species of short interfering RNA (siRNA). The term
"short interfering RNA" or "siRNA" as used herein refers to a
double stranded nucleic acid molecule capable of RNA interference
or "RNAi", as disclosed, for example, in Bass, 2001, Nature 411:
428-429; Elbashir et al., 2001, Nature 411: 494-498; and Kreutzer
et al., International PCT Publication No. WO 00/44895;
Zernicka-Goetz et al., International PCT Publication No. WO
01/36646; Fire, International PCT Publication No. WO 99/32619;
Plaetinck et al., International PCT Publication No. WO 00/01846;
Mello and Fire, International PCT Publication No. WO 01/29058;
Deschamps-Depaillette, International PCT Publication No. WO
99/07409; and Li et al., International PCT Publication No. WO
00/44914. As used herein, siRNA molecules need not be limited to
those molecules containing only RNA, but further encompasses
chemically modified nucleotides and non-nucleotides having RNAi
capacity or activity. Specific siRNA molecules that inhibit Nodal
activity can be designed using methods known to those of skill in
the art or commercially available technology (such as technology
provided by Dharmacon Research, Lafayette, Colo.).
[0068] In another embodiment, the Nodal inhibitors of the invention
include any chemical compounds, nucleic acids, proteins, peptides,
polypeptides, antibodies, or other molecules that inhibit Notch. In
certain embodiments, the Nodal inhibitors are Notch4 inhibitors. In
certain embodiments the Nodal inhibitors are Notch siRNAs. In
certain embodiments, the Nodal inhibitors are Notch4 siRNAs.
[0069] In certain embodiments, the invention provides antibodies or
immunologically functional fragments thereof that selectively bind
to Nodal and methods for selectively inhibiting or interfering with
the activity of Nodal proteins. Standard methods for preparation of
monoclonal and polyclonal antibodies and immunologically active
fragments thereof are well known in the art, for example as
described in Harlow and Lane (1988, ANTIBODIES: A LABORATORY
MANUAL, Cold Spring Harbor Laboratory Press: New York). Methods for
generating antibody fragments, particularly Fab fragments and other
fragments that retain epitope-binding ability and specificity are
also well known, as are fully human antibodies and chimeric
antibodies, including "humanized" antibodies. "Humanized"
antibodies include, for example, antibodies generated in mice that
are "humanized" to reduce negative immune effects that can occur
during administration to human subjects by replacing certain
portions of the mouse antibody with portions of human antibodies.
Thus, the invention encompasses use of antibody inhibitors of Nodal
that include, but are not limited to, single chain antibodies,
single chain Fv antibodies, F(ab) antibodies, F(ab)' antibodies and
(Fab').sub.2 antibodies, chimeric antibodies in which one or more
regions have been replaced by homologous human or non-human
portions, and fully human antibodies. Single chain antibodies are
discussed in detail in International Patent Application Publication
No. WO 88/01649 and U.S. Pat. Nos. 4,946,778 and 5,260,203. Such
inhibitors can be delivered, for example, via a penetratin tag (HIV
or antennaepedia) or by recombinant means (e.g. encoded by a
polynucleotide introduced into a cell in a viral vector).
[0070] In preferred embodiments, methods of the invention comprise
the step of administering a pharmaceutical composition comprising
an effective amount of one or a plurality of Nodal inhibitors
together with a pharmaceutically acceptable diluent, carrier,
solubilizer, emulsifier, preservative and/or adjuvant, wherein the
pharmaceutical composition is capable of inducing a desired
therapeutic effect when properly administered to a patient.
Preferably, acceptable formulation materials are nontoxic to
recipients at the dosages and concentrations employed.
[0071] The expression "effective amount" in reference to a
pharmaceutical composition comprising one or a plurality of Nodal
inhibitors is understood to mean, according to the invention, an
amount of the said pharmaceutical composition that is capable of
preventing or reducing growth of aggressive melanoma cells. For
example, a pharmaceutical composition is therapeutically effective
where a patient who has aggressive melanoma has a reduced number of
melanoma cells and/or reduced metastases of melanoma cells after
treatment with the pharmaceutical composition compared with prior
to said treatment. A pharmaceutical composition administered to a
patient is also therapeutically effective where metastases of
melanoma cells are prevented from occurring in a patient who has
melanoma, has a history of melanoma (e.g. patient is in remission),
or who is considered likely to present with melanoma (e.g. has a
genetic disposition favoring onset of melanoma).
[0072] In certain embodiments, a pharmaceutical composition useful
in the methods of the invention may contain formulation materials
for modifying, maintaining or preserving, for example, the pH,
osmolarity, viscosity, clarity, color, isotonicity, odor,
sterility, stability, rate of dissolution or release, adsorption or
penetration of the composition. In such embodiments, suitable
formulation materials include, but are not limited to, amino acids
(such as glycine, glutamine, asparagine, arginine or lysine);
antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite
or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate,
Tris-HCl, citrates, phosphates or other organic acids); bulking
agents (such as mannitol or glycine); chelating agents (such as
ethylenediamine tetraacetic acid (EDTA)); complexing agents (such
as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or
hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;
disaccharides; and other carbohydrates (such as glucose, mannose or
dextrins); proteins (such as serum albumin, gelatin or
immunoglobulins); coloring, flavoring and diluting agents;
emulsifying agents; hydrophilic polymers (such as
polyvinylpyrrolidone); low molecular weight polypeptides;
salt-forming counterions (such as sodium); preservatives (such as
benzalkonium chloride, benzoic acid, salicylic acid, thimerosal,
phenethyl alcohol, methylparaben, propylparaben, chlorhexidine,
sorbic acid or hydrogen peroxide); solvents (such as glycerin,
propylene glycol or polyethylene glycol); sugar alcohols (such as
mannitol or sorbitol); suspending agents; surfactants or wetting
agents (such as pluronics, PEG, sorbitan esters, polysorbates such
as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin,
cholesterol, tyloxapal); stability enhancing agents (such as
sucrose or sorbitol); tonicity enhancing agents (such as alkali
metal halides, preferably sodium or potassium chloride, mannitol
sorbitol); delivery vehicles; diluents; excipients and/or
pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES,
18.sup.th Edition, (A. R. Gennaro, ed.), 1990, Mack Publishing
Company.
[0073] In certain embodiments, the optimal pharmaceutical
composition will be determined by one skilled in the art depending
upon, for example, the intended route of administration, delivery
format and desired dosage. See, for example, REMINGTON'S
PHARMACEUTICAL SCIENCES, supra. In certain embodiments, such
compositions may influence the physical state, stability, rate of
in vivo release and rate of in vivo clearance of the Nodal
inhibitors.
[0074] In certain embodiments, the primary vehicle or carrier in a
pharmaceutical composition may be either aqueous or non-aqueous in
nature. For example, a suitable vehicle or carrier may be water for
injection, physiological saline solution or artificial
cerebrospinal fluid, possibly supplemented with other materials
common in compositions for parenteral administration. Neutral
buffered saline or saline mixed with serum albumin are further
exemplary vehicles. In preferred embodiments, pharmaceutical
compositions of the present invention comprise Tris buffer of about
pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, and may further
include sorbitol, sucrose, Tween-20 and/or a suitable substitute
therefor. In certain embodiments of the invention, Nodal inhibitor
compositions may be prepared for storage by mixing the selected
composition having the desired degree of purity with optional
formulation agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in
the form of a lyophilized cake or an aqueous solution. Further, in
certain embodiments, the Nodal inhibitor product may be formulated
as a lyophilizate using appropriate excipients such as sucrose.
[0075] The pharmaceutical compositions of the invention can be
selected for parenteral delivery. Alternatively, the compositions
may be selected for inhalation or for delivery through the
digestive tract, such as orally. Preparation of such
pharmaceutically acceptable compositions is within the skill of the
art.
[0076] The formulation components are present preferably in
concentrations that are acceptable to the site of administration.
In certain embodiments, buffers are used to maintain the
composition at physiological pH or at a slightly lower pH,
typically within a pH range of from about 5 to about 8.
[0077] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be provided
in the form of a pyrogen-free, parenterally acceptable aqueous
solution comprising the desired Nodal inhibitor in a
pharmaceutically acceptable vehicle. A particularly suitable
vehicle for parenteral injection is sterile distilled water in
which the Nodal inhibitor is formulated as a sterile, isotonic
solution, properly preserved. In certain embodiments, the
preparation can involve the formulation of the desired molecule
with an agent, such as injectable microspheres, bio-erodible
particles, polymeric compounds (such as polylactic acid or
polyglycolic acid), beads or liposomes, that may provide controlled
or sustained release of the product which can be delivered via
depot injection. In certain embodiments, hyaluronic acid may also
be used to promote sustained duration in the circulation. In
certain embodiments, implantable drug delivery devices may be used
to introduce the desired Nodal inhibitor.
[0078] Pharmaceutical compositions of the invention can be
formulated for inhalation. In these embodiments, Nodal inhibitors
are advantageously formulated as a dry, inhalable powder. In
preferred embodiments, Nodal inhibitor inhalation solutions may
also be formulated with a propellant for aerosol delivery. In
certain embodiments, solutions may be nebulized. Pulmonary
administration and formulation methods therefore are further
described in International Patent Application No. PCT/US94/001875,
which is incorporated by reference and describes pulmonary delivery
of chemically modified proteins.
[0079] It is also contemplated that formulations can be
administered orally. Nodal inhibitors that are administered in this
fashion can be formulated with or without carriers customarily used
in the compounding of solid dosage forms such as tablets and
capsules. In certain embodiments, a capsule may be designed to
release the active portion of the formulation at the point in the
gastrointestinal tract when bioavailability is maximized and
pre-systemic degradation is minimized. Additional agents can be
included to facilitate absorption of the Nodal inhibitor. Diluents,
flavorings, low melting point waxes, vegetable oils, lubricants,
suspending agents, tablet disintegrating agents, and binders may
also be employed.
[0080] A pharmaceutical composition of the invention is preferably
provided to comprise an effective quantity of one or a plurality of
Nodal inhibitors in a mixture with non-toxic excipients that are
suitable for the manufacture of tablets. By dissolving the tablets
in sterile water, or another appropriate vehicle, solutions may be
prepared in unit-dose form. Suitable excipients include, but are
not limited to, inert diluents, such as calcium carbonate, sodium
carbonate or bicarbonate, lactose, or calcium phosphate; or binding
agents, such as starch, gelatin, or acacia; or lubricating agents
such as magnesium stearate, stearic acid, or talc.
[0081] Additional pharmaceutical compositions will be evident to
those skilled in the art, including formulations involving Nodal
inhibitors in sustained- or controlled-delivery formulations.
