U.S. patent application number 14/174610 was filed with the patent office on 2014-06-05 for methods of monitoring angiogenesis and metastasis in three dimensional co-cultures.
This patent application is currently assigned to The United States of America,as represented by the Secretary,Department of Health and Human Services. The applicant listed for this patent is The United States of America,as represented by the Secretary,Department of Health and Human Services, The United States of America,as represented by the Secretary,Department of Health and Human Services. Invention is credited to Frank Cuttitta, Changge Fang, Enrique Zudaire.
Application Number | 20140155292 14/174610 |
Document ID | / |
Family ID | 42826494 |
Filed Date | 2014-06-05 |
United States Patent
Application |
20140155292 |
Kind Code |
A1 |
Fang; Changge ; et
al. |
June 5, 2014 |
METHODS OF MONITORING ANGIOGENESIS AND METASTASIS IN THREE
DIMENSIONAL CO-CULTURES
Abstract
This disclosure relates to fluorescent cell lines and to the use
of such cell lines in monitoring cellular activity, such as
angiogenesis. This disclosure further relates to the use of such
cell lines in a three-dimensional cell culture to monitor
angiogenic and metastatic potential of tumor cells and selecting
personalized therapeutics for treatment of cancer.
Inventors: |
Fang; Changge; (Alexandria,
VA) ; Zudaire; Enrique; (Germantown, MD) ;
Cuttitta; Frank; (Adamstown, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America,as represented by the
Secretary,Department of Health and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America,as
represented by the Secretary,Department of Health and Human
Services
Bethesda
MD
|
Family ID: |
42826494 |
Appl. No.: |
14/174610 |
Filed: |
February 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12802666 |
Jun 10, 2010 |
8679836 |
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14174610 |
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12060752 |
Apr 1, 2008 |
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12802666 |
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60976732 |
Oct 1, 2007 |
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Current U.S.
Class: |
506/10 ;
435/29 |
Current CPC
Class: |
G01N 33/5044 20130101;
G01N 33/5017 20130101; G01N 33/5011 20130101; G01N 33/5005
20130101 |
Class at
Publication: |
506/10 ;
435/29 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A method for monitoring angiogenic or metastatic potential of
tumor cells comprising: preparing a three-dimensional co-culture
comprising: a first layer comprising a neutral polysaccharide
polymer gel in contact with the bottom of a culture dish; a second
layer on top of the first layer, comprising: a solidified gel
matrix; endothelial cells dispersed in the solidified gel matrix;
and tumor cells comprising either a tumor spheroid colony or a
sample of a tumor biopsy, suspended in the solidified gel matrix;
and a third layer on top of the second layer, comprising culture
medium; incubating the three-dimensional co-culture; and detecting
at least one of endothelial cell proliferation, endothelial cell
tubule formation or tumor cell angiotropism of the cells in the
second layer.
2. The method of claim 1, wherein the neutral polysaccharide
polymer gel comprises agarose.
3. The method of claim 1, wherein the endothelial cells stably and
constitutively express a fluorescent protein.
4. The method of claim 3, wherein the tumor cells stably and
constitutively express a fluorescent protein with a different
emission spectrum from the fluorescent protein expressed by the
endothelial cells.
5. The method of claim 3, wherein the second layer further
comprises at least one additional mammalian cell type dispersed in
the solidified gel matrix, and wherein the at least one additional
mammalian cell type stably and constitutively expresses a
fluorescent protein with a different emission spectrum from the
fluorescent protein expressed by the endothelial cells.
6. The method of claim 4, wherein the second layer further
comprises at least one additional mammalian cell type dispersed in
the solidified gel matrix, and wherein the at least one additional
mammalian cell type stably and constitutively expresses a
fluorescent protein with a different emission spectrum from either
of the fluorescent proteins expressed by the endothelial cells or
the tumor cells.
7. The method of claim 1, wherein the second layer further
comprises at least one additional mammalian cell type dispersed in
the solidified gel matrix.
8. The method of claim 7, wherein the at least one additional
mammalian cell type is a cell selected from the group consisting of
macrophage, mast cell, fibroblast, adipocyte, and pericyte.
9. The method of claim 1, wherein the first, second, or third layer
further comprises at least one test agent.
10. The method of claim 9, wherein the test agent is a known or
potential inhibitor of angiogenesis or metastasis.
11. The method of claim 1, wherein the tumor cells are derived from
a subject and the first, second, or third layer further comprises
at least one test agent that has been administered to the subject
as part of a cancer treatment.
12. A method of testing the efficacy of an anti-angiogenic or
anti-metastatic cancer treatment for a subject, comprising
monitoring angiogenic or metastatic potential of tumor cells by the
method of claim 1, wherein the tumor cells are derived from the
subject and the first, second, or third layer comprises at least
one test agent that is a candidate anti-cancer treatment.
13. A method of selecting a personalized anti-angiogenic or
anti-metastatic treatment for cancer in a subject comprising:
preparing multiple three-dimensional co-cultures, each co-culture
comprising: a first layer comprising a neutral polysaccharide
polymer gel in contact with the bottom of a culture dish; a second
layer on top of the first layer, comprising: a solidified gel
matrix; endothelial cells dispersed in the solidified gel matrix;
and tumor cells comprising either a tumor spheroid colony or a
sample of a tumor biopsy, suspended in the solidified gel matrix;
and a third layer on top of the second layer, comprising culture
medium, wherein all but one of the co-cultures further comprises at
least one test agent comprising an anti-angiogenic or
anti-metastatic compound in the first, second, or third layers;
incubating the three-dimensional co-cultures; detecting at least
one of endothelial cell proliferation, endothelial cell tubule
formation or tumor cell angiotropism of the cells in the second
layer; and selecting the at least one test agent having the
greatest effect on at least one of endothelial cell proliferation,
endothelial cell tubule formation or tumor cell angiotropism in
comparison to endothelial cell proliferation, endothelial cell
tubule formation or tumor cell angiotropism in the cells of the
co-culture without the test agent in the medium.
14. The method of claim 13, wherein the neutral polysaccharide
polymer gel comprises agarose.
15. The method of claim 13, wherein the endothelial cells stably
and constitutively express a fluorescent protein.
16. The method of claim 15, wherein the tumor cells stably and
constitutively express a fluorescent protein with a different
emission spectrum from the fluorescent protein expressed by the
endothelial cells.
17. The method of claim 15, wherein the second layer further
comprises at least one additional mammalian cell type dispersed in
the solidified gel matrix, and wherein the at least one additional
mammalian cell type stably and constitutively expresses a
fluorescent protein with a different emission spectrum from the
fluorescent protein expressed by the endothelial cells.
18. The method of claim 16, wherein the second layer further
comprises at least one additional mammalian cell type dispersed in
the solidified gel matrix, and wherein the at least one additional
mammalian cell type stably and constitutively expresses a
fluorescent protein with a different emission spectrum from the
either of the fluorescent proteins expressed by the endothelial
cells or the tumor cells.
19. The method of claim 13, wherein the second layer further
comprises at least one additional mammalian cell type dispersed in
the solidified gel matrix.
20. The method of claim 19, wherein the at least one additional
mammalian cell type is a cell type selected from the group
consisting of macrophage, mast cell, fibroblast, adipocyte, and
pericyte.
21. The method of claim 1, wherein the monoclonal tumor spheroid
colony is produced using a method comprising: preparing a culture
in which monoclonal tumor spheroid colonies are grown, comprising:
a bottom layer comprising about 1% agarose; and a top layer
overlaying the bottom layer, wherein the top layer comprises
isolated tumor cells suspended in about 0.2% of agarose; incubating
the culture to grow monoclonal spheroid colonies; harvesting
monoclonal tumor spheroid colonies from the culture; and
resuspending the monoclonal tumor spheroids in a buffered solution.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This is a continuation of co-pending U.S. application Ser.
No. 12/802,666, filed Jun. 10, 2010; which is a
continuation-in-part of U.S. application Ser. No. 12/060,752, filed
Apr. 1, 2008, now abandoned; which claims the benefit of U.S.
Provisional Application No. 60/976,732, filed Oct. 1, 2007. The
prior applications are incorporated herein by reference in their
entirety.
FIELD OF THE DISCLOSURE
[0002] This disclosure relates to fluorescent cell lines and to the
use of such cell lines in monitoring cellular activity, such as
angiogenesis, as well as their use in three-dimensional cell
cultures, for instance to monitor angiogenic and metastatic
potential of tumor cells. Also described are methods of using such
cells in selecting personalized therapeutics.
BACKGROUND
[0003] Biological processes occurring in any organism involve the
interaction of multiple cell types, biologically relevant factors,
and the organism's environment. Some of the fundamental questions
remaining in biology are related to understanding how the different
cells within organisms communicate and organize to form an
individual. One frequently studied system in which multiple cell
types function together and influence each other is
angiogenesis.
[0004] Angiogenesis is a biological process of generating new blood
vessels from pre-existing blood vessels into a tissue or organ.
Angiogenesis has been intensively studied over the past several
decades because of its fundamental importance in tissue
development, vascular diseases, and cancer. Under normal
physiological conditions, humans or animals undergo angiogenesis
only in very specific restricted situations. For example,
angiogenesis is normally observed in fetal and embryonal
development and formation of the corpus luteum. Post-natal
angiogenesis is an important physiological function in the ovary,
endometrium, placenta, and in wound healing.
[0005] New vessel growth is tightly controlled by many angiogenic
regulators (see for example Folkman, J., Nature Med., 1: 27-31,
1995a), and the switch of the angiogenesis phenotype depends on the
net balance between up-regulation of angiogenic stimulators and
down-regulation of angiogenic suppressors. Pathological
deregulation of angiogenesis is a prominent feature of a number of
human diseases, including atherogenesis, arthritis, psoriasis,
corneal neovascularization, diabetic retinopathy, rheumatoid
arthritis, and cancer, for example during malignant transformation
that facilitates tumor growth and metastasis.
[0006] In cancer, tumors induce angiogenesis by secreting various
growth factors, such as vascular endothelial growth factor (VEGF),
and basic fibroblast growth factor (bFGF) among others. Growth
factors, such as bFGF and VEGF, can induce capillary growth into
the tumor, which is thought to drive tumor expansion by supplying
the tumor with nutrients and/or removing the cellular waste.
[0007] Angiogenesis is also an element of metastasis of a tumor.
Single cancer cells can break away from an established solid tumor,
enter the blood vessel, and be carried to a distant site, where
they can implant and begin the growth of a secondary tumor. It has
even been suggested that the blood vessels in a solid tumor may in
fact be mosaic vessels, comprised of both endothelial cells and
tumor cells. Such mosaicity allows for substantial shedding of
tumor cells into the vasculature.
[0008] Angiogenesis-based anti-tumor therapies typically use
natural and synthetic angiogenesis inhibitors such as angiostatin,
endostatin and tumstatin. Recently the Food and Drug Administration
(FDA) approved an antibody therapy targeting angiogenesis in
colorectal cancer. This therapy is based on a monoclonal antibody
directed against an isoform of VEGF and is marketed under the trade
name Avastin.RTM.. While established anti-angiogenesis therapies
are promising, the need still exists for the development of
additional modulators of angiogenesis.
SUMMARY OF THE DISCLOSURE
[0009] This disclosure relates to an in vitro assay for use in
assessing the cellular activity of cell lines. The assay disclosed
herein uses detectable cell lines from an array of different cell
lines, such as cell lines of different cell types and/or anatomical
origins, such that the effects and interdependency of the different
cell lines can be monitored simultaneously, for example in
real-time multiplex assays. In some examples, the assay uses
multiple different cell lines that contribute to angiogenesis in
vivo, such that the angiogenesis process can be recapitulated in
vitro.
[0010] In some embodiments, the disclosed assay uses mammalian cell
lines that have been stably transfected with mammalian expression
vectors which include nucleic acid sequences encoding proteins that
can be detected by light emitted by the proteins expressed from the
expression vectors, for example a fluorescent protein expressed
from the expression vectors.
[0011] In some of the disclosed embodiments, the in vitro assay is
a multiplex assay method for evaluating cellular activity, in which
a culture is provided that contains one or more different isolated
mammalian cell lines (such as histologically different cell lines)
that stably and constitutively express fluorescent proteins having
different emission spectra, for example the different fluorescent
proteins have different wavelengths of emission maxima, such that
the emission spectra from the different fluorescent proteins is
distinguishable. The culture is assessed for cellular activity by
quantifying fluorescence or detecting a pattern of fluorescence
from fluorescent proteins present in the culture. In some examples,
a culture of two different isolated mammalian cell lines (such as
histologically different cell lines) that stably and constitutively
express fluorescent proteins having different emission spectra is
provided and the cellular activity of one or both of the
fluorescent cell lines present in the culture is assessed by
quantifying fluorescence or detecting a pattern of fluorescence
from fluorescent proteins present in the culture. By extension, the
cellular activity of cell lines present in culture of three, four,
five or even more isolated cell lines expressing fluorescent
proteins with different emission spectra can be assessed by
quantifying fluorescence or detecting a pattern of fluorescence
from the fluorescent proteins present in the culture.