Techniques for formulating a variety of other sustained- or
controlled-delivery means, such as liposome carriers, bio-erodible
microparticles or porous beads and depot injections, are also known
to those skilled in the art. See, for example, International Patent
Application No. PCT/US93/00829, which is incorporated by reference
and describes controlled release of porous polymeric microparticles
for delivery of pharmaceutical compositions. Sustained-release
preparations may include semipermeable polymer matrices in the form
of shaped articles, e.g. films, or microcapsules. Sustained release
matrices may include polyesters, hydrogels, polylactides (as
disclosed in U.S. Pat. No. 3,773,919 and European Patent
Application Publication No. EP 058481, each of which is
incorporated by reference), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 22:547-556),
poly (2-hydroxyethyl-methacrylate) (Langer et al., 1981, J. Biomed.
Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12:98-105),
ethylene vinyl acetate (Langer et al., supra) or
poly-D(-)-3-hydroxybutyric acid (European Patent Application
Publication No. EP 133,988). Sustained release compositions may
also include liposomes that can be prepared by any of several
methods known in the art. See e.g., Eppstein et al., 1985, Proc.
Natl. Acad. Sci. USA 82:3688-3692; European Patent Application
Publication Nos. EP 036,676; EP 088,046 and EP 143,949,
incorporated by reference.
[0082] Pharmaceutical compositions used for in vivo administration
are typically provided as sterile preparations. Sterilization can
be accomplished by filtration through sterile filtration membranes.
When the composition is lyophilized, sterilization using this
method may be conducted either prior to or following lyophilization
and reconstitution. Compositions for parenteral administration can
be stored in lyophilized form or in a solution. Parenteral
compositions generally are placed into a container having a sterile
access port, for example, an intravenous solution bag or vial
having a stopper pierceable by a hypodermic injection needle.
[0083] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or as a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) that is reconstituted prior to
administration.
[0084] Nodal inhibitors useful in the methods of the invention can
be admixed, encapsulated, conjugated or otherwise associated with
other molecules, molecule structures or mixtures of compounds, for
example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption in a patient, using methods that are
well known in the pharmaceutical arts.
[0085] The Nodal inhibitors may be administered orally, topically,
parenterally, by inhalation or spray or rectally in dosage unit
formulations containing conventional non-toxic pharmaceutically
acceptable carriers, adjuvants and vehicles. The term parenteral as
used herein includes percutaneous, subcutaneous, intravascular
(e.g., intravenous), intramuscular, or intrathecal injection or
infusion techniques and the like.
[0086] Compositions intended for oral use may be prepared according
to any method known to the art for the manufacture of
pharmaceutical compositions and such compositions may contain one
or more agents selected from the group consisting of sweetening
agents, flavoring agents, coloring agents and preservative agents
in order to provide pharmaceutically elegant and palatable
preparations. Tablets contain the active ingredient in admixture
with non-toxic pharmaceutically acceptable excipients that are
suitable for the manufacture of tablets. These excipients may be
for example, inert diluents, such as calcium carbonate, sodium
carbonate, lactose, calcium phosphate or sodium phosphate;
granulating and disintegrating agents, for example, corn starch, or
alginic acid; binding agents, for example starch, gelatin or
acacia, and lubricating agents, for example magnesium stearate,
stearic acid or talc. The tablets may be uncoated or they may be
coated by known techniques. In some cases such coatings may be
prepared by known techniques to delay disintegration and absorption
in the gastrointestinal tract and thereby provide a sustained
action over a longer period. For example, a time delay material
such as glyceryl monosterate or glyceryl distearate may be
employed.
[0087] Formulations for oral use may also be presented as hard
gelatin capsules, wherein the active ingredient is mixed with an
inert solid diluent, for example, calcium carbonate, calcium
phosphate or kaolin, or as soft gelatin capsules wherein the active
ingredient is mixed with water or an oil medium, for example peanut
oil, liquid paraffin or olive oil.
[0088] Formulations for oral use may also be presented as
lozenges.
[0089] Aqueous suspensions contain the active materials in
admixture with excipients suitable for the manufacture of aqueous
suspensions. Such excipients are suspending agents, for example
sodium carboxymethylcellulose, methylcellulose,
hydropropyl-methylcellulose, sodium alginate, polyvinylpyrrolidone,
gum tragacanth and gum acacia; dispersing or wetting agents may be
a naturally-occurring phosphatide, for example, lecithin, or
condensation products of an alkylene oxide with fatty acids, for
example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethyleneoxycetanol, or condensation products of ethylene
oxide with partial esters derived from fatty acids and a hexitol
such as polyoxyethylene sorbitol monooleate, or condensation
products of ethylene oxide with partial esters derived from fatty
acids and hexitol anhydrides, for example polyethylene sorbitan
monooleate. The aqueous suspensions may also contain one or more
preservatives, for example ethyl, or n-propyl p-hydroxybenzoate,
one or more coloring agents, one or more flavoring agents, and one
or more sweetening agents, such as sucrose or saccharin.
[0090] Oily suspensions may be formulated by suspending the active
ingredients in a vegetable oil, for example arachis oil, olive oil,
sesame oil or coconut oil, or in a mineral oil such as liquid
paraffin. The oily suspensions may contain a thickening agent, for
example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
and flavoring agents may be added to provide palatable oral
preparations. These compositions may be preserved by the addition
of an anti-oxidant such as ascorbic acid.
[0091] Dispersible powders and granules suitable for preparation of
an aqueous suspension by the addition of water provide the active
ingredient in admixture with a dispersing or wetting agent,
suspending agent and one or more preservatives. Suitable dispersing
or wetting agents or suspending agents are exemplified by those
already mentioned above. Additional excipients, for example
sweetening, flavoring and coloring agents, may also be present.
[0092] Pharmaceutical compositions of the invention may also be in
the form of oil-in-water emulsions. The oily phase may be a
vegetable oil or a mineral oil or mixtures of these. Suitable
emulsifying agents may be naturally-occurring gums, for example gum
acacia or gum tragacanth, naturally-occurring phosphatides, for
example soy bean, lecithin, and esters or partial esters derived
from fatty acids and hexitol, anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters
with ethylene oxide, for example polyoxyethylene sorbitan
monooleate. The emulsions may also contain sweetening and flavoring
agents.
[0093] Syrups and elixirs may be formulated with sweetening agents,
for example glycerol, propylene glycol, sorbitol, glucose or
sucrose. Such formulations may also contain a demulcent, a
preservative, flavoring and/or coloring agents. The pharmaceutical
compositions may be in the form of a sterile injectable aqueous or
oleaginous suspension. This suspension may be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents that have been mentioned above. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a non-toxic parentally acceptable diluent or solvent,
for example as a solution in 1,3-butanediol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or diglycerides. In addition,
fatty acids such as oleic acid find use in the preparation of
injectables.
[0094] The Nodal inhibitors may also be administered in the form of
suppositories, e.g., for rectal administration of the drug. These
compositions can be prepared by mixing the drug with a suitable
non-irritating excipient that is solid at ordinary temperatures but
liquid at the rectal temperature and will therefore melt in the
rectum to release the drug. Such materials include cocoa butter and
polyethylene glycols.
[0095] Nodal inhibitors may be administered parenterally in a
sterile medium. The drug, depending on the vehicle and
concentration used, can either be suspended or dissolved in the
vehicle. Advantageously, adjuvants such as local anesthetics,
preservatives and buffering agents can be dissolved in the
vehicle.
[0096] The formulations can also be preferably applied as a topical
gel, spray, ointment or cream, or as a suppository, containing the
active ingredients in a total amount of, for example, 0.075 to 30%
w/w, preferably 0.2 to 20% w/w and most preferably 0.4 to 15% w/w.
When formulated in an ointment, the active ingredients may be
employed with either paraffinic or a water-miscible ointment
base.
[0097] Alternatively, the active ingredients may be formulated in a
cream with an oil-in-water cream base. If desired, the aqueous
phase of the cream base may include, for example at least 30% w/w
of a polyhydric alcohol such as propylene glycol, butane-1,3-diol,
mannitol, sorbitol, glycerol, polyethylene glycol and mixtures
thereof. The topical formulation may desirably include a compound
which enhances absorption or penetration of the active ingredient
through the skin or other affected areas. Examples of such dermal
penetration enhancers include dimethylsulfoxide and related
analogs.
[0098] The compounds of this invention can also be administered by
a transdermal device. Preferably topical administration will be
accomplished using a patch either of the reservoir and porous
membrane type or of a solid matrix variety. In either case, the
active agent is delivered continuously from the reservoir or
microcapsules through a membrane into the active agent permeable
adhesive, which is in contact with the skin or mucosa of the
recipient. If the active agent is absorbed through the skin, a
controlled and predetermined flow of the active agent is
administered to the recipient. In the case of microcapsules, the
encapsulating agent may also function as the membrane. The
transdermal patch may include the compound in a suitable solvent
system with an adhesive system, such as an acrylic emulsion, and a
polyester patch.
[0099] The oily phase of the emulsions of this invention may be
constituted from known ingredients in a known manner. While the
phase may comprise merely an emulsifier, it may comprise a mixture
of at least one emulsifier with a fat or an oil or with both a fat
and an oil. Preferably, a hydrophilic emulsifier is included
together with a lipophilic emulsifier which acts as a stabilizer.
It is also preferred to include both an oil and a fat. Together,
the emulsifier(s) with or without stabilizer(s) make-up the
so-called emulsifying wax, and the wax together with the oil and
fat make up the so-called emulsifying ointment base which forms the
oily dispersed phase of the cream formulations. Emulsifiers and
emulsion stabilizers suitable for use in the formulation of the
present invention include Tween 60, Span 80, cetostearyl alcohol,
myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate,
among others.
[0100] The choice of suitable oils or fats for the formulation is
based on achieving the desired cosmetic properties, since the
solubility of the active compound in most oils likely to be used in
pharmaceutical emulsion formulations is very low. Thus, the cream
should preferably be a non-greasy, non-staining and washable
product with suitable consistency to avoid leakage from tubes or
other containers. Straight or branched chain, mono- or dibasic
alkyl esters such as di-isoadipate, isocetyl stearate, propylene
glycol diester of coconut fatty acids, isopropyl myristate, decyl
oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate
or a blend of branched chain esters may be used. These may be used
alone or in combination depending on the properties required.
Alternatively, high melting point lipids such as white soft
paraffin and/or liquid paraffin or other mineral oils can be
used.