[0012] In some embodiments, the cellular activity of the cell
line(s) present in the culture is assessed by determining one or
more of the growth rate, migration potential, cell death or tubule
formation potential of the cell lines using the quantified
fluorescence or pattern of fluorescence from the fluorescent
proteins present in the culture. Such assays can be used to measure
cellular activity and interaction within complex biological
systems. Such measurements of cellular activity can even be
obtained and/or measured in a temporal sequence or in real-time as
they occur.
[0013] In some embodiments, the disclosed in vitro assay is used to
determine the effects of an exogenous agent, such as a test agent
(for example a potential modulator of angiogenesis, such as a
potential inhibitor of angiogenesis or a potential stimulator of
angiogenesis), growth factor, biological sample (such as a patient
sample), another cell line (such as one or more fluorescent cell
lines) etc. on the cellular activity of a fluorescent cell line.
Such an assay can be used to screen for modulators of angiogenesis,
for example to identify angiogenesis inhibitors useful in the
treatment of cancer.
[0014] Also disclosed are cell lines have been stably transfected
with mammalian expression plasmids that constitutively express
different fluorescent proteins, for example green fluorescent
protein and related florescent proteins, such as yellow fluorescent
protein, red fluorescent protein, cyan fluorescent protein and the
like. These cell lines are particularly suited for use in the
disclosed methods. Kits for performing the disclosed assays, which
include the disclosed fluorescent cell lines, are also
disclosed.
[0015] This disclosure also relates to methods for monitoring
angiogenic or metastatic potential of tumor cells comprising
preparing a three-dimensional co-culture that is comprised of three
layers. The first layer comprises a neutral polysaccharide polymer
gel in contact with the bottom of the culture dish. The second
layer is on top of the first layer and comprises a solidified gel
matrix, endothelial cells that are dispersed in the solidified gel
matrix; and tumor cells comprising either a tumor spheroid colony
or a sample of a tumor biopsy, and which are also suspended in the
solidified gel matrix, and a third layer comprising culture medium.
Angiogenic or metastatic potential of tumor cells is monitored by
incubating the three-dimensional co-culture; and detecting at least
one of endothelial cell proliferation, endothelial cell tubule
formation or tumor cell angiotropism of the cells in the second
layer. In particular examples of these methods, the first, second,
or third layer further comprises at least one test agent, which in
some embodiments is a known or potential inhibitor of angiogenesis
or metastasis or augmenter of these processes.
[0016] Also disclosed herein are methods of selecting a
personalized anti-angiogenic or anti-metastatic treatment for
cancer in a subject comprising preparing multiple three-dimensional
co-cultures, each co-culture comprising a first layer comprising a
neutral polysaccharide polymer gel in contact with the bottom of a
culture dish; a second layer on top of the first layer, comprising:
a solidified gel matrix; endothelial cells dispersed in the
solidified gel matrix; and tumor cells comprising either a tumor
spheroid colony or a sample of a tumor biopsy, suspended in the
solidified gel matrix; and a third layer comprising culture medium,
wherein all but one of the co-cultures further comprises at least
one test agent comprising an anti-angiogenic or anti-metastatic
compound in the first, second, or third layers. A personalized
anti-angiogenic or anti-metastatic treatment for cancer in a
subject is selected by incubating the three-dimensional
co-cultures; detecting at least one of endothelial cell
proliferation, endothelial cell tubule formation or tumor cell
angiotropism of the cells in the second layer; and selecting the at
least one test agent having the greatest effect on at least one of
endothelial cell proliferation, endothelial cell tubule formation
or tumor cell angiotropism in comparison to endothelial cell
proliferation, endothelial cell tubule formation or tumor cell
angiotropism in the cells of the co-culture without the test
agent.
BRIEF DESCRIPTION OF THE FIGURES
[0017] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0018] FIG. 1A is a digital image of porcine aortic endothelial
(PAE) cells that stably express green fluorescent protein (GFP).
FIG. 1B is a digital image of PAE cells that stably express yellow
fluorescent protein (YFP). FIG. 1C is a digital image of PAE cells
that stably express red fluorescent protein (RFP). FIG. 1D is a
digital image of PAE cells that stably express cyan fluorescent
protein (CFP).
[0019] FIG. 2 as a graph of fluorescence versus cell number showing
linearity of fluorescence versus cell number in mono-cultures of
PAE endothelial cells that stably express YFP and mono-cultures of
human breast adenocarcinoma cell line MCF7 that stably express
RFP.
[0020] FIG. 3A is a graph of fluorescence versus cell number
showing that precise gated fluorescence emission and excitation on
YFP allows discrimination of YFP expressing cells (PAE) from RFP
expressing cells (MCF7) in co-cultures. FIG. 3B is a graph of
fluorescence versus cell number showing that precise gated
fluorescence emission and excitation on RFP allows discrimination
of RFP expressing cells (MCF7) from YFP expressing cells (PAE) in
co-cultures.
[0021] FIG. 4A is a graph of growth curves for PAE endothelial
cells expressing YFP in mono-culture or in co-culture with MCF7
breast cancer cells. FIG. 4B is a graph of growth curves for MCF7
breast cancer cells expressing RFP in mono-culture or in co-culture
with PAE endothelial cells.
[0022] FIG. 5 is a set of digital images of YFP expressing PAE
endothelial cells and RFP expressing MCF7 breast cancer cells in
mono-culture and co-culture.
[0023] FIG. 6 is a bar graph of the migratory potential of serum
obtained from 11 subjects and a negative control.
[0024] FIG. 7A is a set of digital images showing the tubules
formed in cultures of PAE endothelial cells at various suramin
concentrations. FIG. 7B is a graph of the number of long tubules
formed by GFP expressing PAE cells as a function of suramin
concentration. FIG. 7C is a graph of the number of short tubules
formed by GFP expressing PAE cells as a function of suramin
concentration.
[0025] FIG. 8A is a graph showing the migratory potential of
fluorescent endothelial cells in response to increasing
concentrations of sputum obtained from an Idiopatic Pulmonary
Fibrosis (IPF) patient. FIG. 8B is a graph showing the difference
in migration potential of sputum from 13 normal subjects (NV-BAL)
and 13 IPF patients (IPF-BAL) (10% fetal bovine serum (FBS) and
phosphate buffered saline (PBS) are controls).
[0026] FIG. 9 is a graph of the measured fluorescence present in
the cellular growth media as a function of increasing TritonX
concentration.
[0027] FIG. 10 is a flow chart showing an exemplary method for high
throughput screening of test agents (such as small molecules) for
antiangiogenic activity using fluorescent endothelial cells. In one
example, a primary screen of a small molecule library is done using
the disclosed growth and tube formation assays. This primary screen
identifies bioactive compounds, some of which could be cytotoxic. A
counterscreen using the disclosed cell viability assay is used
identify those compounds with cytotoxic activity. Biologically
active compounds which show no cytotoxicity are considered putative
antiangiogenic candidates and can move forward to in vivo
studies.
[0028] FIG. 11 shows an exemplary procedure for determining
antiangiogenic activity of a test agent in a multiwell format. In
this example, assays are performed in 96-well plates which contain
negative controls (column 1) positive controls (column 12) and 80
remaining wells containing the small molecules to be tested. In
some examples, a quality control (Z' score, Zhang et al. J. Biomol.
Screen. 4:67-73, 1999) is applied to every plate. Only plates with
Z values between 0.5 and 1 are considered.
[0029] FIG. 12 shows a montage of micrographs representing an
example of one of the growth assay plates included in an exemplary
high throughput screen. Column 1 contains cell that have not been
stimulated with growth factor (no growth) and column 12 shows
growth of the endothelial cells upon exposure to a growth factor
cocktail. Test wells show different levels of cell growth
(quantification of the fluorescence in each well is done with a
fluorimeter). Wells which contain growth inhibitors are shown in
white boxes (hits are defined using the SASD: sum of the average
squared inside-cluster distances, Gagarin et al, J. Biomol. Screen
11:1-12, 2006).
[0030] FIG. 13A is a graph of cell growth (measured as absolute
fluorescence) as a function of time for the wells shown in FIG. 12.
FIG. 13B is a graph of cell growth (normalized for the growth rate
of the positive control) as a function of time for the wells shown
in FIG. 12.
[0031] FIG. 14 is a schematic representation of the
three-dimensional models for the study of the complex interactions
between different cell types in a three-dimensional environment.
Because the different cell types used are labeled with different
fluorescent proteins it becomes easier to image in real time the
evolution of the model. It becomes also possible to sort apart the
cells and do gene expression analysis on them. Also, these models
allow for screening of drugs (antiangiogenic, antitumoral, etc) in
a more complex in vitro system.
[0032] FIG. 15 is a digital image of a screen shot of the main GUI
window of the AngioApplication.TM..
[0033] FIG. 16 is a digital image of a screen shot of the settings
window of AngioApplication.TM..
[0034] FIG. 17A is a digital image of a screen shot of the
AngioApplication.TM. tubule analysis screen showing a sample under
fluorescent illumination. FIG. 17B is a digital image of a screen
shot of the AngioApplication.TM. tubule analysis screen showing a
sample under bright field illumination.
[0035] FIG. 18 is a digital image of a screen shot of the
AngioApplication.TM. custom open dialog.
[0036] FIG. 19 is a digital image of a screen shot of the excel
output of the AngioApplication.TM..
[0037] FIG. 20 is a drawing showing several interactive cellular
components involved with tumor associated
angiogenesis/lymphangiogenesis.
[0038] FIG. 21 shows 2D and 3D co-culture approaches to simulate
the in vivo angiogenic interactions between tumor and endothelial
cells.
[0039] FIG. 22 is a series of photomicrographs of 2D co-cultures of
endothelial cells (PAE) and indicated tumor cell lines. PAE cells
were grown on top of matrigel layered on top of a layer of the
indicated tumor cell line. PAE seeding density was approximately
18,000 cells/well. Cultures were incubated for six hours.
[0040] FIG. 23 is a series of photomicrographs of 2D co-cultures
comparing the tube formation response of different endothelial
cells (PAE, HMEC or LEC-1) vs. different tumor cells (A549,
CRL-1721 or 92-1). Endothelial cells (at approximately 18,000/well)
were plated on top of matrigel solidified on top of a monolayer of
indicated tumor cell line.
[0041] FIG. 24 is a series of photographs of human/rat xenograft
tumors (A549 (left), PC-12 (middle), and 92.1 (right)) excised from
nude mice. Top row shows whole tumors and peripheral
vascularization. Bottom row shows tumors sectioned to display
angiogenesis through tumor core.
[0042] FIG. 25 is a series of confocal photomicrographs of 3D
co-cultures of HMEC-1 endothelial cells (yellow) and MCF-7 tumor
xenografts (dashed blue ellipse). Left and right panels are
magnified 10.times.. Middle panel is magnified 5.times..
Endothelial cells (about 21,000/well) were mixed with single
ringlet of xenograft core biopsy in molten matrigel that was
allowed to solidify on top of agarose-coated 96 well plates.
[0043] FIG. 26 is a confocal photomicrograph of a nine-day 3D
co-culture of PAE endothelial cells (red) and 92-1 ocular melanoma
(blue).
[0044] FIG. 27 is a confocal photomicrograph of a nine-day 3D
co-culture of BEC-1 endothelial cells (red) and rat
pheochromocytoma PC-12 (blue).
[0045] FIG. 28 is a confocal photomicrograph showing peritumoral
vascularization in a twelve-day 3D co-culture of PAE endothelial
cells (red) and NSCLC A549 (blue).
[0046] FIG. 29 shows confocal photomicrographs of twenty-day 3D
co-cultures of HMEC-1 endothelial cells (red) and human tumor cells
(blue). Co-cultures with ocular melanoma 92.1 (left) and lung
cancer A549 (right) are shown.
[0047] FIG. 30 shows confocal photomicrographs of six-day 3D
co-cultures of PAE (red) and leiomyosarcoma HTB-88 core biopsy
xenograft (blue dashed ellipses). Peripheral (top) or central
(bottom) xenograft tissue was cultured with PAE. Co-cultures were
incubated with (left) or without (right) Avastin.RTM..
[0048] FIG. 31 shows a series of confocal photomicrographs of
five-day 3D co-cultures of HMEC-1 (yellow) and human leiomyosarcoma
HTB-88 core biopsy xenograft (blue dashed ellipses). Peripheral
ringlet xenograft tissue/HMEC-1 co-cultures were incubated with
Avastin.RTM., Thalidomide, Sunitinib or Fumagilin.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
I. Abbreviations
[0049] 2D: two-dimensional
[0050] 3D: three-dimensional
[0051] ATCC: American Type Culture Collection
[0052] bFGF: basic fibroblast growth factor
[0053] BEC: brain endothelial cells
[0054] BME: Basement Membrane Extract
[0055] DMSO: dimethyl sulfoxide
[0056] EC50: The term half maximal effective concentration
[0057] EBM-2: endothelial basal medium-2
[0058] FBS: fetal bovine serum
[0059] FDA: Food and Drug Administration
[0060] GFP: green fluorescent protein.
[0061] HMEC-1: human microvascular endothelial cell
[0062] IPF: Idiopatic Pulmonary Fibrosis
[0063] LEC: lymphatic endothelial cells
[0064] PAE: porcine aortic endothelial cells
[0065] PBS: phosphate buffered saline
[0066] RFP: red fluorescent protein
[0067] VEGF: endothelial growth factor
[0068] YFP: yellow fluorescent protein
II. Terms
[0069] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology can be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0070] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Although methods and materials similar or
equivalent to those described herein can be used in the practice or
testing of this disclosure, suitable methods and materials are
described below. The term "comprises" means "includes." The
abbreviation, "e.g." is derived from the Latin exempli gratia, and
is used herein to indicate a non-limiting example. Thus, the
abbreviation "e.g." is synonymous with the term "for example."
[0071] To facilitate review of the various embodiments of the
disclosure, the following explanations of specific terms are
provided:
[0072] Animal: A living multi-cellular vertebrate organism, a
category that includes, for example, mammals and birds. The term
mammal includes both human and non-human mammals. Similarly, the
term "subject" includes both human and veterinary subjects, for
example, humans, non-human primates, dogs, cats, horses, pigs,
rats, mice, and cows.
[0073] Angiogenesis: A biological process leading to the generation
of new blood vessels through sprouting and/or growth from
pre-existing blood vessels. The process can involve the migration
and proliferation of endothelial cells from preexisting vessels.
Angiogenesis occurs during pre-natal development, post-natal
development, and in the adult. In the adult, angiogenesis occurs
during the normal cycle of the female reproductive system, wound
healing, and during pathological processes such as cancer (for a
review see Battegay, J. Molec. Med. 73(7): 333-346, 1995).
[0074] Angiogenic activity: The ability of an agent to promote or
inhibit angiogenesis. Angiogenic activity can be measured in an
angiogenesis assay, for example using the fluorescent cell lines
and assays disclosed herein.
[0075] Angiogenic factor: A molecule that affects angiogenesis, for
example by stimulating or inhibiting angiogenesis. Numerous
experiments have suggested that tissues secrete factors that
promote angiogenesis under conditions of poor blood supply during
normal and pathological angiogenesis processes. The formation of
blood vessels is initiated and maintained by a variety of factors
secreted either by a cell (such as a tumor cell) or by accessory
cells. Many different growth factors and cytokines have been shown
to exert chemotactic, mitogenic, modulatory or inhibitory
activities on endothelial cells, smooth muscle cell and fibroblasts
and can, therefore, be expected to participate in an angiogenic
process. For example, factors modulating growth, chemotactic
behavior and/or functional activities of vascular endothelial cells
include aFGF, bFGF, angiogenin, angiotropin, epithelial growth
factor, IL-8, and vascular endothelial growth factor (VEGF) among
others.
[0076] Because many angiogenic factors are mitogenic and
chemotactic for endothelial cells, their biological activities
(such as angiogenic activities) can be determined in vitro by
measuring the induced migration of endothelial cells or the effect
of these factors on endothelial cell proliferation using the cell
lines assays and methods disclosed herein. For example, migration
assays and other assays, such as tubule formation assays and growth
assays can also be used to determine angiogenic activity, for
example the angiogenic activity in the presence of a test agent,
such as a potential angiogenesis inhibitor.
[0077] Angiogenic potential: The ability of a factor, such as a
compound or cell type, such as a tumor cell, to stimulate
angiogenesis in an endothelial cell line.
[0078] Angiotropism: The movement of a tumor cell along a vascular
highway. Such movement is a hallmark of metastasis of a tumor. In
particular examples, angiotropism is observable as the migration of
tumor cells in vitro along endothelial tubules.
[0079] Biological sample: A sample obtained from a plant or animal
subject about which information is desired, for example,
information about the samples ability to promote cellular growth,
tubule formation, and/or cellular migration. As used herein,
biological samples include all clinical samples, including, but not
limited to, cells, tissues, and bodily fluids, such as: blood;
derivatives and fractions of blood, such as serum, and lymphocytes
(such as B cells, T cell, and subfractions thereof); extracted
galls; biopsied or surgically removed tissue, including tissues
that are, for example, unfixed, frozen, fixed in formalin and/or
embedded in paraffin; tears; milk; skin scrapes; surface washings;
urine; sputum; cerebrospinal fluid; prostate fluid; pus; bone
marrow aspirates; middle ear fluids, bronchoalveolar levage,
tracheal aspirates, sputum, nasopharyngeal aspirates, oropharyngeal
aspirates, or saliva. In particular embodiments, the biological
sample is obtained from an animal subject, such as in the form of
middle ear fluids, bronchoalveolar levage, tracheal aspirates,
sputum, nasopharyngeal aspirates, oropharyngeal aspirates, or
saliva. In particular embodiments, the biological sample is
obtained from a subject, such as blood or serum. In other
embodiments, the biological sample is a sample of tissue removed
from a tumor (e.g., a tumor biopsy). A patient sample is a sample
obtained from a subject, such as a mammalian subject, for example a
human subject under medical care.
[0080] Cellular activity: The activity of a particular cell line,
such as the ability of the cell to divide, migrate in response to
stimulus, or to form three dimensional structures, such as tubules.
The cellular activity of a particular cell line can be assessed
using in vitro assays, for example the assays disclosed herein.
[0081] Cancer: A malignant disease characterized by the abnormal
growth and differentiation of cells. "Metastatic disease" refers to
cancer cells that have left the original tumor site and migrate to
other parts of the body for example via the bloodstream or lymph
system.
[0082] Examples of hematological tumors include leukemias,
including acute leukemias (such as acute lymphocytic leukemia,
acute myelocytic leukemia, acute myelogenous leukemia and
myeloblastic, promyelocytic, myelomonocytic, monocytic and
erythroleukemia), chronic leukemias (such as chronic myelocytic
(granulocytic) leukemia, chronic myelogenous leukemia, and chronic
lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's
disease, non-Hodgkin's lymphoma (indolent and high grade forms),
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, myelodysplastic syndrome, hairy cell leukemia, and
myelodysplasia.
[0083] Examples of solid tumors, such as sarcomas and carcinomas,
include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,
osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma,
Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
lymphoid malignancy, pancreatic cancer, breast cancer (such as
adenocarcinoma), lung cancers, gynecological cancers (such as,
cancers of the uterus (e.g., endometrial carcinoma), cervix (e.g.,
cervical carcinoma, pre-tumor cervical dysplasia), ovaries (e.g.,
ovarian carcinoma, serous cystadenocarcinoma, mucinous
cystadenocarcinoma, endometrioid tumors, celioblastoma, clear cell
carcinoma, unclassified carcinoma, granulosa-thecal cell tumors,
Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma),
vulva (e.g., squamous cell carcinoma, intraepithelial carcinoma,
adenocarcinoma, fibrosarcoma, melanoma), vagina (e.g., clear cell
carcinoma, squamous cell carcinoma, botryoid sarcoma), embryonal
rhabdomyosarcoma, and fallopian tubules (e.g., carcinoma)),
prostate cancer, hepatocellular carcinoma, squamous cell carcinoma,
basal cell carcinoma, adenocarcinoma, sweat gland carcinoma,
medullary thyroid carcinoma, papillary thyroid carcinoma,
pheochromocytomas sebaceous gland carcinoma, papillary carcinoma,
papillary adenocarcinomas, medullary carcinoma, bronchogenic
carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,
choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor,
seminoma, bladder carcinoma, and CNS tumors (such as a glioma,
astrocytoma, medulloblastoma, craniopharyogioma, ependymoma,
pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,
menangioma, melanoma, neuroblastoma and retinoblastoma), and skin
cancer (such as melanoma and non-melanoma).
[0084] Cell culture: The process by which either prokaryotic or
eukaryotic cells are grown under controlled conditions. In practice
the term "cell culture" has come to refer to the culturing of cells
derived from multicellular eukaryotes, especially animal cells,
such as mammalian cells, for example the fluorescent cells
disclosed herein. Mammalian cells are grown and maintained at an
appropriate temperature and gas mixture (typically, 37.degree. C.,
5% CO.sub.2) in a cell incubator. Culture conditions vary widely
for each cell type, and variation of conditions for a particular
cell type can result in different phenotypes being expressed. Aside
from temperature and gas mixture, the most commonly varied factor
in culture systems is the growth medium. Recipes for growth media
can vary in pH, glucose concentration, growth factors, and the
presence of other nutrient components. The growth factors used to
supplement media are often derived from animal blood, such as calf
serum.
[0085] Some cells naturally live without attaching to a surface,
such as cells that exist in the bloodstream. Others require a
surface, such as most cells derived from solid tissues. Cells grown
unattached to a surface are referred to as suspension cultures.
Other adherent cultures cells can be grown on tissue culture
plastic, which may be coated with extracellular matrix components
(for example collagen or fibronectin) to increase its adhesion
properties and provide other signals needed for growth. Co-culture
refers to the culture of more than one cell line (such as more than
one of the disclosed cell lines), more than one cell type, or a
cell line and a tissue sample, such a sample of a tumor biopsy, in
a single vessel. A 2-Dimensional (2D) co-culture is a co-culture
wherein the different cell lines or cell types are not cultured
within the same dimension or layer of the culture, and are
separated for example, by a gelled layer of gel matrix. A
3-Dimensional (3D) co-culture is a co-culture wherein the different
cell lines or cell types are cultured together within a
three-dimensional gel matrix.
[0086] Chemical stimulus: A chemical signal that stimulates an
activity of a cell, or cell line, for example the cell lines
disclosed herein. Examples of chemical stimuli include growth
factors, such as bFGF and VEGF.
[0087] Chemotherapeutic agents: Any chemical agent with therapeutic
usefulness in the treatment of diseases characterized by abnormal
cell growth. Such diseases include tumors, neoplasms, and cancer as
well as diseases characterized by hyperplastic growth such as
psoriasis. In one embodiment, a chemotherapeutic agent is an
angiogenesis inhibitor. Chemotherapeutic agents are described for
example in Slapak and Kufe, Principles of Cancer Therapy, Chapter
86 in Harrison's Principles of Internal Medicine, 14th edition;
Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology
2nd ed., 2000 Churchill Livingstone, Inc; Baltzer and Berkery.
(eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis,
Mosby-Year Book, 1995; Fischer Knobf, and Durivage (eds): The
Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book,
1993. Combination chemotherapy is the administration of more than
one agent to treat cancer, for example an alkylating agent and an
angiogenesis inhibitor.
[0088] Contacting: The placement in direct physical association,
including both in solid and in liquid form. Contacting can occur in
vivo, for example by administering an agent to a subject, or in
vitro for example with isolated cells or cell-cultures, for example
cell-cultures of the disclosed fluorescent cell lines.
"Administrating" to a subject includes topical, parenteral, oral,
intravenous, intra-muscular, sub-cutaneous, inhalational, nasal, or
intra-articular administration, among others.
[0089] Control: A reference standard. A control can be a known
value indicative of basal cellular activity, such as basal
migratory potential, doubling time, tubule formation potential and
the like, or a control cell-culture, such as a culture including at
least one of the disclosed fluorescent cell lines, not treated with
an exogenous agent, such as a test agent, one or more cell lines
(such as the fluorescent cell lines disclosed herein), angiogenic
factor, angiogenic inhibitor, or the like. A difference between a
test sample and a control can be an increase or conversely a
decrease. The difference can be a qualitative difference or a
quantitative difference, for example a statistically significant
difference. In some examples, a difference is an increase or
decrease, relative to a control, of at least about 10%, such as at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 100%, at least about 150%,
at least about 200%, at least about 250%, at least about 300%, at
least about 350%, at least about 400%, at least about 500%, or
greater then 500%.
[0090] Disperse: Distribute throughout a medium, such as a gel
matrix of the disclosed 3D co-cultures. In particular examples,
cells that are dispersed in a medium are distributed evenly
throughout the medium. However, dispersal of cells in a medium does
not require absolute even distribution of cells.
[0091] EC50: The term half maximal effective concentration (EC50)
refers to the concentration of a drug which induces a response
halfway between the baseline and maximum. EC50 is commonly used as
a measure of drug potency.
[0092] Encoding: Unless evident from its context, includes nucleic
acid sequences, such as RNA and DNA sequences, that encode a
polypeptide, as well as RNA and DNA sequences that are transcribed
into proteins, such as fluorescent proteins, for example nucleic
acid sequences that encode green fluorescent protein, red
fluorescent protein, yellow fluorescent protein, cyan fluorescent
protein and the like.
[0093] Electromagnetic radiation: A series of electromagnetic waves
that are propagated by simultaneous periodic variations of electric
and magnetic field intensity, and that includes radio waves,
infrared, visible light, ultraviolet light, X-rays and gamma rays.
In particular examples, electromagnetic radiation is emitted by a
laser, which can possess properties of monochromaticity,
directionality, coherence, polarization, and intensity. Lasers are
capable of emitting light at a particular wavelength (or across a
relatively narrow range of wavelengths), for example such that
energy from the laser can excite one fluorophore with a specific
excitation wavelength but not excite a second fluorophore with a
specific excitation wavelength difference and distinct from the
excitation wavelength on the first fluorophore.