[0101] For therapeutic purposes, the Nodal inhibitors of this
invention are ordinarily combined with one or more adjuvants
appropriate to the indicated route of administration. If
administered by mouth, the compounds may be admixed with lactose,
sucrose, starch powder, cellulose esters of alkanoic acids,
cellulose alkyl esters, talc, stearic acid, magnesium stearate,
magnesium oxide, sodium and calcium salts of phosphoric and
sulfuric acids, gelatin, acacia gum, sodium alginate,
polyvinylpyrrolidone, and/or polyvinyl alcohol, and then tableted
or encapsulated for convenient administration. Such capsules or
tablets may contain a controlled-release formulation as may be
provided in a dispersion of active compound in hydroxypropylmethyl
cellulose. Formulations for parenteral administration may be in the
form of aqueous or non-aqueous isotonic sterile injection solutions
or suspensions. These solutions and suspensions may be prepared
from sterile powders or granules having one or more of the carriers
or diluents mentioned for use in the formulations for oral
administration. The compounds may be dissolved in water,
polyethylene glycol, propylene glycol, ethanol, corn oil,
cottonseed oil, peanut oil, sesame oil, benzyl alcohol, sodium
chloride, and/or various buffers. Other adjuvants and modes of
administration are well and widely known in the pharmaceutical
art.
[0102] Dosage levels of the order of from about 0.1 mg to about 140
mg per kilogram of body weight per day are useful in the treatment
of the above-indicated conditions (about 0.5 mg to about 14 g per
patient per day). The amount of active ingredient that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. Dosage unit forms will generally contain between
from about 1 mg to about 500 mg of an active ingredient. The daily
dose can be administered in one to four doses per day. In the case
of skin conditions, it may be preferable to apply a topical
preparation of compounds of this invention to the affected area two
to four times a day.
[0103] It will be understood, however, that the specific dose level
for any particular patient will depend upon a variety of factors
including the activity of the specific compound employed, the age,
body weight, general health, sex, diet, time of administration,
route of administration, and rate of excretion, drug combination
and the severity of the particular disease undergoing therapy.
[0104] For administration to non-human animals, the composition may
also be added to the animal feed or drinking water. It may be
convenient to formulate the animal feed and drinking water
compositions so that the animal takes in a therapeutically
appropriate quantity of the composition along with its diet. It may
also be convenient to present the composition as a premix for
addition to the feed or drinking water.
[0105] Dosing frequency will depend upon the pharmacokinetic
parameters of the particular Nodal inhibitor used in the
formulation. Typically, a clinician administers the composition
until a dosage is reached that achieves the desired effect. The
composition may therefore be administered as a single dose, or as
two or more doses (which may or may not contain the same amount of
the desired molecule) over time, or as a continuous infusion via an
implantation device or catheter. Further refinement of the
appropriate dosage is routinely made by those of ordinary skill in
the art and is within the ambit of tasks routinely performed by
them. Appropriate dosages may be ascertained through use of
appropriate dose-response data. In certain embodiments, Nodal
inhibitors can be administered to patients throughout an extended
time period.
[0106] Pharmaceutical compositions and/or Nodal inhibitors can be
administered alone or in combination with other therapeutic agents,
in particular, in combination with other chemotherapeutic
agents.
[0107] In addition, the invention provides methods for monitoring
the effectiveness of a pharmaceutical composition as an agent for
treating aggressive melanoma in a patient comprising the steps of:
(a) obtaining a sample of skin cells from a patient; (b) assaying
the skin cells for the presence of Nodal; (c) administering an
amount a pharmaceutical composition to the patient; (d) repeating
step (a) using a subsequently-collected biological sample obtained
from the patient; and (e) comparing the amount of Nodal detected in
the skin cells from step (a) with the amount of Nodal detected in
the skin cells from step (c), wherein the effectiveness of the
pharmaceutical composition is monitored by detecting changes in the
amount of Nodal in the subsequently-collected skin cells compared
with the skin cells taken in step (a).
[0108] Unless otherwise required by context, singular terms used
herein shall include pluralities and plural terms shall include the
singular.
EXAMPLES
[0109] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting the invention.
Example 1
3-D Matrices Preconditioned by Human Embryonic Stem Cells Promote
Epigenetic Changes in Aggressive Tumor Cells
[0110] As illustrated in FIG. 1, H1 or HSF-6 human embryonic stem
cells (hESCs), 5.times.10.sup.4 cells in compact colonies, were
seeded onto a 3-D matrix comprised of growth factor-reduced
Matrigel (BD Biosciences) in the presence of conditioned stem cell
medium for 3 to 4 days. Subsequently, the hESCs were removed from
their 3-D matrix with NH.sub.4OH followed by thorough washes with
double-distilled H.sub.2O, PBS, and complete medium, leaving a
denuded, preconditioned, 3-D matrix (CMTX Matrigel). Onto this
preconditioned matrix were seeded human amelanotic metastatic
cutaneous melanoma cells (C8161), 2.5.times.10.sup.5 cells/6-well
dish, for 3 to 4 days. At the end of this incubation period,
analyses of potential changes in morphology, gene and protein
expression, and behavioral function(s) were performed on the
melanoma cells exposed to the hESC preconditioned matrix
microenvironment. Preconditioning of an extracellular matrix
exerted a dramatic effect on melanoma cell morphology (shown in
FIG. 2). On a control unconditioned Matrigel matrix, C8161 melanoma
cells (FIG. 2A) grew into overconfluent monolayers, whereas
undifferentiated H1 (FIG. 2B) and HSF-6 (FIG. 2C) human embryonic
stem cells (hESCs) formed compact colonies of cells with a high
nucleus-to-cytoplasm ratio. However, C8161 melanoma cells seeded
onto the 3-D matrices preconditioned by the human embryonic stem
cells acquired an altered phenotype manifested by the formation of
spheroids similar to the colonies formed by human embryonic stem
cells (FIG. 2D-F). In contrast, the conditioned media from human
embryonic stem cells did not exert an epigenetic change on the
C8161 cells (FIG. 2G), suggesting that hESCs influence melanoma
cell phenotype through the alteration of the immediate
microenvironment.
[0111] To further analyze epigenetic changes in the phenotype of
C8161 cells exposed to the human embryonic stem cell
microenvironment, Western blot and RT-PCR analyses of a melanocyte
marker, Melan-A were performed (FIGS. 3A and 3B). Melan-A was
absent both in H1 human embryonic stem cells (indicating their lack
of a differentiated pigment cell phenotype) and in C8161 melanoma
cells (illustrating their dedifferentiated phenotype) on Matrigel.
However, Melan-A expression was induced in amelanotic C8161
melanoma cells exposed to the H1 preconditioned Matrigel matrix
(FIG. 3A), demonstrating the epigenetic induction of a
melanocyte-specific phenotype marker, similar to control
melanocytes (HEMn) on Matrigel. By contrast, C8161 melanoma cells
exposed to 3-D matrices preconditioned by normal HEMn were not
induced to change their morphology or to express Melan-A (FIG. 3B).
Thus, the normal melanocyte microenvironment does not share the
ability of the hESC microenvironment to epigenetically reprogram
metastatic melanoma cells to express a melanocyte-like phenotype.
This was confirmed by the results of FIG. 3C, showing
semi-quantitative RT-PCR analysis which demonstrates that a
collagen I 3-D matrix preconditioned by human melanocytes (HEMn18
or HEMn20 CMTX) does not change the expression of the genes tested,
indicating that the benign melanocyte microenvironment does not
epigenetically influence metastatic melanoma cells to change their
plastic, molecular phenotype.
Example 2
[0112] Aggressive Tumor Cells are Less Invasive and Tumorigenic
Following Culture on hESC Microenvironments
[0113] The aggressiveness of tumor cells is correlated with their
ability to invade through the extracellular matrix; thus, the
effect of hESC microenvironments on melanoma cell invasion was
investigated. As illustrated in FIG. 4A, the in vitro invasiveness
of aggressive C8161 cells was significantly inhibited following
culture on matrices preconditioned by hESCs, suggesting
suppressive, anti-invasion cues associated with this human
embryonic microenvironment.
[0114] Comparable results were found in vivo tumor formation. A
microenvironment of human embryonic stem cells (H9 CMTX) was
prepared as described above. C8161 human cutaneous melanoma cells
were exposed to the H9 CMTX or Matrigel for 3 to 4 days prior to
transplantation in a mouse model. Nude immunocompromised mice
received an injection of the C8161 cells subcutaneously into the
midscapular region (to mimic spontaneous metastatic dissemination
found in human cancers). The animals were injected using a 25 or
27-gauge needle with 2.5.times.10.sup.5 tumor cells/mouse in 0.05
ml RPMI media.
[0115] Tumor size was monitored on alternate days and was measured
using a microcaliper. At the time of necropsy (19 days after
injection), the mice were euthanized using CO.sub.2 compressed gas
asphyxiation followed by cervical dislocation and the tumor and
major organs were removed and prepared for histology. The sections
were stained with anti-Nodal antibodies (R&D Systems) to
determine Nodal expression in the tumors (see Example 6). As shown
in FIGS. 4A and 4B, melanoma cells were less invasive and
tumorigenic in vivo following culture on hESC
microenvironments.
Example 3
Characterization of Nodal Signaling Pathway Members in Human Normal
Cells, Metastatic Cancer Cells, Human Embryonic Stem Cells and
Other Stem Cell Types: Aggressive Tumors Express Nodal but Not
Lefty
[0116] In order to elucidate the expression of key components of
the Nodal signaling pathway in normal, neoplastic and stem cell
types, Western blot analyses were conducted which revealed that in
a manner similar to hESCs, metastatic melanoma (C8161) and breast
carcinoma (MDA-MB-231) cells express Nodal protein at approximately
48 kDA (FIG. 5A). This is in contrast to corresponding normal cell
types [melanocytes, myoepethial cells (Hs 578 Bst) and primary
human mammary epithelial cells (HMEpC)], in which Nodal was not
detected.
[0117] The Lefty proteins (Lefty-A, Lefty-B), divergent members of
the TGF-.beta. superfamily, spatially and temporally antagonize
Nodal in embryological systems (Tabibzadeh et al., 2006, Stem Cells
24:1998-2006). Moreover, the Lefty genes are downstream targets of
Nodal signaling, thereby providing a powerful negative-feedback
loop for this pathway. Id. Using Western blot analysis it was
determined that hESCs express Lefty protein at approximately 42, 34
and 28 kDAs. In contrast, Lefty is not expressed by metastatic
breast carcinoma and melanoma cells or by corresponding normal
somatic cell types (FIG. 5A). Real time RT-PCR analysis confirmed
these results as Lefty mRNA expression was exclusive to the hESC
cell lines (FIG. 5B).
[0118] Nodal propagates its signal by binding to heterodimeric
complexes between type I (ALK 4/7) and type II (ActRIIB)
activin-like kinase receptors. Genetic studies in zebrafish and
mice have determined that Cripto, an Epidermal Growth
Factor-Cripto-1/FRL1/cryptic (EGF-CFC) family member, directly
associates with ALK 4 and Nodal and that these associations
facilitate the ability of Nodal to propagate its signal (Schier et
al.; Yeo et. al., 2001, Mol. Cell 7:949-957). Using Western blot
analysis and immunofluorescence microscopy, it was determined that
hESCs uniformly express high levels of Cripto at approximately 35
kDA; however, only a subpopulation of metastatic human melanoma
(C8161) and breast carcinoma (MDA-MB-231) cells express a
relatively low level of Cripto (FIGS. 5A,C).
[0119] In order to analyze the expression of Nodal, Lefty and
Cripto in other human stem cell types and in first trimester human
cytotrophoblast cells (HTR-8/SVneo), Western blot analyses were
conducted which revealed that umbilical cord derived mesenchymal
stem cells (MSC; SC00125) and adult MSCs do not express Nodal and
Cripto, and that although amniotic fluid-derived stem cells
(GM00473, GM00957A) and cytotrophoblast cells express Cripto, only
the latter developmental cell type expresses an appreciable amount
of Nodal (FIG. 5D). Of note, in contrast to hESCs, none of the
other stem cell lines examined expressed an appreciable level of
Lefty protein or mRNA (FIG. 5B).
[0120] In summary, like hESCs, cancer cells express Nodal, while
unlike hESCs, they do not express Lefty. C8161 cells (human
metastatic melanoma cells) and MDA-MB-231 cells (human metastatic
breast carcinoma cells) expressed Nodal and Cripto (at a low
level), and they did not express Lefty. Expression of Nodal, Lefty,
and Cripto was undetectable in normal human melanocytes, Hs 578 Bst
normal human myoepithelial cells, and HMEpC normal human mammary
epithelial cells.
Example 4
[0121] Nodal Expression Correlates with Tumor Progression
[0122] Human melanoma specimens were screened for the presence of
Nodal protein. Formalin-fixed, paraffin-embedded archival tissue
was obtained from patients with primary or metastatic cutaneous
melanoma (Loyola University Chicago, Ill.). Immunohistochemical
staining was performed on a HNS 710i Automated Immunostainer
(Richard-Allan Scientific (RAS), Kalamazoo, Mich.) with the
Multi-Species HRP/AEC Detection Systems. Following
deparaffinization in xylene, ethanol degradation, and antigen
retrieval with citrate buffer, four blocking steps were applied:
0.03% hydrogen peroxide, Avidin and Biotin blocks (Avidin/Biotin
blocking kit, Vector Laboratories, Inc., Burlingame, Calif.), and a
Serum-Free protein block. Anti-Nodal antibody (20 .mu.g/mL,
R&D) was applied for 90 minutes. Slides were rinsed in TBS-T,
incubated with biotinylated anti-goat IgG (2 .mu.g/ml, Vector
Labs), washed with TBS-T and incubated with the streptavidin
peroxidase reagent for 15 minutes. Color was produced with AEC
(red) substrate (RAS) and counterstaining with Mayer's hematoxylin.
Samples were dehydrated in reagent grade alcohol and cover slipped
with permanent mounting medium. Negative control reactions were
conducted with ChromPure Goat IgG (Jackson Labs), isotype matched
and used at the same concentration as the Nodal antibody.
[0123] The immunohistochemistry demonstrated that Nodal is absent
in normal skin (FIG. 6A) and is weakly expressed or absent in
primary melanomas (FIG. 6B). In the primary lesions, Nodal
immunostaining was generally confined to small clusters of tumor
cells in the vertical growth phase and was rarely observed in
radial lesions (n=5; FIGS. 6C and D). In contrast, Nodal protein
was expressed in 60% of the cutaneous melanoma metastases examined
(n=10; FIGS. 6E and F). Immunostaining was heterogeneous, varying
among patients in extent, intensity and localization. For example,
Nodal was found localized to cell membranes, and was expressed
diffusely in the cytoplasm (FIG. 10). Western blot analyses
similarly revealed that 45% of metastatic melanomas tested were
positive for Nodal (n=22). This is in contrast to normal skin
(n=9), and melanocytes (n=5), neither of which expressed Nodal
(data summarized in Table 1). Collectively, these results
demonstrated that Nodal expression was positively correlated with
melanoma progression.
TABLE-US-00001 TABLE 1 Nodal Signal Nodal Staining (IHC) Case Type
(Western) Primary - Normal Skin - Primary - Normal Skin - Primary
-* Normal Skin - Primary -* Normal Skin - Primary -* Normal Skin -
Metastasis -* Normal Skin - Metastasis ++ Normal Skin - Metastasis
+ Normal Skin - Metastasis + Normal Skin - Metastasis - Melanocytes
- Metastasis - Melanocytes - Metastasis - Melanocytes - Metastasis
++ Melanocytes - Metastasis ++ Melanocytes - Metastasis ++
Dendritic Cells - Dendritic Cells - Metastatic Melanoma -
Metastatic Melanoma - Metastatic Melanoma + Metastatic Melanoma +
Metastatic Melanoma - Metastatic Melanoma - Metastatic Melanoma -
Metastatic Melanoma + Metastatic Melanoma - Metastatic Melanoma -
Metastatic Melanoma + Metastatic Melanoma - Metastatic Melanoma +
Metastatic Melanoma - Metastatic Melanoma - Metastatic Melanoma +
Metastatic Melanoma - Metastatic Melanoma + Metastatic Melanoma +
Metastatic Melanoma + Metastatic Melanoma - Metastatic Melanoma +
++ Represents strong positive staining for Nodal encompassing
>75% of the tumor mass + Represents positive staining for Nodal
encompassing >50% of the tumor mass or the detection of Nodal
using Western Blot analyses -* Represents Nodal staining in a small
subpopulation (<10%) of the tumor mass - Represents the absence
of Nodal
[0124] As with the positive correlation of Nodal expression with
melanoma progression, such that Nodal protein is not expressed in
normal melanocytes or radial growth phase melanomas, but is present
in more aggressive vertical growth phase and metastatic lesions,
immunohistochemical analysis of a human breast tissue microarray
(TMA) revealed that Nodal protein is similarly absent in normal
breast tissue, and that its expression is positively correlated
with breast carcinoma progression (FIG. 7).
[0125] The expression and prevalence of Nodal staining in breast
tissue was designated as none, weak (<25%), moderate (25-75%) or
strong (>75%). DCIS is ductal carcinoma in situ and IDC is
invasive ductal carcinoma. Spearman's rank correlation showed a
significant positive correlation between breast cancer progression
and Nodal expression (P<0.05) (data summarized in Table 2).
TABLE-US-00002 TABLE 2 None Weak Moderate Strong Total Benign 26 1
0 0 27 DCIS 3 0 0 0 3 IDC 24 1 3 5 33
Example 5
[0126] Localization of Nodal and Lefty in hESC Matrices
[0127] Immunofluorescence localization with confocal microscopy was
performed in order to visualize the deposition of Lefty into the
microenvironment of hESCs. Utilizing this methodology, it was
determined that Lefty protein localizes to the areas where hESCs
are in contact with the underlying Matrigel matrix, and that
hESC-derived Lefty permeates into the underlying matrix (FIG. 8).
This is in contrast to Nodal protein, which localizes to the
surface of hESC colonies. These results were confirmed with Western
blot analyses, which demonstrated that Lefty protein can be
detected in Matrigel conditioned by H9 hESCs (H9 CMTX; FIG. 8), but
that Nodal protein is not detectable in this H9 CMTX. Furthermore,
neither Nodal nor Lefty were found in unconditioned control
Matrigel alone.
Example 6
[0128] Nodal Expression Down-Regulated in Aggressive Tumor Cells
Exposed to hESC Conditioned Matrix
[0129] A determination of the effects of H9 CMTX on Nodal
expression in metastatic melanoma (C8161) and breast carcinoma
(MDA-MB-231) cells was undertaken. As illustrated in FIG. 9, the
microenvironment of human embryonic stem cells (hESCs) leads to the
reduction of Nodal expression and tumorgenicity in plastic
metastatic melanoma and breast cancer cells exposed to the
embryonic preconditioned matrix. Western blot analyses (see below)
revealed that the microenvironments of hESCs reduced the expression
of Nodal protein in multipotent melanoma (C8161) and breast
carcinoma (MDA-MB-231) cells (FIG. 9A). Exposure to H9 CMTX
down-regulates Nodal protein expression in both melanoma and breast
carcinoma cells, and this effect is reversible over time (FIG. 9B).
Exposure to H9 CMTX similarly abrogates Nodal mRNA expression in
the melanoma and breast carcinoma cells (FIG. 9C).
[0130] FIG. 9D shows immunohistochemical analysis of Nodal staining
in an orthotopic tumor derived from C8161 cells pre-exposed to
Matrigel or a hESC conditioned matrix (H9 CMTX) (from in vivo
experiment in Example 2). The C8161 cells exposed to the H9 CMTX
expressed much less Nodal.
[0131] As a functional correlate, it was determined that exposure
of C8161 and MDA-MB-231 cells to H9 CTMX results in a significant
reduction in their ability to undergo anchorage independent growth,
and that this inhibition of in vitro clonogenicity can be partially
rescued by the inclusion of recombinant Nodal (100 ng/mL) (FIG.
9E). Of note, using Western blot analysis in conjunction with real
time RT-PCR, the ability to inhibit Nodal expression in cancer
cells was shown to be exclusive to the microenvironment of hESCs
(FIGS. 9F & 9G). For example, exposure of C8161 cells to
matrices conditioned by melanocytes, amniotic fluid derived stem
cells (GM00473, GM00957A), or cytotrophoblast cells (HTR-8/SVneo)
did not inhibit Nodal protein or mRNA expression (FIGS. 9F &
9G), thus illuminating the exclusivity of the epigenetic influence
of the hESC microenvironment.