[0094] Emission or emission signal: The light of a particular
wavelength generated from a source. In particular examples, an
emission signal is emitted from a fluorophore, such as a
fluorescent protein, after the fluorophore absorbs light at its
excitation wavelength(s).
[0095] Excitation or excitation signal: The light of a particular
wavelength necessary and/or sufficient to excite an electron
transition to a higher energy level. In particular examples, an
excitation is the light of a particular wavelength necessary and/or
sufficient to excite a fluorophore, such as a fluorescent protein,
to a state such that the fluorophore will emit a different (such as
a longer) wavelength of light then the wavelength of light from the
excitation signal.
[0096] Exogenous agent: An exogenous agent is any agent external to
a target cell line(s) that is to be studied, and it includes small
molecules, proteins, biological samples (such as patient samples)
and other cells or cell lines, such as fluorescent cell lines other
than the target cell line, for example a different type of cell
that can by identified as different by a distinguishable
fluorescent signal. In particular examples, the exogenous agent is
a test agent such as a small molecule, protein or nucleic acid, but
which is not a cell or tissue sample.
[0097] Expression: With respect to a gene sequence, refers to
transcription of the gene and, as appropriate, translation of the
resulting mRNA transcript to a protein. Thus, expression of a
protein coding sequence, such as the expression of a fluorescent
protein, results from transcription and translation of the coding
sequence for that protein. Constitutive expression refers to the
expression of a gene product, such as a protein, for example a
fluorescent protein, in a substantial continuous manner, such that
the expression is not interrupted. An example of constitutive
expression is continuous expression in the absence of an exogenous
stimulating agent, such as an agent used to activate a promoter.
Stable expression refers to expression that is not lost or reduced
substantially over time, for example expression that does not
diminish through multiple passages of a cell line, for example a
cell line constitutively expressing a fluorescent protein.
[0098] Expression control sequences: Nucleic acid sequences that
regulate the expression of a heterologous nucleic acid sequence to
which it is operatively connected. Expression control sequences are
operatively connected to a nucleic acid sequence when the
expression control sequences control and regulate the transcription
and, as appropriate, translation of the nucleic acid sequence. Thus
expression control sequences can include appropriate promoters,
enhancers, transcription terminators, a start codon (i.e., ATG) in
front of a protein-encoding gene, splicing signal for introns,
maintenance of the correct reading frame of that gene to permit
proper translation of mRNA, and stop codons. The term "control
sequences" is intended to include, at a minimum, components whose
presence can influence expression, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0099] A promoter is a minimal sequence sufficient to direct
transcription. Also included are those promoter elements which are
sufficient to render promoter-dependent gene expression
controllable for cell-type specific, tissue-specific, or inducible
by external signals or agents; such elements may be located in the
5' or 3' regions of the gene. Both constitutive and inducible
promoters are included (see e.g., Bitter et al., Methods in
Enzymology 153:516-544, 1987). For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like
may be used. In one embodiment, when cloning in mammalian cell
systems, promoters derived from the genome of mammalian cells
(e.g., metallothionein promoter) or from mammalian viruses (e.g.,
the retrovirus long terminal repeat; the adenovirus late promoter;
the vaccinia virus 7.5K promoter; the SV40 viral promoter; the CMV
promoter and the like) can be used. Promoters produced by
recombinant DNA or synthetic techniques may also be used to provide
for transcription of the nucleic acid sequences, for example when
incorporated into a vector, such as a mammalian expression
vector.
[0100] Fluorescent property: A characteristic of a fluorescent
molecule, such as a fluorescent protein, for example green
fluorescent protein, red fluorescent protein, yellow fluorescent
protein, cyan fluorescent protein and the like. Examples of
fluorescent properties include the molar extinction coefficient at
an appropriate excitation wavelength, the fluorescence quantum
efficiency, the shape of the excitation spectrum or emission
spectrum (the "fluorescence spectrum," the excitation wavelength
maximum and emission wavelength maximum, the ratio of excitation
amplitudes at two different wavelengths, the ratio of emission
amplitudes at two different wavelengths, the excited state
lifetime, or the fluorescence anisotropy. Quantifying fluorescence
refers to the determination of the amount of fluorescence generated
by a fluorophore, for example a fluorescent protein, which can be
the quantity of photons emitted by a fluorophore. In some examples,
fluorescence is quantified by measuring the intensity of a
fluorescence signal at a particular wavelength, for example the
wavelength of the emission maxima of a particular fluorophore, such
as a fluorescent protein. Fluorescence intensity can also be
quantified at a wavelength that is not the emission maxima of a
particular fluorophore, for example to avoid emission spectra that
overlap and thereby interfere with the emission maxima of a
particular fluorophore, such as a particular fluorescent protein.
In some examples, a fluorescence signal is emitted by a population
of fluorescent proteins, for example fluorescent proteins present
in a population of cells containing such fluorescent proteins. Such
a signal can be quantified, for example to determine the number, or
relative number of cells that emit such a fluorescent signal.
Detecting a pattern of fluorescence refers to the correlation of a
fluorescent signal to a specific location to determine the location
where a fluorescence signal, such as a fluorescent signal of a
particular wavelength, originates. In some examples, a pattern of
fluorescence determines the location and or shape of the cells that
emit a fluorescence signal, such as cells containing a fluorescent
protein, for example to determine the number of the total area of
the tubules, the total number of tubules, number of nodes, number
of branch points, the number of tubes per node, or node area formed
by such cells using the methods disclosed herein.
[0101] Fluorescent protein: A protein capable of emission of a
detectable fluorescent signal. Fluorescent proteins can be
characterized by the wavelength of their emission spectrum. For
example green fluorescent protein (GFP) has a fluorescent emission
spectrum in the green part of the visible spectrum. In addition to
green-fluorescent proteins, fluorescent proteins are known which
fluoresce in other regions of the visible spectrum, for example
blue-fluorescent proteins, cyan-fluorescent proteins,
yellow-fluorescent proteins, orange-fluorescent proteins,
red-fluorescent proteins, and far-red fluorescent proteins.
Examples of fluorescent proteins can be found in the following
patent documents: U.S. Pat. Nos. 5,804,387; 6,090,919; 6,096,865;
6,054,321; 5,625,048; 5,874,304; 5,777,079; 5,968,750; 6,020,192;
6,146,826; 6,969,597; 7,150,979; 7,157,565; and 7,166,444; and
published international patent applications WO 07/085,923; WO
07/052,102, WO 04/058973, WO 04/044203, WO 03/062270; and WO
99/64592. Additional examples of fluorescent proteins are available
from Clontech, Laboratories, Inc. (Mountain View, Calif.) under the
trade name Living Colors.RTM.. Nucleic acids encoding such
fluorescent proteins can be incorporated into mammalian expression
vectors for use in producing the disclosed fluorescent cell
lines.
[0102] Gel Matrix: A semi-solid cell culture media that is derived
from extracellular matrix proteins or any suitable equivalent
synthetic gel product. Gel matrices are fluid at 4.degree. C. and
gel at 37.degree. C. In particular examples a gel matrix is a
commercially available medium such as BD Matrigel.TM. Matrix (BD
Bioscience), Cultrex.RTM. BME (Trevigen), or Geltrex.RTM.
(Invitrogen.RTM.). Other basement membrane extracts that can
function as a support matrix scaffolding include human
placenta-derived BME (Vivo Biosciences, Inc) and synthetic BME
(available from Glycosan Biosystems).
[0103] Gene: A nucleic acid sequence that encodes a polypeptide
under the control of a regulatory sequence, such as a promoter or
operator. A gene includes an open reading frame encoding a
polypeptide of the present disclosure, as well as exon and
(optionally) intron sequences. An intron is a DNA sequence present
in a given gene that is not translated into protein and is
generally found between exons. The coding sequence of the gene is
the portion transcribed and translated into a polypeptide (in vivo,
in vitro or in situ) when placed under the control of an
appropriate regulatory sequence. The boundaries of the coding
sequence can be determined by a start codon at the 5' (amino)
terminus and a stop codon at the 3' (carboxyl) terminus. If the
coding sequence is intended to be expressed in a eukaryotic cell, a
polyadenylation signal and transcription termination sequence can
be included 3' to the coding sequence.
[0104] Transcriptional and translational control sequences include,
but are not limited to, DNA regulatory sequences such as promoters,
enhancers, and terminators that provide for the expression of the
coding sequence, such as expression in a host cell. A
polyadenylation signal is an exemplary eukaryotic control sequence.
A promoter is a regulatory region capable of binding RNA polymerase
and initiating transcription of a downstream (3' direction) coding
sequence.
[0105] Growth rate: The expansion of the number of cells of a
specified cell line through cell division as a function of time. In
one example the growth rate is the rate at which a cell line grown
in culture doubles.
[0106] Preferred mammalian codon(s): The subset of codons from
among the set of all possible codons encoding an amino acid that
are most frequently used in proteins expressed in mammalian cells.
Table 1 summarizes preferred mammalian codons for each amino
acid:
TABLE-US-00001 TABLE 1 Amino Acid Preferred codons Gly GGC, GGG Glu
GAG Asp GAC Val GUG, GUC Ala GCC, GCU Ser AGC, UCC Lys AAG Asn AAC
Met AUG Ile AUC Thr ACC Trp UGG Cys UGC Tyr UAU, UAC Leu CUG Phe
UUC Arg CGC, AGG, AGA Gln CAG His CAC Pro CCC
In some embodiments, the nucleotide sequence encoding the amino
acid sequence of a fluorescent protein has been codon optimized for
expression in a mammalian cell. By codon optimized it is meant that
at least some of the codons that encode the fluorescent protein
have been exchanged for codons that are preferentially used by
mammalian cells, for example the codons listed in Table 1.
Typically the exchange of codons does not alter the amino acid
sequence of the resulting fluorescent protein relative to the
fluorescent protein encoded by the nucleic acid sequence with
unexchanged codons.
[0107] High throughput technique: Through this process one can
rapidly identify active compounds, antibodies or genes which affect
a particular biomolecular pathway, for example pathways in
angiogenesis. In certain examples, combining modern robotics, data
processing and control software, liquid handling devices, and
sensitive detectors, high throughput techniques allows the rapid
screening of potential pharmaceutical agents in a short period of
time.
[0108] Histology: The study of the microscopic anatomy and
classification of tissue, including the histology of mammalian
cells, such as cells and cell lines from mammalian tissues.
Histological typing refers to the categorizing of tissue into
histological types, for example by microanatomical origin (such as
connective tissue, nerves, muscles, and circulatory cells, among
others) or cell-types (such as epithelial cells, stromal cells
among others). Cells can be classified as being of different
histological types by virtue of the staining and/or reaction with
antibodies, or by characteristic microanatomical features. Cells of
different histological types interact differently with different
stains and/or antibodies. Methods for histological typing are well
known in the art. Histology can be use to determine if cells are of
different types. Thus, in some examples different cell lines are
histologically different cell lines.
[0109] Immortalized cell or cell line: A cell or cell line that has
acquired the ability to proliferate indefinitely either through
random mutation or deliberate modification, such as artificial
expression of the telomerase gene. There are numerous well
established immortalized cell lines representative of particular
cell types.
[0110] Inhibitor (for example, of angiogenesis): A substance
capable of inhibiting to some measurable extent, for example
angiogenesis. In disclosed examples inhibition is measured in the
assays disclosed herein.
[0111] Isolated: An "isolated" biological component (such as a cell
(or cell line), nucleic acid, peptide or protein) has been
substantially separated, produced apart from, or purified away from
other biological components in the cell of the organism in which
the component naturally occurs, for instance, other chromosomal and
extrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides
and proteins that have been "isolated" thus include nucleic acids
and proteins purified by standard purification methods. The term
also embraces nucleic acids, peptides and proteins prepared by
recombinant expression in a cell as well as chemically synthesized
nucleic acids. The term "isolated" or "purified" does not require
absolute purity; rather, it is intended as a relative term. Thus,
for example, an isolated peptide preparation is one in which the
peptide or protein is more enriched than the peptide or protein is
in its natural environment within a cell. Preferably, a preparation
is purified such that the protein or peptide represents at least
50% of the total peptide or protein content of the preparation. In
addition, the term "isolated" can also be applied to a cell or a
cell line, for example an isolated cell or cell line is one that is
removed from its original host. Isolated cells or cell lines can be
placed back in a host, even the host from which they were
originally isolated.
[0112] Metastatic potential: The ability of cancer cells to leave
the original tumor site and migrate to other parts of the body, for
example via the bloodstream or lymphatic system. In particular
examples, the metastatic potential of a tumor cell is indicated by
movement of a tumor cell along pre-vascular endothelial tubules in
vitro (angiotropism).
[0113] Migration potential: The ability of cells, such as the cell
line disclosed herein, to translocate in response to a chemical
stimulus, such as a growth factor. Migration potential can be
determined with the assays disclosed herein.
[0114] Mimetic: The ability for a composition or an environment to
resemble another composition or environment. In particular
examples, an in vitro cell culture provides a mimetic to an in vivo
context when the cells of the culture behave in a manner that
correlates to their in vivo behavior.