Western Blot Analyses
[0132] Protein lysates were prepared and quantified as previously
described in Hess et al., 2001, Cancer Res. 61:3250-3255. Equal
amounts of protein were separated by SDS-polyacrylamide gel
electrophoresis under reducing conditions, and the resolved
proteins were transferred onto Immobilon-P membranes (Millipore
Corp., Bedford, Mass.). Membranes were blocked in 1% TBS, 0.1%
Tween 20 (TBS-T) and 5% dry milk powder or 3% gelatin (for Nodal
Westerns). Blots were incubated with anti-Nodal or anti-Lefty
antibodies (Polyclonal rabbit anti-Nodal (H-110) 1:500 Santa Cruz
Biotechnology, Santa Cruz, Calif.; Polyclonal goat anti-Lefty 1:500
R&D Systems, Minneapolis, Minn.), washed in TBS-T, and
incubated with the appropriate horseradish peroxidase-labeled
secondary antibody. Secondary antibodies were detected by enhanced
chemiluminescence (Super Signal; Pierce, Rockford, Ill.) and
exposure to autoradiography film (Molecular Technologies, St Louis,
Mo.). Nodal protein was detected as two major bands at .about.48
and .about.35 kDa representing precursor and pro-Nodal
respectively. Nodal often appeared as multiple bands, likely due to
degradation of protein modifications. All experiments were done at
least three times.
Example 7
[0133] Lefty is a Major hESC-Derived Factor Responsible for
Inhibiting Nodal Expression and Clonogenicity in Metastatic Cancer
Cells
[0134] As noted previously, the microenvironment of human embryonic
stem cells (hESCs) leads to the reduction of Nodal expression and
tumorgenicity in plastic metastatic melanoma and breast cancer
cells exposed to the embryonic preconditioned matrix. It was
determined that there is an abundance of the Nodal inhibitor Lefty
within the hESC conditioned matrices (FIG. 10A; CMTX). By contrast,
Lefty protein was absent in the C8161 cells on its own conditioned
matrix (FIG. 10A; C8161+CMTX).
[0135] Cancer cells were also exposed to Matrigel conditioned by
hESCs in which Lefty protein expression was knocked down with
FITC-tagged Morpholino oligonucleotides specific for Lefty-A and
Lefty-B (MO.sup.LEFTY). The fluorescently-tagged Morpholinos could
be detected microscopically in over 75% of the hESC colonies
treated (FIG. 10B), and Western blot analysis confirmed the
efficient knock down of Lefty protein in hESCs for up to 3 days
(FIG. 10C). The expression of Oct-3/4 and Nanog, representative of
hESC pluripotency, was not affected during this time, and the
morphology of the hESC colonies was not altered. Thus, although
MO.sup.LEFTY efficiently knocked down Lefty protein expression in
the hESCs, it did not induce stem cell differentiation (FIG. 10D).
Real time RT-PCR analysis revealed that exposure of metastatic
melanoma cells to Matrigel conditioned by H9 hESCs treated with
MO.sup.LEFTY did not result in an abrogation of Nodal expression
(FIG. 10E). In fact this "H9 Lefty-deficient" matrix up-regulated
Nodal mRNA expression in the C8161 cells (FIG. 10E).
[0136] Additionally, Dynabeads covalently coupled to anti-Lefty
antibody were utilized to isolate Lefty from hESCs cultured on a
feeder-free Matrigel matrix. This purified hESC-derived Lefty was
subsequently seeded into fresh Matrigel and the effects of the
"Lefty-containing" matrix on cancer cell phenotype were examined.
Western blot analysis revealed that hESC-derived Lefty abrogates
and diminishes Nodal protein expression in metastatic melanoma
(C8161) and breast carcinoma (MDA-MB-231) cells, respectively (FIG.
10F).
[0137] Also, exposure of C8161 and MDA-MB-231 cells to "H9
Lefty-containing" matrix was found to significantly reduce
anchorage independent growth, and this inhibition of in vitro
clonogenicity could be completely rescued by the inclusion of
recombinant Nodal (100 ng/mL) (FIG. 10G).
Example 8
[0138] rLefty Capable of Inhibiting Nodal at Elevated
Concentrations
[0139] As shown in the Western blot in FIG. 11, addition of rLefty
to C8161 cells reduces Nodal expression at the concentrations
shown. rLeftyB can inhibit Nodal protein in C8161 cells, but at a
higher doses than its hESC counterpart. These results are
consistent with prior findings (Tabibzadeh, S et al., 2006, Stem
Cells 24: 1998-2006).
Example 9
[0140] Lefty Derived from hESC's, Unlike Recombinant Lefty, is
Glycosylated
[0141] In an effort to understand the disparate results between
hESC-derived Lefty and rLefty on Nodal signaling, an analysis of
glycoprotein content in rLefty-B, rLefty-A and a lysate from the H9
hESCs plus their conditioned matrix was undertaking. It was found
that in contrast to the rLefty proteins, H9-derived Lefty is
heavily glycosylated (FIG. 12).
Example 10
[0142] Nodal Inhibition and the Microenvironment of hESCs Abrogate
Tumorigenicity In Vivo
[0143] The effects of the hESC microenvironment on the in vivo
tumorigenicity of melanoma and breast carcinoma cells were examined
using orthotopic mouse models. Exposure of metastatic melanoma
(C8161) and breast carcinoma (MDA-MB-231) cells to H9 CMTX resulted
in a significant reduction in tumorigenicity as compared to cells
exposed to unconditioned Matrigel (FIGS. 4B & 13A). In order to
further substantiate the role of Nodal in tumorigenicity, and to
illuminate a potential mechanism for the tumor-suppressive
properties of the hESC microenvironment, previously shown to
diminish Nodal expression (FIG. 9), orthotopic mouse models were
utilized to examine the effects of: (a) Ectopic Nodal expression on
the tumorigenic potential of C81-61 cells (isogenetically matched
non-tumorigenic variants of the metastatic C8161 melanoma cell
line); and (b) Nodal inhibition on MDA-MB-231 breast carcinoma
tumorigenicity. The control C81-61 melanoma cell line was unable to
form palpable tumors when 500,000 cells were injected, and using
gross observation and histology 5.5 weeks after inoculation, these
tumor cells could not be detected at the site of injection. In
contrast, 100% of C81-61 cells transfected with a Nodal expression
vector formed palpable tumors within 3.5 weeks of injection (FIG.
13B). Palpable tumors arose within 2.5 weeks after the injection of
500,000 control MDA-MB-231 cells, and knocking down Nodal
expression with Nodal Morpholinos (MO.sup.Nodal) resulted in a
significant reduction in MDA-MB-231 tumorigenicity (FIG. 13C) when
the same number of cells were injected.
[0144] In order to establish a mechanism by which exposure to the
hESC microenvironment abrogates tumorigenicity, the effects of this
treatment on the in vivo tumor cell proliferation-to-apoptosis
ratio were analyzed. Using immunohistochemical staining for Ki67 as
a measure of proliferation, and terminal deoxynucleotidyl
transferase biotin-dUTP nick-end labeling (TUNEL) as a measure of
apoptosis, it was determined that Nodal knock down and exposure to
hESC CMTX correspondingly decreased the ratio of proliferation to
apoptosis in metastatic C8161 melanoma cells and in metastatic
MDA-MB-231 breast carcinoma cells (FIGS. 13DE & 13E). Moreover,
an in vitro analysis of cell proliferation demonstrated reduced
proliferation in C8161 and MDA-MB-231 cells treated with
MO.sup.Nodal relative to cells treated with MO.sup.Control (FIG.
13F).
Example 11
Nodal Signaling Essential for Tumor Formation; Inhibitors of Nodal
Signaling Reduce Agressiveness and Tumorigenicty
[0145] The role of Nodal signaling in tumor formation was analyzed,
and it was found that downregulation of Nodal signaling results in
acquisition of a melanocyte-like phenotype and loss of the
dediffernentiated, plastic phenotype.
[0146] Administration of an anti-Nodal Morpholino (MO Nodal) also
resulted in down-regulation of Nodal, and an in vivo reduction in
tumor formation. An in vitro colony forming assay was used to
analyze colony-forming ability of poorly aggressive C81-61 cells,
aggressive C8161 cells, C8161 cells treated with MO.sub.Nodal and
C8161 cells treated with MO.sub.Nodal and rescued with recombinant
Nodal (100 ng/mL). The assay was conducted using 50,000 cells
suspended in 0.35% agarose in RPMI containing 10% serum, which were
plated into 6-well dishes on 0.5% agar in the same medium. Colonies
grew, and pictures were taken at day 7. After 2 weeks, colonies
were stained with Crystal Violet and counted.
[0147] Utilizing the in vitro assay, it was found that C8161 cells
were able to form colonies in soft agar within 7 days, and that
their less aggressive isogenic counterparts (C81-61) were not
clonogenic (FIG. 14A). Nodal inhibition with MO.sub.Nodal
significantly diminished the ability of C8161 cells to undergo
anchorage-independent growth. Even after 2 weeks, MO.sub.Nodal
reduced the colony formation of the C8161 cells by 57% (n=16,
p<0.001), a phenomenon that was rescued by the inclusion of
rNodal (100 ng/mL). Interestingly, rNodal did not induce colony
formation in the C81-61 cell line. For the clonogenic assays,
statistical significance was determined using a test. In all cases,
differences were statistically significant at p<0.05.
[0148] As a corollary to these findings, an orthotopic mouse model
was used to examine the effect of Nodal inhibition on melanoma
tumor formation. For the experimental tumourigenesis model, 5 week
old nude mice (Harlan, Madison, Wis.) were injected subcutaneously
with 250,000 C8161 cells, treated with control or anti-Nodal
Morpholinos, in 50 .mu.L of complete RPMI. Tumor measurements were
taken on days 3, 7, 14 and 17 post-injection, and mice were
sacrificed on day 17. In vivo tumor formation in a mouse injected
with C8161 cells treated with either MO.sup.Control or MO.sub.Nodal
is shown in FIG. 14B. For the orthotopic mouse tumor formation
studies, statistical significance was determined using the
Kruskal-Wallis One Way Analysis of Variance on Ranks, followed by
Dunn's method.
[0149] Palpable subcutaneous tumors arose within 7 days following
the injection of only 250,000 control C8161 cells. In contrast,
knocking down Nodal expression resulted in a significant reduction
in C8161 tumorigenicity (FIG. 12B; n=5, p<0.05) when the same
number of cells were injected. This reduction in tumorgenicity was
characterized by 30% diminution of tumor incidence as well as a
decrease in tumor growth.
[0150] Immunohistochemistry was used to analyze tumors from the
mice, which showed that the tumors that formed in the MO.sub.Nodal
treatment group started to regain Nodal expression by day 17 (FIG.
14C).
[0151] Additionally, poorly aggressive melanoma cells (C81-61)
cells acquired tumorigenic potential when transfected with Nodal
cDNA. C81-61 cells were transfected with either an empty vector or
a Nodal expression construct (n=5). As shown in FIG. 14D, such
cells demonstrated tumor growth.
[0152] Administration of an ALK 4/5/7 inhibitor resulted in a
reduction of the expression of various vasculogenic mimicry
plasticity biomarkers. FIG. 15A shows Western blot analyses of
Nodal, phosphorylated SMAD-2, total SMAD 2/3, and Actin in C8161
cells 48 hours after administration of either vehicle or an ALK
4/5/7 inhibitor (SB431542, 1 .mu.M, 10 .mu.M). All Nodal bands
represent the pro-protein. FIG. 15B shows Western blot analyses of
Nodal, Tyrosinase and Actin in C8161 cells 24 hours after the
administration of either vehicle or different concentrations of ALK
inhibitor while FIG. 15C shows a Western analyses for VE-Cadherin,
Keratin 18 and Actin in C8161 cells cultured on 3-D collagen I
matrices for 6 days in the presence of vehicle or different
concentrations of ALK inhibitor. FIG. 15D shows a reduction in
invasive ability and FIG. 15E shows an abrogation of vasculogenic
mimicry following down-regulation of Nodal with ALK 4/5/7
inhibitor.
[0153] The experiments described above revealed that metastatic
melanomas express the embryonic morphogen Nodal, that Nodal is
essential for tumor formation, and that its effects can be
mitigated through Nodal pathway inhibition, either directly or
indirectly (e.g. through ALK inhibition).
Example 12
Nodal Expression is Down-Regulated by Notch Inhibition
[0154] To address the possible molecular mechanisms underlying the
reprogramming of melanoma cells exposed to the hESC matrix
microenvironments, an analysis of the Nodal promoter was initiated,
a putative binding sequence effector for the Notch pathway (CBF-1)
was discovered, and the possibility of molecular cross-talk between
the Notch and Nodal pathways was investigated. Nodal expression in
metastatic melanoma cells treated with Notch siRNAs was knocked
down, particularly with Notch 4 siRNA. Conversely, Notch expression
was relatively unaffected by knockdown of Nodal, suggesting that
Notch is upstream of Nodal with possible molecular cross-talk.
[0155] As shown in FIG. 16A, Nodal expression was knocked down in
C8161 cells 72 hrs following the administration of Notch siRNA.
Real-time RT-PCR and Western blot analyses confirmed the silencing
of each Notch at this time point. In contrast, as shown in FIG.
16B, Notch expression is relatively unaffected by the knockdown of
Nodal (via treatment with the Nodal inhibitor SB431542) in C8161
cells.
Example 13
Hypermethylation Plays a Role in the Nodal Pathway in Aggressive
Tumor Cells
[0156] Hypermethylation of Nodal was observed in the highly
metastatic C8161 cells, but not in the isogenically matched C81-61
melanoma cells, nor in melanocytes or hESCs (H9). Sequencing based
methylation analyses, therefore, could be used to indicate the
methylation status in human tumors, and hence serve as a valuable
prognostic marker for disease state.
[0157] Nodal's methylation status is supported by work in
Feinberg's laboratory showing that CTCF binding site methylation
separates enhancers from promoters. (Gius, et. al., 2004, Cancer
Cell 6:361-371) In that work, it was found that azacytidine shut
down expression of as many genes as it activated; it is known now
that this subset of genes contains CTCF binding sites within the
promoter CpG island. In particular for Nodal, the sequence is
CCGCGCTGGGTGCCCAG [SEQ ID NO: 1]. The consensus that was identified
in genes activated by methylation is CCGCGN(N)GG(G)(N)GCC(N)CAG
[SEQ ID NO:2], and Feinberg has directly demonstrated methylation
dependent activation, with CTCF insulator binding abrogation in
several promoters with this consensus sequence. Paradoxically, when
this site is methylated, CTCF can no longer bind, and the promoter
is enabled. This is a major imprinting mechanism, and has
significant implications for how Nodal may be regulated during both
cancer and development.
[0158] Referring to FIG. 17, although culture of the C8161 cells in
the presence of a hESC microenvironment (H9 CMTX) globally
increases methylation by only 6.8%, the shaded region has a 32%
increase in methylation when cells are cultured on H9 CMTX versus
Matrigel alone. Sequence alignment revealed that the differentially
methylated cytosines are associated with putative transcription
factor binding sites. The shaded area contains consensus sequences
for EBP, Sp1 and AP-2alpha, and the 32% increase in methylation in
this region may indicate a silencing of the Nodal gene in the tumor
cells exposed to the hESC CMTX.
Example 14
Exemplary Assays and Procedures
General Maintenance of Cell Lines
[0159] The derivation and phenotypic characteristics of the human
melanoma cell lines have been previously described. Seftor, et.
al., 2002, Clin. Experim. Metastas. 19:233-246; Seftor, et. al.,
2005 Cancer Res. 65:10164-10169. The melanoma cell lines are
maintained in RPMI 1640 medium (Invitrogen) supplemented with 10%
fetal bovine serum (FBS, Gemini Bioproducts) and 0.1% gentamycin
sulfate with the exception of C81-61 cells which are maintained in
Ham's F10 medium supplemented with 15% FBS, 1.times.Mito+ (BD
Bioscience) and gentamycin sulfate. Normal human melanocytes are
purchased (Cascade Biologics) or isolated from neonatal foreskins.
Seftor, et. al., 2005. A single cell suspension is prepared, added
to plastic flasks for the adherence of melanocytes and the cells
propagated in Medium 254 with Human Melanocyte Growth Supplement
(Cascade Biologics) including, 100 units/ml penicillin, 100
.mu.g/ml streptomycin, and 250 ng/mL amphotericin B. The human
embryonic stem cell lines are cultured as previously described.
Thomson, et. al., 1998, Science 282:1145-1147. Briefly, cells are
grown in 6-well plates precoated with 0.1% porcine gelatin and
containing 1.9.times.105 irradiated mouse embryonic fibroblasts
(strain CF-1; ATCC) per well. The cells are maintained in medium
containing DMEM/F12 (1:1), 20% knock-out serum replacement,
non-essential minimal amino acids, L-glutamine (Invitrogen),
.beta.-mercaptomethanol, and 4 ng/ml FGF-2 (R&D Systems), and
are split with collagenase (1 mg/ml) before the colonies begin to
overlap. The cultures are determined to be free of mycoplasma
contamination using a PCR-based assay (Roche). The normal human
neonatal epidermal melanocytes (HEMn-LP; Cascade Biologics,
Portland Oreg.), myoepithelial cells (Hs 578 Bst; American Type
Culture Collection (ATCC), Manassas, Va.) and primary mammary
epithelial cells (HMEpC; Cell Applications Inc., San Diego Calif.)
were maintained as per distributor instructions. Live umbilical
cord blood stem cells (SC00125; New Jersey Stem Cell Resource at
Coriell Institute for Medical Research) amniotic fluid derived stem
cells (GM00473, GM00957A) and adult bone marrow derived mesenchymal
stem cells (Stem Cell Technologies, Vancouver BC, Canada) were
maintained under the recommended conditions. The HTR-8/SVneo is a
well characterized immortalized human extravillous cytotrophoblast
cell line, and was maintained as previously described Graham et
al., 1993, Exp. Cell Res. 206:204-211. Recombinant Nodal and Lefty
(R&D Systems) were diluted as per manufacturer suggestions. The
expression vector for wild type Nodal was kindly provided by Dr.
Daniel Constam (Swiss Institute for Experimental Cancer Research
(ISREC), Epalinges, Switzerland) and was transfected into C81-61
cells as previously described. Le Good et al., 2005, Curr. Biol.
15:31-36.
Preparation/Preconditioning of 3-D Human Matrices
[0160] 25-30 .mu.l of a defined human matrix (50 .mu.g/ml human
laminin; 50 .mu.g/ml human collagen IV in a 3 mg/ml human collagen
I base; Sigma) are either spread onto coverslips or directly placed
into 12-well culture dishes and polymerized with an application of
100% ethanol at room temperature. After extensive washes with PBS,
hESC's are seeded onto the 3-D matrix in complete stem cell medium.
After 3-4 days images are captured digitally using a Zeiss Televal
inverted microscope and Hitachi HV-C20 CCD camera. The cells are
then removed with 20 mM NH.sub.4OH followed by thorough washes with
sterile water, PBS and then complete medium. The conditioned matrix
is then analyzed by 2-D LDS-PAGE and Western blot directly, or
reseeded with melanoma cells and incubated for an additional 3
days. The cells re then harvested for further biochemical,
molecular and functional analyses.
3-D Conditioned Matrix Experiments
[0161] Conditioned matrices were prepared using hESCs, melanocytes,
myoepithelial cells, amniotic fluid derived stem cells, or
trophoblast cells on growth factor-reduced Matrigel (14 mg/mL; BD
Biosciences) as previously described. Postovit et al., 2006, Stem
Cells 24: 501-505. In all cases, cells were 80-100% confluent
during the conditioning of the matrix. Alternatively, hESC-derived
Lefty protein was seeded into Matrigel prior to polymerization.
Human melanoma (C8161) or breast carcinoma (MDA-MB-231) cells,
2.5.times.10.sup.5 cells/6-well dish, were subsequently exposed to
this preconditioned matrix for 3 to 4 days.
Invasion/Migration Assay
[0162] The Membrane Invasion Culture System (MICS) chamber is used
to evaluate the degree of tumor cell invasion through matrices in
vitro (both stimulated and unstimulated) as described previously.
Hendrix, M. J. C. et. al., 1992, J. Natl. Cancer Inst.,
84:165-174.
Determination of Cell Viability and Proliferation
[0163] Cell proliferation is assayed by immunohistochemical
staining of BrdU incorporation into newly synthesized DNA of
replicating cells at various time points (BrdU Labeling and
Detection Kit III; Roche). Assessment of proliferation index is
monitored by Ki-67 expression.
Proliferation Assay
[0164] 1.5.times.10.sup.4 cells were plated in individual wells of
a 24-well dish under standard tissue culture conditions and cell
counts were taken daily following harvesting.
Soft Agar Clonogenic Assay
[0165] Clonogenicity of cells are assessed as previously described.
Hamburger, A. W., and Salmon, S. E., 1977, Science 197:461-463.
Each parameter is tested in triplicate for clone formation in soft
agar. Briefly, 104 cells are plated in 60 mm Petri dishes in
complete medium placed over the soft agar. On specific days after
the cells are plated, phase contrast images of the colonies are
taken using a Zeiss Axiovert 25 with an Hitachi HCV-20 color
camera.
Anchorage Independent Growth Assays
[0166] Anchorage independent growth assays were conducted as
previously described. Topczewska et al., 2006, Nat. Med. 12:
925-932.