[0115] Mixed cell population: A population of cells, such as cells
in culture, that contains two or more different types of cells,
such as histologically different cell lines. Examples of different
types of cells include cells of different embryonic origin (such as
cells originating from the ectoderm, endoderm, or mesoderm), cells
from different cellular locations (such as cells from epithelium,
endothelium, or stroma), cells from different tissues or organs
(such as cells from pulmonary myocardial, neural, vascular, skin,
bone, or skeletal or smooth muscle tissue).
[0116] Neoplasm or tumor: Any new and abnormal growth; particularly
a new growth of tissue in which the growth is uncontrolled and
progressive. A neoplasm, or tumor, serves no useful function and
grows at the expense of the healthy organism.
[0117] In general, tumors appear to be caused by abnormal
regulation of cell growth. Typically, the growth of cells in the
body is strictly controlled; new cells are created to replace older
ones or to perform new functions. If the balance of cell growth and
death is disturbed, a tumor may form. Abnormalities of the immune
system, which usually detects and blocks aberrant growth, also can
lead to tumors. Other causes include radiation, genetic
abnormalities, certain viruses, sunlight, tobacco, benzene, certain
poisonous mushrooms, and aflatoxins.
[0118] Tumors are classified as either benign (slow-growing and
usually harmless depending on the location), malignant
(fast-growing and likely to spread and damage other organs or
systems) or intermediate (a mixture of benign and malignant cells).
Some tumors are more common in men or women, some are more common
amongst children or elderly people, and some vary with diet,
environment and genetic risk factors.
[0119] Symptoms of neoplasms depend on the type and location of the
tumor. For example, lung tumors can cause coughing, shortness of
breath, or chest pain, while tumors of the colon can cause weight
loss, diarrhea, constipation and blood in the stool. Some tumors
produce no symptoms, but symptoms that often accompany tumors
include fevers, chills, night sweats, weight loss, loss of
appetite, fatigue, and malaise.
[0120] Blood vessels supply tumors with nutrients and oxygen. Tumor
growth is dependent on the generation of new blood vessels that can
maintain the needs of the growing tumor, and many tumors secrete
substances (angiogenic factors) that are able to induce
proliferation of new blood vessels (angiogenesis). Anti-tumor
therapies include the use of angiogenesis inhibitors, which reduce
the formation of blood vessels in the tumor, effectively starving
the tumor and/or cause the tumor to drown in its own waste.
[0121] Neovascularization: The growth of new blood vessels.
Neovascularization can be the proliferation of blood vessels in
tissue not normally containing them, or the proliferation of blood
vessels in an ischemic or otherwise damaged tissue.
Neovascularization can be pathological when it is unwanted or
mediates a pathological process, for example when it occurs in the
retina or cornea.
[0122] Neutral: A molecule is neutral when its overall charge is
neither positive nor negative. One example of a neutral
polysaccharide is agarose.
[0123] Nucleic acid: A deoxyribonucleotide or ribonucleotide
polymer in either single (ss) or double stranded (ds) form, and can
include analogues of natural nucleotides that hybridize to nucleic
acids in a manner similar to naturally occurring nucleotides. In
some examples, a nucleic acid is a nucleotide analog.
[0124] Unless otherwise specified, any reference to a nucleic acid
molecule includes the reverse complement of nucleic acid. Except
where single-strandedness is required by the text herein (for
example, a ssRNA molecule), any nucleic acid written to depict only
a single strand encompasses both strands of a corresponding
double-stranded nucleic acid. For example, depiction of a
plus-strand of a dsDNA also encompasses the complementary
minus-strand of that dsDNA. Additionally, reference to the nucleic
acid molecule that encodes a specific protein, or a fragment
thereof, encompasses both the sense strand and its reverse
complement.
[0125] Operably connected or operably linked: A first nucleic acid
sequence is operably connected to a second nucleic acid sequence
when the first nucleic acid sequence is placed in a functional
relationship with the second nucleic acid sequence. For instance, a
promoter is operably connected to a coding sequence, such as the
coding sequence of a fluorescent protein, for example GFP, if the
promoter affects the transcription or expression of the coding
sequence. Generally, operably connected DNA sequences are
contiguous.
[0126] Passaging cells: Passaging or splitting cells involves
transferring a small number of cells into a new vessel. Cells can
be cultured for a longer time if they are split regularly, as it
avoids the senescence associated with prolonged high cell density.
Suspension cultures are easily passaged with a small amount of
culture containing a few cells diluted in a larger volume of fresh
media. For adherent cultures, cells first need to be detached;
which is typically done with a mixture of trypsin-EDTA. A small
number of detached cells can then be used to seed a new
culture.
[0127] Pharmaceutical agent or drug: A chemical compound or
composition capable of inducing a desired therapeutic or
prophylactic effect when properly administered to a subject (such
as the inhibition of angiogenesis), alone or in combination with
another therapeutic agent(s) or pharmaceutically acceptable
carriers. Pharmaceutical agents include, but are not limited to,
angiogenic factors, for example bFGF, and VEGF, and anti-angiogenic
factors, such as inhibitors of bFGF, or VEGF. For example, suitable
anti-angiogenic factors include, but are not limited to, SU5416,
which is a specific VEGF-R antagonist, SU6668 which blocks the
receptors for VEGF, bFGF, and PDGF and Avastin.RTM.. See, for
example, Liu et al., Seminars in Oncology 29 (Suppl 11): 96-103,
2002; Shepherd et al., Lung Cancer 34:S81-S89, 2001.
[0128] Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers of use are conventional. Remington's
Pharmaceutical Sciences, by E.W. Martin, Mack Publishing Co.,
Easton, Pa., 15th Edition, 1975, describes compositions and
formulations suitable for pharmaceutical delivery of the
compositions disclosed herein.
[0129] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(such as powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0130] Primary cells: Cells that are cultured directly from a
subject. With the exception of some derived from tumors, most
primary cell cultures have limited lifespan. After a certain number
of population doublings cells undergo the process of senescence and
stop dividing, while generally retaining viability.
[0131] Protein coding sequence or a sequence that encodes a
peptide: A nucleic acid sequence that is transcribed (in the case
of DNA) and is translated (in the case of mRNA) into a peptide in
vitro or in vivo when placed under the control of appropriate
regulatory sequences. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxy) terminus. A coding
sequence can include, but is not limited to, cDNA from prokaryotic
or eukaryotic mRNA, genomic DNA sequences from prokaryotic or
eukaryotic DNA, and even synthetic DNA sequences. A transcription
termination sequence is usually located 3' to the coding
sequence.
[0132] Signal: A detectable change or impulse in a physical
property that provides information. In the context of the disclosed
methods, examples include electromagnetic signals, such as light,
for example light of a particular quantity or wavelength, for
example a wavelength of light emitted from a fluorescent
protein.
[0133] Recombinant: A recombinant nucleic acid or protein is one
that has a sequence that is not naturally occurring (for example a
vector, such as a vector encoding a fluorescent protein, such as
GFP and the like) or has a sequence that is made by an artificial
combination of two otherwise separated segments of sequence. This
artificial combination can be accomplished, for example, by
chemical synthesis or by the artificial manipulation of isolated
segments of nucleic acids or proteins, for example, by genetic
engineering techniques.
[0134] Test agent: Any agent that that is tested for its effects,
for example its effects on a cell. In some embodiments, a test
agent is a chemical compound, such as a chemotherapeutic agent or
even an agent with unknown biological properties.
[0135] Therapeutically effective amount: A dose sufficient to have
a therapeutic effect, for example to inhibit to some degree
advancement, or to cause regression of the disease, or which is
capable of relieving symptoms caused by the disease. For example, a
therapeutically effective amount of an angiogenesis inhibitor can
vary from about 0.1 nM per kilogram (kg) body weight to about 1
.mu.M per kg body weight, such as about 1 nM to about 500 nM per kg
body weight, or about 5 nM to about 50 nM per kg body weight. The
exact dose is readily determined by one of skill in the art based
on the potency of the specific compound, the age, weight, sex and
physiological condition of the subject.
[0136] Treating: Inhibiting the full development of a disease or
condition, for example, in a subject who is at risk for a disease
such as cancer. "Treatment" refers to a therapeutic intervention
that ameliorates a sign or symptom of a disease or pathological
condition after it has begun to develop. The term "ameliorating,"
with reference to a disease or pathological condition, refers to
any observable beneficial effect of the treatment. The beneficial
effect can be evidenced, for example, by a delayed onset of
clinical symptoms of the disease in a susceptible subject, a
reduction in severity of some or all clinical symptoms of the
disease, a slower progression of the disease, an improvement in the
overall health or well-being of the subject, or by other parameters
well known in the art that are specific to the particular disease.
A "prophylactic" treatment is a treatment administered to a subject
who does not exhibit signs of a disease or exhibits only early
signs for the purpose of decreasing the risk of developing
pathology. A personalized treatment is a treatment that is tailored
to a particular subject based on the characteristics of the subject
and optionally also the particular disease.
[0137] Transduced Transformed, Transfected: A virus or vector
"transduces" or "transfects" a cell when it transfers nucleic acid
into the cell. A cell is "transformed" by a nucleic acid transduced
into the cell when the DNA becomes stably replicated by the cell,
either by incorporation of the nucleic acid into the cellular
genome, or by episomal replication. As used herein, the term
transformation encompasses all techniques by which a nucleic acid
molecule might be introduced into such a cell, including
transfection with viral vectors, transformation with plasmid
vectors, and introduction of naked DNA by electroporation,
lipofection, and particle gun acceleration.
[0138] Tumor spheroid colony: An in vitro clonal expansion of a
single parental tumor cell and which has the three dimensional
characteristics of an in vivo tumor. Tumor spheroids include not
only morphogenic capacities and histotypic reorganization of an in
vivo tumor, but also maintain its functional activities and gene
expression patterns (Hauptmann et al., Int. J. Cancer. 61:819-825,
1995). Additionally, tumor spheroids provide a simple geometry for
modeling the effects of anticancer treatments (Buffa et al., Int J
Radiat Oncol Biol Phys. 49:1109-1118, 2001).
[0139] Tumor biopsy: A section of tumor tissue removed from a whole
tumor, for example a tumor from a subject. Tumor biopsies contain a
mixed population of cells including tumor and stromal cells. In
particular examples, a tumor biopsy is a tumor tissue plug, which
is a section of a punch biopsy (periphery, mid-section, and central
core) from a tumor, such as a tumor from a subject or a xenograft
tumor.
[0140] Tubule formation potential: The ability of a cell line to
form a tube-like structure in vitro, for example a structure
similar to a blood vessel, such as a capillary. Tubule formation
potential can be determined by determining the pattern displayed by
cells which have been induced to form tubules, for example by
determining the pattern of fluorescence from cells expressing
fluorescent proteins, such as the cell lines disclosed herein. In
particular examples, tubule formation potential is a characteristic
of endothelial cells. In other examples it is a characteristic of
other cell types including certain tumor cells lines (e.g.
MDA-MB-435) and pericytes.
[0141] Vector: A nucleic acid molecule that can be introduced into
a cell, thereby producing a transformed cell. A vector can include
nucleic acid sequences that permit it to replicate in a cell, such
as an origin of replication, for example a SV40 origin for
replication in mammalian cells and a pUC origin of replication for
propagation in E. coli, and can also include one or more selectable
marker genes, such as antibiotic resistance genes, such as the
kanamycin resistance gene and the neomycin resistance gene. Other
genetic elements and protein coding sequences can also be included
in the vector, such as sequences encoding a fluorescent protein,
for example GFP or the like, promoters for the expression of
proteins, such as the SV40 early promoter and the immediate early
promoter of cytomegalovirus (PCMV IE), Kozak translation initiation
sequences, and polyadenylation signals, such as the SV40
polyadenylation signal.
III. Description of Several Embodiments
[0142] Understanding biological processes that underlie cellular
organization, such as in organ development and the pathogenesis of
diseases such as cancer, would be facilitated by methods for
studying these complex interactions in vitro. In vitro assays are
needed to investigate the relationship between multiple cellular
components involved in the biological processes (such as
angiogenesis), investigate new combinatorial approaches to boost
the efficiency of existing therapeutics, and to facilitate the
discovery of new potential single and/or combination drugs.
Disclosed herein is an in vitro assay that meets these needs.
[0143] A basic component of this in vitro assay is the immortalized
fluorescent cell lines disclosed herein. The disclosed fluorescent
cell lines represent an array of different cell types, such as cell
types that contribute to biological processes, such as
angiogenesis, in vivo. The disclosed fluorescent cell lines are
derived from different anatomical origins which are known to be
relevant during the angiogenesis process. In particular examples,
the cells are mammalian cell lines, such as human cell lines, and
may be immortalized cell lines. The disclosed cell lines have been
stably transfected with mammalian expression plasmids that
constitutively express different fluorescent proteins, for example
green fluorescent protein and related florescent proteins, such as
yellow fluorescent protein, red fluorescent protein, cyan
fluorescent protein and the like.