Differentiation Assays of Stem Cell Populations and
Clonally-Derived Melanoma Stem Cells
[0167] Stem cells from the various 3-D preconditioned matrices are
harvested and replated on an appropriate ECM in a specified
differentiation media, as previously described. Hendrix et. al.,
2003, Nature Rev. Cancer 3:411-421; Hsu et. al., 2004, Methods Mol.
Med. 107:13-28; Pittenger et. al., 1999, Science 284:143-147
Experimental Orthotopic Tumor Models
[0168] 5 week old mice were injected subcutaneously with 250,000
C8161 or 500,000 C81-61 human cutaneous melanoma cells in 50 .mu.L
of complete RPMI; or 500,000 MDA-MB-231 cells in 50 .mu.L of
complete RPMI were injected into the mammary fat pad of 8 week old
mice. When tumors became palpable measurements were taken twice per
week.
2-D L(ithium)DS-PAGE and Western Blot
[0169] Analysis of extracellular matrix components before and after
conditioning by the different cells is performed using Invitrogen's
3-10 pH IPG strips in the first dimension and 4-12% Bis/Tris
LDS-polyacrylamide gradient gels in the second dimension using MES
(proteins up to .about.100 kDa) or MOPS (proteins >100 kDa)
reservoir buffers, as per the manufacturer's protocols. The gels
are stained with Sypro Red then electroblotted onto Immobilon P
membranes (Millipore) for Western analysis using specific
extracellular matrix antibodies (Chemicon; R&D Systems; Life
Technologies).
Recovery of Lefty from Cell Conditioned Matrices
[0170] M-280 tosylactivated Dynabeads (Dynal Biotech) are
covalently coupled to anti-Lefty antibody (M-20:sc7408; Santa Cruz
Biotechnology, Inc.) at a final concentration of 8 .mu.g
antibody/1.times.107 beads as per the manufacturer's protocol. The
cell conditioned matrix is solubilized in RIPA buffer, sonicated,
centrifuged and the supernatant mixed by rotation with the beads
for 1 hour at 4.degree. C. After washing twice with PBS, Lefty is
recovered using either 50 mM glycine-HCl (pH 3.0), then normalized
to pH 7.4 with a 0.1 volume of 1 M Tris pH 8.5, or 0.2 M Tris pH8.5
plus 0.5 M NaCl.
RNA Extraction and Reverse Transcriptase Polymerase Chain Reaction
(RT-PCR)
[0171] Total RNA was isolated using TRIzol reagent (Invitrogen) and
1 .mu.g was reverse transcribed as previously described. Topczewska
et al., 2006. Real-time PCR was performed as previously described
(Id.) using TaqMan.RTM. gene expression human primer/probe sets for
the following genes: VEGF (Hs00173626_ml), TSP-1 (Hs00170236_ml),
Ki67 (Hs00606991_ml), Leftyl/B (Hs00764128_sl), Nodal
(Hs00250630_sl). Target gene expression was normalized to the
endogenous control gene GAPDH (GAPDH: 4333764F), RPLPO (RPLPO:
4333761F) and/or 18S rRNA (Hs99999901_sl). Data was analyzed using
Applied Biosystems Sequence Detection Software (Version 1.2.3).
FACs Analysis/Sorting
[0172] Fluorescence Activated Cell (FAC) analyses and sorting are
conducted using the BD FACsAria. Prior to FAC analysis, cells are
incubated with antibodies as per manufacturer instructions and
intracellular proteins detected in cells that have been previously
permeabilized. The FAC protocols are optimized for both cell
surface proteins (such as CD34) and intracellular proteins (such as
keratins). Live cell sorts are conducted using a 100 .mu.m nozzle
and aseptic technique. Successful live sorts are performed against
cell surface proteins, fluorescently labeled cells and cells
containing fluorescent anti-sense Morpholinos.
Glycoprotein Determination
[0173] Protein lysates underwent SDS-polyacrylamide gel
electrophoresis and transfer and were stained for glycoproteins
using the Pro-Q Emerald 300 staining kit (Molecular Probes). After
drying the blot, glycoproteins were visualized using an ultraviolet
tansilluminator and an image of the green fluorescing proteins
captured using a color CCD camera (Toshiba) equipped with a deep
yellow #15 filter. The blot was then rehydrated as per the
manufacturer's instructions and Lefty protein was detected with
immunoblotting.
IVIS Imaging System 200 Series
[0174] Real-time biophotonic imaging of GFP-labeled tumor cells in
the mouse model(s) is performed using a Xenogen IVIS Imaging System
200 Series imager. This system contains a custom lens and improved
resolution with single cell sensitivity for in vitro analyses. A
laser scanner and associated software provides an ability to
perform 3-D surface topography for single-view diffuse tomographic
reconstructions of internal sources in order to track tumor
formation and metastatic potential of GFP-labeled tumor cells.
Quantitative in vivo assays are performed using dual reporters to
differentiate increases in cell proliferation from increases in
specific gene expression.
Laser Capture Microdissection
[0175] The Veritas Laser Capture Microdissection (LCM) system
(Arcturus) combines a three objective lens microscope (up to
40.times.) for visualizing a sample mounted on a slide and
selecting the areas of interest, a UV laser for cutting around the
perimeter of the areas of interest, and a IR laser that melts and
thereby sticks the surface of a collector cap to these areas, or
individual cells for isolation. The Veritas LCM can be used for
isolating live cells cultured on 3-D matrices which have been cast
in the etched space of a specially made membrane containing slide
compatible with the Veritas system (PEN frame slides). Captured
material is subsequently lysed for RNA isolation (Picopure,
Arcturus) and downstream applications including Q-PCR and
microarray gene expression analysis.
Confocal Immuno-Microscopy
[0176] Immuno-confocal microcopy is performed using a Zeiss LSM 510
META Confocal Microscope. Prior to analysis, 3-D cultures or tissue
sections are incubated with specific antibodies against target
proteins as per protocols previously established in the
laboratory.
In Situ Hybridization
[0177] 3-D cultures or tissue samples are placed on subbed
microscope slides, and prepared as previously described. Kulesa,
et. al., 2000, Develop. 127 (13):2843-2852.
Nodal and Lefty Knockdown
[0178] Nodal, and Lefty protein expression were inhibited using
anti-sense Morpholino oligonucleotides (Gene Tools Inc., Philomath,
Oreg.). The Morpholino sequences were selected based on
manufacturer's recommendations (21-25 mer antisense). Fluoroscein
(FITC)-conjugated control (5'-CCTCTTACCTCAGTTACAATTTATA-3') [SEQ ID
NO: 19], Nodal (5'-AAGCAGCACCTCCAGCCCTTATATC-3') [SEQ ID NO: 20],
Lefty-A (5'-GCCACATGGTGCTGCCCTGGG-3') [SEQ ID NO: 21], and Lefty-B
(5'CTGCATGGTGCTGCCCTGGAGGA-3') [SEQ ID NO: 22]. Morpholinos (20
.mu.M) were delivered using the scrape method. Topczewska et al.,
2006. Cancer cells were sorted for FITC and were recovered for 1
day prior to experimentation.
Knockdown of Gene Expression by siRNA
[0179] Cells are plated in 6-well tissue culture plates and allowed
to grow to 50% confluence in serum containing, antibiotic-free
medium. The cells are then transfected with 10 or 100 nM of a
gene-specific siRNA or a non-specific siRNA control using
oligoFECTAMINE according to manufacturer's specifications
(Invitrogen). The cells are then harvested 3 days post transfection
and assessed for gene expression by RT-PCR, Q-PCR and Western blot
analysis, as well as functional assays. Quantitative PCR (Q-PCR):
Total RNA is isolated from cells using Trizol RNA isolation reagent
(Invitrogen) according to manufacturer's specifications. Reverse
transcription of the total RNA is performed in a Robocycler
gradient 96 thermocycler (Stratagene) using the Advantage PCR kit
according to the manufacturer's instructions (Clontech). Q-PCR is
performed using a 7500 Real Time PCR System (Applied Biosystems)
and TaqMan.RTM. gene expression primer/probe sets (Applied
Biosystems). Briefly, 5 .mu.l cDNA, 1.25 .mu.l
20.times.Assays-on-Demand Gene Expression Assay Mix and 12.5 .mu.l
2.times.TaqMan.RTM. Universal PCR Master Mix in a total of 25 .mu.l
are amplified with the following thermocycler protocol: 1 cycle at
50.degree. C. for 2 min; 1 cycle at 95.degree. C. for 10 min; and
40 cycles at 95.degree. C. for 15 seconds/60.degree. C. for 1 min.
All data is analyzed with the Sequence Detection Software (version
1.2.3, Applied Biosystems), and expression of each target gene
normalized to an endogenous control gene. Each experiment is
repeated twice and each sample is performed in triplicate.
Microarray Analysis
[0180] Microarray and bioinformatics analyses of the cells is
performed using the U133A Human Genome Array from Affymetrix as a
cooperative agreement with Translational Genomics (TGen; Phoenix,
Ariz.; Dr. Jeffrey Trent).
Comparative Genomic Hybridization Analysis: Genomic DNA
Isolation
[0181] Genomic DNA is isolated from cells using the PUREGENE DNA
isolation kit (Gentra Systems). Five .mu.g of gDNA is digested with
EcoRI, extracted with phenol:chloroform, ethanol precipitated, and
resuspended in sterile distilled water, as previously described.
O'Hagan et. al., 2003, Cancer Res. 53:5352-5356.
Statistical Analysis
[0182] All statistical analyses are performed using Microsoft
Excel's spreadsheet software with the majority of statistics
consisting of a "one-way analysis of variance" (ANOVA)
determination with a value of p<0.05 deemed significant. For the
orthotopic mouse tumor formation studies, we determined statistical
significance using the Kruskal-Wallis One Way Analysis of Variance
on Ranks, followed by Dunn's method or a one way analysis of
variance (ANOVA) followed by the Student-Newman-Keuls method for
pairwise multiple comparisons. For the clonogenic and proliferation
assays we determined statistical significance using ANOVA followed
by the Student-Newman-Keuls method for pairwise multiple
comparisons. For the correlation of breast cancer stage and Nodal
expression, a Spearman Rank Order Correlation was employed. In all
cases, differences were statistically significant at P<0.05.
Analysis of DNA methylation by sequencing of sodium
bisulfite-treated DNA
[0183] Genomic DNA is obtained by digestion with proteinase K
(Quiagen) followed by phenol/chloroform extraction, and is
subjected to sodium bisulfite treatment to modify unmethylated
cytosine to uracil using the `CpGenome.TM. DNA Modification Kit`
(Chemicon International). Bisulfite-treated DNA is amplified by a
nested-PCR protocol using the primers described in Table 1.