[0144] The fluorescence signals produced by the cell lines
expressing green fluorescent protein and related fluorescent
proteins can be used for real time direct estimation of cell
numbers, because the fluorescent signal from a population of cells
stably and constitutively expressing a fluorescent protein is
proportional to the number of such cells. In addition, because
these fluorescent cell lines emit a detectable signal that can be
localized in space, the individual cells can be localized in space,
for example to determine a pattern of fluorescence attributable to
the cells. These features can be used in a number of different
assays including growth assays, migration assays, tubule formation
assays, cell viability assays, and the like. The use of fluorescent
cells in such assays, for example the assays disclosed herein, not
only eliminates the need for expensive commercially available kits
(for example kits needed to generate and end point readable signal,
for example a chemical agent that renders the cell, or cell
morphology detectable) but also simplifies procedures by
considerably shortening the protocol time, because no additional
agents need to be added to generate a signal. Additionally, because
no detection reagents have to be added to the cells in culture the
fluorescent cell lines and assays disclosed herein avoided
putatively harmful interactions between those chemicals and the
cellular components under study.
[0145] Deregulation of angiogenesis plays a major role in a number
of human diseases. A dramatic increase in the research effort in
the field of angiogenesis has resulted in a substantial
understanding of the angiogenic process and subsequently the
development of new therapeutics to modulate angiogenesis. Although
angiogenesis inhibitors are among the most promising drug
candidates for cancer, the existing "single drug, non-personalized"
approach has proven to be problematic regarding extending patient
survival time and the development of drug resistant tumor
clones.
[0146] The inability to develop more successful therapies is
hampered by insufficient knowledge about the interactions between
the multiple cellular components involved in the angiogenesis
process and the inability to evaluate angiogenic potential in
individual patients. Hence, one of the major problems confronting
clinicians today is the ability to assess angiogenic/antiangiogenic
therapy effectiveness in a mixed cell environment, such as the
mixed cell environment responsible for angiogenesis. The disclosed
assays are particularly suited to the investigation of angiogenesis
especially in a mixed cell environment, such as a mixed cell
environment approximating in vivo conditions (for example a mixed
cell population shown in FIG. 14).
[0147] The disclosed assays can be used to monitor the effects of a
drug, such as an existing angiogenesis inhibitor or other
chemotherapeutic agent, on a patient sample, for example, by
determining the effect of a patient sample, patient serum, plasma,
tumor cells, on the fluorescent cell lines disclosed herein. Using
the disclosed assays, a patient sample from a patient with cancer
could have elevated level of proangiogenic factors, such as growth
factors. Using the disclosed assays, it is possible to monitor the
patient, via monitoring a sample obtained from a patient, to
determine if a particular treatment is having a desired effect, for
example reducing the level of proangiogenic factors present in the
sample. Thus, a particular therapy can be developed for an
individual patient, for example a personalized combinatorial drug
therapy which would be effective for that individual.
[0148] Also disclosed herein are methods for monitoring angiogenic
or metastatic potential of tumor cells, the methods comprising
preparing a three-dimensional co-culture that is comprised of three
layers. The first layer comprises a neutral polysaccharide polymer
gel in contact with the bottom of the culture dish. The second
layer is on top of the first layer and comprises a solidified gel
matrix, endothelial cells that are dispersed in the solidified gel
matrix; and tumor cells comprising either a tumor spheroid colony
or a sample of a tumor biopsy, and which are also suspended in the
solidified gel matrix, and a third layer comprising culture medium.
Angiogenic or metastatic potential of tumor cells is monitored by
incubating the three-dimensional co-culture; and detecting at least
one of endothelial cell proliferation, endothelial cell tubule
formation or tumor cell angiotropism of the cells in the second
layer. In particular examples, the neutral polysaccharide polymer
gel comprises agarose. In other examples, the endothelial cells
stably and constitutively express a fluorescent protein. In still
other examples, the tumor cells stably and constitutively express a
fluorescent protein with a different emission spectrum from the
fluorescent protein expressed by the endothelial cells. In yet
further examples, the second layer further comprises at least one
additional mammalian cell type dispersed in the solidified gel
matrix, such as a cell type selected from the group consisting of
macrophage, mast cell, fibroblast, adipocyte, and pericyte. In
still further examples, the additional mammalian cell type stably
and constitutively expresses a fluorescent protein with a different
emission spectrum from the fluorescent protein expressed by the
endothelial cells.
[0149] In particular examples of the disclosed methods for
monitoring angiogenic or metastatic potential of tumor cells, the
first, second, or third layer further comprises at least one test
agent, which in some examples is a known or potential inhibitor or
promoter of angiogenesis or metastasis.
[0150] In other examples of the disclosed methods, the tumor cells
are derived from a subject and the first, second, or third layer
further comprises at least one test agent that has been
administered to the subject as part of a cancer treatment.
[0151] Further disclosed herein are methods of testing the efficacy
of an anti-angiogenic or anti-metastatic cancer treatment for a
subject, comprising monitoring angiogenic or metastatic potential
of tumor cells by the above described methods utilizing a three
dimensional co-culture, wherein the tumor cells are derived from
the subject and the first, second, or third layer comprises at
least one test agent that is a candidate anti-cancer treatment.
[0152] Additionally disclosed herein are methods of selecting a
personalized anti-angiogenic or anti-metastatic treatment for
cancer in a subject comprising preparing multiple three-dimensional
co-cultures, each co-culture comprising a first layer comprising a
neutral polysaccharide polymer gel in contact with the bottom of a
culture dish; a second layer on top of the first layer, comprising:
a solidified gel matrix; endothelial cells dispersed in the
solidified gel matrix; and tumor cells comprising either a tumor
spheroid colony or a sample of a tumor biopsy, suspended in the
solidified gel matrix; and a third layer on top of the second layer
comprising culture medium, wherein all but one of the co-cultures
further comprises at least one test agent comprising an
anti-angiogenic or anti-metastatic compound in the first, second,
or third layers. A personalized anti-angiogenic or anti-metastatic
treatment for cancer in a subject is selected by incubating the
three-dimensional co-cultures; detecting at least one of
endothelial cell proliferation, endothelial cell tubule formation
or tumor cell angiotropism of the cells in the second layer; and
selecting the at least one test agent having the greatest effect on
at least one of endothelial cell proliferation, endothelial cell
tubule formation or tumor cell angiotropism in comparison to
endothelial cell proliferation, endothelial cell tubule formation
or tumor cell angiotropism in the cells of the co-culture without
the test agent. In particular examples, the neutral polysaccharide
polymer gel comprises agarose. In some examples, the endothelial
cells stably and constitutively express a fluorescent protein. In
other examples, the tumor cells stably and constitutively express a
fluorescent protein with a different emission spectrum from the
fluorescent protein expressed by the endothelial cells. In
particular examples, the second layer further comprises at least
one additional mammalian cell type dispersed in the solidified gel
matrix, which in some examples, stably and constitutively expresses
a fluorescent protein with a different emission spectrum from the
fluorescent protein expressed by the endothelial cell, and in
further examples is a cell type selected from the group consisting
of macrophage, mast cell, fibroblast, adipocyte, and pericyte.
A. Assays
[0153] Aspects of this disclosure relate to an assay method, such
as a multiplex assay method, for evaluating cellular activity, for
example the cellular activity of the disclosed fluorescent cell
lines. The disclosed method involves providing an in vitro culture
of one or more cell lines, such as an in vitro mixture of one or
more of the fluorescent cell lines disclosed herein, for example
cell lines of different histological types, for example different
types of mammalian cells, such as mammalian somatic cells. Examples
include porcine aortic endothelial cell line PAE, human lymphatic
endothelial cell line LEC-1, human microvascular endothelial cell
line HMEC-1, or rhesus macaque choroidal endothelial cell line
RF/6A (ATTC CRL-1780) (which has marker characteristics of a
pericyte line (SMA, TIMP-3, NG2, PDGFR-.beta., etc); epithelial
cell lines, such as human adenocarcinoma cell line A549;
adenocarcinoma cell lines, such as human breast adenocarcinoma cell
line MCF7; or mast cell cell lines, such as human mast cell line
HMC-1, among others. These particular cell lines are examples of
different cell lines believed to play a role in angiogenesis.
[0154] In some embodiments, an in vitro cell line mixture is
provided which contains a first isolated mammalian cell line stably
and constitutively expressing a first fluorescent protein and one
or more additional isolated mammalian cell lines stably and
constitutively expressing fluorescent proteins having an emission
spectrum different from the emission spectrum of the first
fluorescent protein and having different histological types. The in
vitro cell line mixture is cultured and the cellular activity of
the first isolated mammalian cell line or one or more addition
isolated mammalian cell lines present in the culture is assessed by
quantifying fluorescence or detecting a pattern of fluorescence
from the first fluorescent protein or the fluorescent protein
expressed by the one or more additional mammalian cell lines. By
providing a mixture of isolated mammalian cell lines of different
histological types each expressing a different fluorescent protein
with different emission spectra, it is possible to assess the
cellular activity of the multiple cell lines present in the
mixture, for example simultaneously or serially. Thus, in some
embodiments, the cellular activity of the first isolated mammalian
cell line present in the mixture is assessed and the cellular
activity of the additional isolated mammalian cell lines present in
the cell line mixture is assessed by quantifying fluorescence or
detecting a pattern of fluorescence from the first fluorescent
protein and the fluorescent proteins present in the additional
isolated cell lines present in the culture.
[0155] In some embodiments, an in vitro cell line mixture is
provided which contains a first isolated mammalian cell line stably
and constitutively expressing a first fluorescent protein and a
second isolated mammalian cell line stably and constitutively
expressing a second fluorescent protein, in which the first and
second fluorescent proteins have different emission spectra and the
first isolated mammalian cell line and the second isolated
mammalian cell line are different cell lines, for example cell
lines of different histological types. In some embodiments, the
cellular activity of the first isolated mammalian cell line present
in the mixture is assessed by quantifying fluorescence (for example
by quantifying the fluorescence intensity at a particular
wavelength, such as the emission maxima) or detecting a pattern of
fluorescence from the first fluorescent protein (such as the
pattern of fluorescence of the fluorescent proteins present in the
fluorescent cell line, for example the location of the fluorescence
in two or three dimensional space). In some embodiments, the
cellular activity of the first isolated mammalian cell line present
in the mixture is assessed and the cellular activity of the second
isolated cell line present in the cell line mixture is assessed by
quantifying fluorescence or detecting a pattern of fluorescence
from the first fluorescent protein and the second fluorescent
protein.
[0156] In some embodiments, an in vitro cell line mixture is
provided which contains at least three, such as three, four, five,
six or more, different isolated mammalian cell lines, such as
different cell lines each having a different histological type,
wherein each isolated mammalian cell line stably and constitutively
expresses a different fluorescent protein having an emission
spectrum distinguishable from the other fluorescent proteins. In
such a mixture each cell line is uniquely associated with a
particular fluorescent protein having a different emission spectrum
from the other fluorescent proteins so that the individual cell
lines present in the mixture can be distinguished, such that the
fluorescence from the individual cell line can be quantified and/or
the pattern of fluorescence detected. Thus, the quantified
fluorescence or pattern of fluorescence attributable to a specific
fluorescent cell line can be determined.
[0157] In some embodiments, assessing cellular activity includes
determining the growth rate, migration potential, and/or tubule
formation potential of the first isolated mammalian cell line
and/or additional isolated cell lines present in the in vitro
mixture using the quantified fluorescence or the pattern of
fluorescence from the fluorescent proteins in the mixture, such as
first fluorescent protein and/or the additional fluorescent
proteins, such as a second, third forth, fifth, six, etc.
fluorescent proteins present in the in vitro mixture. In some
examples, the number of dead cells present in the mixture is
determined by determining the quantified fluorescence present in
the media of the cell-mixture. Exemplary methods for determining
the growth rate, cell death, migration potential, or tubule
formation potential of the isolated cell lines disclosed herein are
given below.
[0158] i. Growth Assay
[0159] Using the disclosed fluorescent cell lines, a real time
growth assay has been developed and applied to mono- or
multiple-cell cultures (co-culture). As disclosed herein the
fluorescence signal emitted by a culture of the disclosed
fluorescent cell lines is proportional to the number of fluorescent
cells present in the culture (see for example FIG. 2). In other
words, the fluorescence signal, for example measured as the
intensity of the emission maxima, from a population of fluorescent
cells of one type in a culture will double as the number of
fluorescent cells of that type in the culture doubles. Conversely,
the fluorescence signal, for example measured as the intensity of
the emission maxima, from a population of cells of one type in a
culture will be reduced to half if the number of cells of that type
in the culture is divided in half. These properties can be used to
measure the effect of an exogenous agent, such as one or more
additional cell lines, or a test agent, on the fluorescent cells in
culture. It should be noted that at some point the total
fluorescence of a culture may reach signal saturation, such that
the signal reaches a plateau as a function of cell number.
[0160] In some embodiments, the effect of an additional cell line
(for example a different cell line) on a first fluorescent cell
line is determined (this can be extended to multiple cell lines and
even one or more fluorescent cell lines, or combinations thereof,
for example in a multiplex assay). For example, as shown in FIG. 4,
the effect of the yellow fluorescent PAE cell line on the red
fluorescent MCF7 cell line has been measured.