TABLE-US-00003 TABLE 1 Primers used for amplifications after DNA
bisulfite conversion. Primer Sequence NODAL gene CpG1 (52 CpGs)
NODAL-1 EF 5' - TTT TAG AAG GGA GTG AAT TGG -3' (SEQ ID NO: 3)
NODAL-1 ER 5' - AAA AAA TAA AAA CTT CTA ATC TCC - 3' (SEQ ID NO: 4)
NODAL-1 IF 5' - AGT ATT TTA GTA AAT TTT TTA TTG -3 ' (SEQ ID NO: 5)
NODAL-1 IR 5' - ATT AAT ATT ACT ATA ATA ATT TAA TC - 3' (SEQ ID NO:
6) NODAL gene CpG2 (47 CpGs) NODAL-2 EF 5' - TAA TTT TAT AAG ATT
GGA GAT TAG - 3' (SEQ ID NO: 7) NODAL-2 ER 5' - TAC TAA AAC CCA AAA
TAT AAA AAC - 3' (SEQ ID NO: 8) NODAL-2 IF 5' - TTT AAA TTA AAA TTT
AGA GAT AAT GG - 3' (SEQ ID NO: 9) NODAL-2 IR 5' - ACT TTC AAA CCT
AAC CAA CCC - 3' (SEQ ID NO: 10) LEFTY 1 (B) (61 CpGs) LEFTY1 EF 5'
- TAG TTT TTA AGG TTT AGG GTG TG - 3' (SEQ ID NO: 11) LEFTY1 ER 5'
- TAC TAA CCC TAC TCT TAT CCC - 3' (SEQ ID NO: 12) LEFTY1 IF 5' -
AG TTT TAG TTG GGG TTT TTT AAG - 3' (SEQ ID NO: 13) LEFTY1 IR 5' -
TTA AAA ACC AAC ACA CAC CTA C - 3' (SEQ ID NO: 14) LEFTY 2 (A) (66
CpGs) LEFTY2 EF 5' - TAG TTT TTG AGG TTT AGG GTG TG - 3' (SEQ ID
NO: 15) LEFTY2 ER 5' - TAT CTC CTA ACC TAA CTA CC - 3' (SEQ ID NO:
16) LEFTY2 IF 5' - AG TTT TAG TTG GGG TTT TTT AAG - 3' (SEQ ID NO:
17) LEFTY2 IR 5' - CTC AAT AAC CCT ACC ATC CTC - 3' (SEQ ID NO: 18)
* EF/R = external primer set; IF/R = internal primer set
[0184] PCR is performed in a volume of 25 .mu.l containing PCR
Buffer (Qiagen); 1.5 mM of MgCl2 (Qiagen); 200 .mu.M of dNTPs
(Invitrogen); 0.32 .mu.M of each primer and 1 U of Hot Start Taq
Plus DNA Polymerase (Qiagen). The PCR conditions are: 94.degree. C.
for 10 min, 94.degree. C. for 3 min, 48.degree. C. for 3 min,
72.degree. C. for 2 min one cycle; 94.degree. C. for 3 min,
50.degree. C. for 3 min, 72.degree. C. for 2 min five cycles and
94.degree. C. for 1 min, 52.degree. C. for 1 min, 72.degree. C. for
1 min 35 cycles for the first reaction and the same annealing
temperatures (48.degree., 50.degree. and 52.degree. C.) for the
nested reaction. Amplified products are purified using the Gel
Purification Kit (Qiagen) and are ligated to a vector using the
TOPO TA Cloning Kit (Invitrogen). Twenty four positive clones are
sequenced for each sample using the vector's forward and reverse
primers. DNA sequencing reactions are performed using the `DNA
dRhodamine Terminator Cycle Sequencing Ready reaction` kit (Applied
Biosystems) and an ABI3730x1 sequencer (Applied Biosystems)
according to the manufacturer's instructions.
Immunoblotting
[0185] Protein lysates were prepared and quantified as previously
described in Hess et al., 2001, Cancer Res. 61:3250-3255. Equal
amounts of protein were separated by SDS-polyacrylamide gel
electrophoresis under reducing conditions, and the resolved
proteins were transferred onto Immobilon-P membranes (Millipore
Corp., Bedford, Mass.). Membranes were blocked in 1% TBS, 0.1%
Tween 20 (TBS-T) and 5% dry milk powder or 3% gelatin (for Nodal
Westerns). Blots were incubated with primary antibody (Table 2),
washed in TBS-T or TBS-T containing 0.5M NaCl for the Nodal
Westerns, and incubated with the appropriate horseradish
peroxidase-labeled secondary antibody. Secondary antibodies were
detected by enhanced chemiluminescence (Super Signal; Pierce,
Rockford, Ill.) and exposure to autoradiography film (Molecular
Technologies, St Louis, Mo.). Nodal protein was detected as two
major bands at .about.48 and .about.35 kDa representing precursor
and pro-Nodal respectively. Nodal often appeared as multiple bands,
likely due to degradation of protein modifications.
TABLE-US-00004 TABLE 2 Antibodies Utilized for Western Blot (WB),
Immunohistochemical (IHC) and Immunofluorescence (IF) Analyses.
Concentration Antibody & Use Company Polyclonal goat anti- 2
.mu.g/mL, IF R&D Systems, mNodal 2 .mu.g/mL, IHC Minneapolis,
MN Polyclonal rabbit anti- 1:500, WB Santa Cruz Biotechnology,
Nodal (H-110) Santa Cruz, CA Polyclonal goat anti- 1:500, WB Santa
Cruz Biotechnology, Lefty 1:50, IF Santa Cruz, CA Monoclonal Mouse
anti- 1 .mu.g/mL, WB R&D Systems, Cripto 10 .mu.g/mL, IF
Minneapolis, MN Polyclonal Goat anti- 1:20, IHC Santa Cruz
Biotechnology, Ki67 Santa Cruz, CA Monoclonal mouse anti- 1:5000,
WB Chemicon International, Actin Temecula, CA
Immunofluorescence
[0186] Cells were fixed with 4% paraformaldehyde, made permeable
with 20 mM Hepes, 0.5% TritonX-100 and blocked with serum-free
protein block (DAKO, Carpinteria, Calif.). Primary antibodies were
diluted in antibody dilutent (DAKO) to the concentrations outlined
in SI Table 2, and appropriate fluorochrome-conjugated secondary
antibodies were used according to manufacturer recommendations. For
certain images, nuclei were stained with DAPI (0.1 mg/mL; Molecular
Probes), and images were obtained using confocal microscopy (Zeiss
510 META, Carl Zeiss Inc.).
Immunohistochemistry
[0187] Formalin-fixed, paraffin-embedded archival tissue was
obtained from patients with primary or metastatic cutaneous
melanoma (Loyola University Chicago, Ill.). Immunohistochemical
staining was performed on a HNS 710i Automated Immunostainer
(Richard-Allan Scientific (RAS), Kalamazoo, Mich.) with the
Multi-Species HRP/AEC Detection Systems. Following
deparaffinization in xylene, ethanol degradation, and antigen
retrieval with citrate buffer, four blocking steps were applied:
0.03% hydrogen peroxide, Avidin and Biotin blocks (Avidin/Biotin
blocking kit, Vector Laboratories, Inc., Burlingame, Calif.), and a
Serum-Free protein block. Anti-Nodal antibody (20 .mu.g/mL, R&D
Systems, Minneapolis, Minn.) was applied for 90 minutes. Slides
were rinsed in TBS-T, incubated with biotinylated anti-goat IgG (2
.mu.g/ml, Vector Labs), washed with TBS-T and incubated with the
streptavidin peroxidase reagent for 15 minutes. Color was produced
with AEC (red) substrate (RAS) and counterstaining with Mayer's
hematoxylin. Samples were dehydrated in reagent grade alcohol and
cover slipped with permanent mounting medium. Negative control
reactions were conducted with ChromPure Goat IgG (Jackson Labs),
isotype matched and used at the same concentration as the Nodal
antibody. Immunohistochemical staining for Nodal in a breast
carcinoma progression TMA (CBL-TMA-029; Creative Biollabs, Port
Jefferson Station, N.Y.) was performed as previously described.
Topczewska et al., 2006. Tissues from the orthotopic tumor models
were formalin-fixed and paraffin-embedded and immunohistochemical
staining on this tissue was conducted using a Ki67-specific
antibody (Table 2) or ChromPure Goat IgG (Jackson Labs) as
previously described. Topczewska et al., 2006. TUNEL assays to
measure apoptosis were conducted as per instructions (Upstate).
[0188] It should be understood that the foregoing disclosure
emphasizes certain specific embodiments of the invention and that
all modifications or alternatives equivalent thereto are within the
spirit and scope of the invention as set forth in the appended
claims. The combination of particular aspects of the various
embodiments of the invention is included in the scope of the
invention. All patents, patent applications, and other scientific
or technical writings referred to anywhere herein are incorporated
by reference in their entirety.
Sequence CWU 1
1
22117DNAArtificialSynthetic 1ccgcgctggg tgcccag
17218DNAArtificialSynthetic 2ccgcgnnggg ngccncag
18321DNAArtificialSynthetic 3ttttagaagg gagtgaattg g
21424DNAArtificialSynthetic 4aaaaaataaa aacttctaat ctcc
24524DNAArtificialSynthetic 5agtattttag taaatttttt attg
24626DNAArtificialSynthetic 6attaatatta ctataataat ttaatc
26724DNAArtificialSynthetic 7taattttata agattggaga ttag
24824DNAArtificialSynthetic 8tactaaaacc caaaatataa aaac
24926DNAArtificialSynthetic 9tttaaattaa aatttagaga taatgg
261021DNAArtificialSynthetic 10actttcaaac ctaaccaacc c
211123DNAArtificialSynthetic 11tagtttttaa ggtttagggt gtg
231221DNAArtificialSynthetic 12tactaaccct actcttatcc c
211323DNAArtificialSynthetic 13agttttagtt ggggtttttt aag
231422DNAArtificialSynthetic 14ttaaaaacca acacacacct ac
221523DNAArtificialSynthetic 15tagtttttga ggtttagggt gtg
231620DNAArtificialSynthetic 16tatctcctaa cctaactacc
201723DNAArtificialSynthetic 17agttttagtt ggggtttttt aag
231821DNAArtificialSynthetic 18ctcaataacc ctaccatcct c
211925DNAArtificialSynthetic 19cctcttacct cagttacaat ttata
252025DNAArtificialSynthetic 20aagcagcacc tccagccctt atatc
252121DNAArtificialSynthetic 21gccacatggt gctgccctgg g
212223DNAArtificialSynthetic 22ctgcatggtg ctgccctgga gga 23
* * * * *