[0161] In some embodiments, the disclosed growth assay is used to
assess if the presence of one or more additional cell lines, such
as one or more of the fluorescent cell lines disclosed herein,
affects the growth rate of a fluorescent cell line of interest. A
fluorescent cell line of interest can be grown in co-culture with
one or more additional cell lines and the growth of the fluorescent
cell line of interest can be determined. For example, using the
difference between the fluorescence signal of the fluorescent cell
line of interest and a control indicates that the one or more
additional cell lines can be used to determine if the one or more
additional cell lines affects the growth rate of the fluorescent
cell line of interest.
[0162] In some embodiments, the difference between the fluorescence
signal (such as the intensity of the fluorescence signal at a
particular wavelength, for example the emission maxima of the
fluorescence signal) attributable to the fluorescent cell line of
interest grown in co-culture with one or more additional cell lines
relative to a control is at least about 10%, meaning that the
growth rate of the cell line of interest is either reduced or
increased by at least about 10%, such as at least about 20%, at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, at least about 100%, at least about 150%, at least about 200%,
at least about 250%, at least about 300%, at least about 350%, at
least about 400%, at least about 500%, or greater then 500%. In
some embodiments, the difference is a statistically significant
difference. Thus, the presence of one or more additional cell lines
can induce a statistically significant difference in the growth
rate of a fluorescent cell line of interest, as compared to the
control, such as value indicative of the basal rate of growth of
the fluorescent cell line, or the fluorescent cell line of interest
grown in the absence of the other cells or cell lines, for example
grown in mono-culture. In some examples, the additional cell line
(or additional cell lines) will have a negative impact on the first
fluorescent cell line, such that the number of cells of the first
fluorescent cell line is reduced as a function of time relative to
a control. In some examples, the additional cell line (or
additional cell lines) will have a positive impact on the first
fluorescent cell line, such that the number of cells of the first
fluorescent cell line present in a cell culture increases as a
function of time relative to a control. It is also contemplated
that the fluorescent cell line of interest can be co-cultured with
primary cells, such as primary cells obtained from a subject, for
example tumor cells, and the effect of the primary cells on the
growth rate of the fluorescent cell line of interest
determined.
[0163] In some embodiments, multiple fluorescent cell lines are
grown in co-culture. Thus, the effect of each fluorescent cell line
on the other fluorescent cell line(s) present can be determined,
for example in a multiplex assay. For example, using appropriate
filters or FACS analysis among other techniques, fluorescent cell
lines expressing different fluorescent proteins, such as red,
green, yellow, cyan and the like fluorescent proteins can be
discriminated and the fluorescent signal attributable to the
different cell lines determined. Thus, the growth rates of
individual fluorescent cell lines can be determined from a
mono-culture and/or a co-culture of two or more fluorescent cell
lines. Such analysis greatly enhances the information that can be
obtained about the individual fluorescent cell lines.
[0164] In addition to determining the effect of cell lines on a
fluorescent cell line of interest, the growth assays can be used to
determine if an exogenous agent, such as a test agent, for example
a chemical agent, affects the growth of a fluorescent cell line of
interest. This can also be extended to multiple cell lines (either
fluorescent or not grown in co-culture, for example in a multiplex
assay). In some embodiments, the disclosed growth assay is used to
determine if an exogenous agent, such as a test agent (for example
a potential modulator of angiogenesis, such as a potential
inhibitor of angiogenesis or a potential stimulator of
angiogenesis), growth factor, patient sample, etc. affects the
growth rate of a fluorescent cell line of interest, such as one or
more of the fluorescent cell lines disclosed herein. In addition,
the differential effect of the exogenous agent on the different
cell lines can be determined, as can the combinatorial effect of
the exogenous agent and the cells on a cell line of interest.
[0165] A fluorescent cell line of interest can be contacted with an
exogenous agent and the impact of the exogenous agent on the growth
of the fluorescent cell line of interest can be determined. For
example, a difference between the fluorescence signal of the
fluorescent cell line of interest and a control indicates that the
exogenous agent, such as a test agent (for example a potential
modulator of angiogenesis, such as a potential inhibitor of
angiogenesis or a potential stimulator of angiogenesis), growth
factor, patient sample, different cell line, etc. is a modulator
(such as an inducer or inhibitor) of angiogenesis. Thus, in several
embodiments, one or more of the disclosed fluorescent cell lines
growing in culture are contacted with a test agent (or test agents)
to determine if the test agent is a modulator of angiogenesis.
Exemplary test agents are given below. Following contact with the
exogenous agent, the fluorescence of the culture can be measured
versus time and/or concentration to determine the impact of the
exogenous agent on the one or more fluorescent cell lines present
in the culture. For example, the fluorescence signal generated by a
fluorescent cell line of interest (such as the intensity of the
fluorescence signal at a particular wavelength, for example the
emission maxima of the fluorescence signal) can be measured to
determine if the fluorescence signal attributable to the
fluorescent cell line of interest (such as the intensity of the
fluorescence signal at a particular wavelength, for example the
emission maxima of the fluorescence signal) is increasing as a
function of concentration of the exogenous agent, time, or both,
for example by comparison with a control, such as a value
indicative of the basal rate of growth of the fluorescent cell line
of interest or the fluorescent cell line of interest not contacted
with the exogenous agent. In several embodiments, the control is a
known value indicative of normal growth of the fluorescent cell
line of interest, for example the doubling time of cellular number.
In some embodiments, the control is the fluorescence signal of a
culture of cells (typically, but not necessarily, a culture of the
fluorescent cell line of interest) not contacted with the exogenous
agent.
[0166] In some embodiments, an exogenous agent, such as a test
agent, decreases the growth rate of the fluorescent cell line of
interest. A test agent exhibiting such an activity is identified as
an inhibitor of angiogenesis and would be of use in treating a
disease or condition in which normal angiogenesis is increased, for
example cancer. In some embodiments, a decrease in the growth rate
of the fluorescent cell line of interest relative to a control is
at least about a 30%, at least about a 40%, at least about a 50%,
at least about a 60%, at least about a 70%, at least about a 80%,
at least about a 90%, at least about a 100%, at least about a 150%,
at least about a 200%, at least about a 250%, at least about a
300%, at least about a 350%, at least about a 400%, at least about
a 500% decrease. Because the fluorescence signal attributable to a
fluorescent cell line of interest is proportional to the number of
cells of the cell line of interest present, the percentage decrease
can be measured as a percentage decrease in the fluorescent signal,
for example the fluorescence intensity at a particular wavelength,
such as the emission maxima, attributable to the cell line of
interest. In additional embodiments, the decrease is a
statistically significant decrease as compared to a control.
[0167] In other embodiments, the exogenous agent, such as a test
agent, increases the growth of the fluorescent cell line as
compared to a control. A test agent exhibiting such an activity is
identified as a stimulator of angiogenesis and would be of use in
treating a disease or condition in which normal angiogenesis is
inhibited. In some embodiments, an increase in the growth of the
fluorescent cell line is at least about a 30%, at least about a
40%, at least about a 50%, at least about a 60%, at least about a
70%, at least about a 80%, at least about a 90%, at least about a
100%, at least about a 150%, at least about a 200%, at least about
a 250%, at least about a 300%, at least about a 350%, at least
about a 400%, at least about a 500% increase as compared to
control. Because the fluorescence signal attributable to a
fluorescent cell line of interest is proportional to the number of
cells of the cell line of interest present, the percentage increase
can be measured as a percentage increase in the fluorescent signal,
for example the fluorescence intensity at a particular wavelength,
such as the emission maxima, attributable to the cell line of
interest. In additional embodiments, the increase is a
statistically significant increase as compared to a control.
[0168] ii. Tubule Formation Assay
[0169] Similarly to the growth assay, cultures of fluorescent cell
lines expressing different fluorescent proteins, such as the
fluorescent cell lines disclosed herein can be applied to tubule
formation assays. Formation of new blood vessels is fundamental to
angiogenesis and is the focus of many drug screening and cell
signaling studies. Blood vessel development is a significant event
in the development and growth of solid tumors, and is involved in
wound healing, retinopathy and macular degeneration. The disclosed
fluorescent cell lines, and in particular the disclosed endothelial
fluorescent cell lines are ideal for use in assays for assessing
the degree of blood vessel formation using in vitro cell culture
assays (see for example Auerbach et al. 2003. Clinical Chemistry
49:1, 32-40. 2. Taraboletti and Giavazzi, 2004 EJC. 40, 881-889).
Because no fluorescent/colorimetric staining is needed, the tubule
formation assay can be followed over time and can be directly
visualized used in existing instrumentation, such as the BD
Pathway.TM. Bioimager (BD Bioscience, San Jose, Calif.). This
allows for the study of the interaction between different cells
types in this angiogenesis in vitro assay. In addition, the tubule
formation potential can also be determined for a co-culture of a
fluorescent cell line of interest with primary cells, such as
primary cells obtained from a subject, for example tumor cells.
[0170] Tubule Formation assays are typically based on the ability
of endothelial cells, such as the fluorescent endothelial cells
disclosed herein, to form distinct blood-vessel-like tubules in an
extracellular matrix (such as BD Matrigel.TM. Matrix available from
BD Bioscience, BME available from Trevigen, or Geltrex.TM.
available from Invitrogen.RTM., and the like). In other examples,
tubule formation assays involve observation of the tubule forming
potential of certain tumor cell lines (e.g. MDA-MB-435) and
pericytes. The cells are visualized under microscopy, such as
fluorescence microscopy in the case of the fluorescent cell
disclosed herein, and the ability of a fluorescent cell line of
interest to form tubules (also called the tubule formation
potential) is determined. The determination of tubule formation can
be performed by manual tracing or by automated confocal imaging
system, for example using a BD Pathway.TM. Bioimager in conjunction
with AngioApplication.TM.. Using the disclosed fluorescent cell
lines, tubule formation assays can be performed on live cells, for
example to avoid artifact that may arise from fixation artifacts,
such as the disruption of tubules. Several parameters can be
measured in tubule formation assays, such as the total area of the
tubules, the total number of tubules, number of nodes, number of
branch points, the number of tubes per node, and/or node area. In
some embodiments, the tubule formation potential is determined by a
computer implemented method, for example using the program
AngioApplication.TM..
[0171] The fluorescent cell lines disclosed herein can be used to
determine the effects of an exogenous agent, such as cell lines and
test agents, on tubule formation. In some embodiments, multiple
fluorescent cell lines are grown in co-culture. Thus, the effect of
each fluorescent cell line on the other fluorescent cell line(s)
present can be determined, or the differential effect of an
exogenous agent, such as a test agent, or patient sample, on the
different cell lines can be assessed in a multiplex assay. For
example using appropriate filters, the fluorescent signal from
fluorescent cell lines expressing different fluorescent proteins,
such as red, green, yellow, cyan fluorescent proteins can be
discriminated and the fluorescent signal attributable from the
different fluorescent cell lines determined. Thus, the tubule
formation potential of individual cell lines can be determined from
a mono-culture or even a co-culture, for example a co-culture of
more than one fluorescent cell line.
[0172] When grown in co-culture, a difference between the tubule
formation potential of the fluorescent cell line of interest from a
control, such a mono-culture of the fluorescent cell line of
interest indicates that the other cell line(s) is a modulator of
angiogenesis, as evidenced by the difference in tubule formation
potential. In some embodiments, the difference between the tubule
formation potential, for example as measured by the number of least
one of the total area of the tubules, the total number of tubules,
number of nodes, number of branch points, the number of tubes per
node, or node area formed in the co-culture of the fluorescent cell
line of interest relative to a control is at least about 10%, such
as at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 100%, at least about
150%, at least about 200%, at least about 250%, at least about
300%, at least about 350%, at least about 400%, at least about
500%, or greater then 500%. In some embodiments, the difference is
a statistically significant difference. Thus, a cell line can
induce a statistically significant difference in the tubule
formation potential of a fluorescent cell line of interest, such as
one of the disclosed fluorescent cell lines. Taking a combinatorial
approach the impact of multiple different cell lines either alone
or in combination on the tubule formation potential of the
fluorescent cell line of interest can be determined. In some
examples, the presence of one or more additional cell lines
increases the tubule formation potential of the fluorescent cell
line of interest, for example as measured by the total area of the
tubules, the total number of tubules, number of nodes, number of
branch points, the number of tubes per node, or node area formed by
the fluorescent cell line of interest. These cell lines would be
identified as positive regulators of angiogenesis. In some
examples, the presence of one or more additional cell lines
decreases the tubule formation potential of the fluorescent cell
line of interest, for example as measured by the total area of the
tubules, the total number of tubules, number of nodes, number of
branch points, the number of tubes per node, or node area formed by
the fluorescent cell line of interest. These cell lines would be
identified as negative regulators of angiogenesis.
[0173] Utilizing the disclosed fluorescent cell lines, tubule
formation assays can also be used to screen for a biological effect
of a test agent, such as the effect of potential modulators of
angiogenesis. In some embodiments, a fluorescent cell line of
interest (or multiple cell lines of interest in a multiplex assay)
can be contacted with an exogenous agent, such as a cell line or
test agent, and the impact of the exogenous agent on tubule
formation potential can be determined. Exemplary test agents are
given below. For example using the difference between the total
area of the tubules, the total number of tubules, number of nodes,
number of branch points, the number of tubes per node, and/or node
area between a fluorescent cell line of interest and a control are
used to determine if an exogenous agent, such as a test agent,
impacts the ability of a fluorescent cell line of interest to form
tubules. A difference between the tubule formation potential of a
fluorescent cell line of interest contacted with an exogenous agent
and a control (such as a control culture exposed to the exogenous
agent) indicates that the exogenous agent is a modulator of
angiogenesis. In some embodiments, the difference between the
tubule formation potential of the fluorescent cell line contacted
with an exogenous agent relative to a control is at least about
10%, such as at least about 20%, at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, at least about 100%, at least
about 150%, at least about 200%, at least about 250%, at least
about 300%, at least about 350%, at least about 400%, at least
about 500%, or greater then 500%. In some embodiments, the
difference is a statistically significant difference. Thus, an
exogenous agent can induce a statistically significant difference
in the tubule formation potential of the fluorescent cell line of
interest contacted with the test agent, as compared to the control,
such as the fluorescent cell line of interest not contacted with
the exogenous agent.
[0174] In one embodiment, the exogenous agent decreases ability of
a fluorescent cell line of interest to form tubules. A test agent
exhibiting such an activity is identified as an inhibitor of
angiogenesis and would be of use in treating a disease or condition
in which normal angiogenesis is increased, for example cancer. In
some embodiments, a decrease in the tubule formation potential of
the fluorescent cell line of interest is at least about a 30%, at
least about a 40%, at least about a 50%, at least about a 60%, at
least about a 70%, at least about a 80%, at least about a 90%, at
least about a 100%, at least about a 150%, at least about a 200%,
at least about a 250%, at least about a 300%, at least about a
350%, at least about a 400%, at least about a 500% decrease as
compared to control. In additional embodiments, the decrease is a
statistically significant decrease as compared to a control.
[0175] In another embodiment, the exogenous agent increases the
potential of a fluorescent cell line of interest to form tubules as
compared to a control, such as the fluorescent cell line of
interest that has not been contacted with the exogenous agent. A
test agent exhibiting such an activity is identified as a
stimulator of angiogenesis and would be of use in treating a
disease or condition in which normal angiogenesis is inhibited. In
some embodiments, an increase in the growth of the fluorescent cell
line is at least about a 30%, at least about a 40%, at least about
a 50%, at least about a 60%, at least about a 70%, at least about a
80%, at least about a 90%, at least about a 100%, at least about a
150%, at least about a 200%, at least about a 250%, at least about
a 300%, at least about a 350%, at least about a 400%, at least
about a 500% increase as compared to control. In additional
embodiments, the increase is a statistically significant increase
as compared to a control.
[0176] iii. Migration Assay
[0177] Another assay that can be used with the disclosed
fluorescent cell lines is a cellular migration assay. These assays
assess cellular migration in a controlled environment, such as a
differential migration of the cell line, (or multiple cell lines in
a multiplex assay) as determined by fluorescent signals (such as
the intensity of a fluorescent signal of a particular color, or at
a particular wavelength, such as the emission maxima of a
particular fluorescent protein) in a location that is associated
with migration to a particular location.
[0178] In one example, a cellular migration assay determines the
ability of cells to migrate up or down a chemical gradient.
Migration "up" a chemical gradient refers to migration from a
region of lower chemical concentration of a chemical to a region of
higher chemical concentration (for example migration toward a
higher concentration of a chemical attractant or away from a lower
concentration of the chemical attractant), while migration "down" a
chemical gradient refers to migration from a region of higher
chemical concentration to a region of lower chemical concentration
(for example migration away from a higher concentration of a
chemical repellent toward a lower concentration of the chemical
repellent). Such migration is typically referred to as chemotaxis.
Cells, such as the fluorescent cell lines disclosed herein, respond
to chemical signals in their environment by the stimulation of
concerted movement either toward a chemical attractant or away from
a chemical repellent. In mammalian cells, such as the fluorescent
cell lines disclosed herein, typical chemo-attractants include
factors excreted by cells, for example factors found in serum, such
as growth factors and the like.
[0179] The disclosed fluorescent cells can be used in any cell
migration assay format, such as the ChemoTx.TM. system (NeuroProbe,
Rockville, Md.) transwell system or any other suitable device or
system. In some examples, a cell migration assay is carried out as
follows. A culture of a fluorescent cell line of interest (such as
any of the disclosed fluorescent cell lines or a mixture of such as
fluorescent cell lines) is placed into a first chamber of a cell
migration apparatus, and an exogenous agent (such as a
chemoattractant) is placed in a second chamber that is adjacent to
and in communication with the first chamber of the cell migration
apparatus, so that cellular migration from the first chamber to the
second chamber can be detected. The chambers may be separated by a
membrane or filter that permits passage of cells from one chamber
to the other chamber. The membrane or filter is configured such
that the passive diffusion of the cells across the membrane or
filter is minimized. In one example, the first chamber is the upper
chamber of the apparatus and the second chamber is the lower
chamber of the apparatus. In some examples the upper chamber is
omitted and the cells are placed directly on a membrane or filter
in communication with the lower chamber. The ability of a
fluorescent cell line such as the fluorescent cell lines disclosed
herein to be stimulated to migrate can be determined. Typical
migration assays have "unknown" sites (with cell suspension above
the filter and a solution containing the chemotactic factor below
it) and "negative control" sites (with cell suspension above the
filter and suspension media, but no chemotactic factor, below).
Random migration of unstimulated cells will account for some of the
cells that pass through the filter. Migrated cells at the negative
control sites show the extent of unstimulated random migration,
which can then be differentiated from chemotactic migration, or
chemotaxis. Cells that stably express a fluorescent protein, such
as the disclosed fluorescent cells can be read in a microplate with
a fluorescence microplate reader. Thus, the number of fluorescent
cells present in either the upper chamber, lower chamber, or both
chambers can be determined, for example as a function of time.
[0180] In some embodiments, the disclosed migration assay is used
to determine if an exogenous agent affects or differentially
affects the migration of one or more of the fluorescent cell line
of interest, such as one or more of the fluorescent cell lines
disclosed herein. A fluorescent cell line of interest can be
contacted with exogenous agent and the impact of the exogenous
agent on the migration of the fluorescent cell line of interest can
be determined. For example, a difference between the number of
cells that migrate between a fluorescent cell line of interest
contacted with an exogenous agent and a control indicates that the
exogenous agent, such as a test agent, cell line, growth factor,
etc., is a modulator of cellular migration. In other embodiments,
differences in migration among different cell lines in the
migration assay provide an indication of differential migration of
the different cell lines in response to the exogenous agent. In
some embodiments, the difference between the number of cells that
migrate of the fluorescent cell line contacted with an exogenous
agent relative to a control, (for example as measured by the
fluorescence intensity of a fluorescent protein stably and
constitutively expressed by the cells) is at least about 10%, such
as at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, at least
about 80%, at least about 90%, at least about 100%, at least about
150%, at least about 200%, at least about 250%, at least about
300%, at least about 350%, at least about 400%, at least about
500%, or greater then 500%. In some embodiments, the difference is
a statistically significant difference. Thus, an exogenous agent
can induce a statistically significant difference in the migration
of a fluorescent cell line of interest contacted with the exogenous
agent, as compared to the control, such as the fluorescent cell
line of interest not contacted with the exogenous agent or a
different cell line that has been mixed with the cell line of
interest.
[0181] In one embodiment, the exogenous agent decreases the ability
of a fluorescent cell line of interest to migrate. A test agent
with such an activity is identified as an inhibitor of angiogenesis
and would be of use in treating a disease or condition in which
normal angiogenesis is increased, for example cancer. In some
embodiments, a decrease in migration of the fluorescent cell line
of interest is at least about a 30%, at least about a 40%, at least
about a 50%, at least about a 60%, at least about a 70%, at least
about a 80%, at least about a 90%, at least about a 100%, at least
about a 150%, at least about a 200%, at least about a 250%, at
least about a 300%, at least about a 350%, at least about a 400%,
at least about a 500% decrease as compared to control. In
additional embodiments, the decrease is a statistically significant
decrease as compared to a control.
[0182] In another embodiment, the exogenous agent increases the
migration of the fluorescent cell line of interest as compared to a
control. A test agent with such as activity is identified as a
stimulator of angiogenesis and would be of use in treating a
disease or condition in which normal angiogenesis is inhibited. In
some embodiments, an increase in migration of the fluorescent cell
line is at least about a 30%, at least about a 40%, at least about
a 50%, at least about a 60%, at least about a 70%, at least about a
80%, at least about a 90%, at least about a 100%, at least about a
150%, at least about a 200%, at least about a 250%, at least about
a 300%, at least about a 350%, at least about a 400%, at least
about a 500% increase as compared to control. In additional
embodiments, the increase is a statistically significant increase
as compared to a control.
[0183] iv. Cell Viability Assay
[0184] Another example of an assay that can be used with the
disclosed fluorescent cell lines is a cell viability assay. These
assays are based on the release of fluorescent protein from the
cytoplasm of fluorescent cell lines that constitutively express
fluorescent protein that occurs when the integrity of the cell
membrane of the cells is compromised, for example when the cell
dies, such as when the cell is exposed to a cytotoxic agent, such
as a test agent that is cytotoxic to the cell. Upon exposure to a
cytotoxic agent the fluorescent protein is liberated to the culture
media and it can be measured, for example using a fluorimeter. The
greater the amount of fluorescent protein liberated from the cells
present in the culture, the greater the intensity of the
fluorescence present in the media. The measured fluorescence in the
media corresponds to number of dead cells.
[0185] In some embodiments, the cell viability assay is used to
determine if an exogenous agent is cytotoxic to one or more of the
fluorescent cell lines of interest, such as one or more of the
fluorescent cell lines disclosed herein. A fluorescent cell line of
interest can be contacted with exogenous agent and the impact of
the exogenous agent on the death of the fluorescent cell line of
interest can be determined. For example, an increase in the
relative florescence present in the media of between a fluorescent
cell line of interest contacted with an exogenous agent and a
control indicates that the exogenous agent, such as a test agent,
cell line, growth factor, etc., is cytotoxic to the cell line of
interest. In other embodiments, differential cytotoxicity of an
exogenous agent to different cell lines in the cell viability assay
provides an indication that a specific exogenous agent is
preferentially cytotoxic to one cell line but not other cell lines
present in the culture. Such information is useful for screening
agents that are preferentially or differentially cytotoxic to a
specific cell-type, for example to the exclusion of other cell
types. For example, in a mixed cell population a test agent could
be screened to determine if it was cytotoxic (for example
differentially cytotoxic) to diseased cells (such as tumor cells)
present in the mixed cell population, but not normal cells present
in the mixed cell population.
[0186] In some embodiments, the difference between the fluorescence
of the media of a fluorescent cell line contacted with an exogenous
agent relative to a control, (for example as measured by the
fluorescence intensity of a fluorescent protein liberated from the
cell line into the media) is at least about 10%, such as at least
about 20%, at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 100%, at least about 150%, at least
about 200%, at least about 250%, at least about 300%, at least
about 350%, at least about 400%, at least about 500%, or greater
then 500%. In some embodiments, the difference is a statistically
significant difference. Thus, an exogenous agent can induce a
statistically significant difference in the number of cells that
die as a the migration of a fluorescent cell line of interest
contacted with the exogenous agent, as compared to the control,
such as the fluorescent cell line of interest not contacted with
the exogenous agent or a different cell line that has been mixed
with the cell line of interest.
[0187] The fluorescent cell lines of the present invention can be
used in the above-discussed assays to monitor endothelial cell
responses to various exogenous agents, including test agents.
However, as described herein, endothelial cell responses to tumor
cells in two-dimensional culture assays are not correlative with
observations of tumor-stimulated angiogenesis in the in vivo whole
animal context. In order to recreate the in vivo tumor
microenvironment in an in vivo context, a three-dimensional
co-culture assay was developed, as discussed in detail below.
B. Immortalized Fluorescent Cell lines
[0188] Disclosed herein are immortalized mammalian cell lines that
stably express a fluorescent protein. The disclosed fluorescent
cell lines are produced in disclosed examples by transfecting
mammalian expression vectors for fluorescent proteins, such as
green, yellow, red and blue fluorescent proteins and the like into
a variety of cell lines such as cell lines derived from both
vascular and lymphatic endothelial cells as well as inflammatory
cells (such as monocytes and mast cells) and tumor cells (such as
tumor cells from lung, breast, and the like). The transfected cells
are selected for stable (through antibiotic resistance) and
high-homogeneous expression (through flow cytometry cell sorting)
of the fluorescent proteins.
[0189] In some embodiments, the disclosed mammalian cell line is
stably transfected with a mammalian expression vector that includes
a nucleotide sequence encoding the amino acid sequence of a
fluorescent protein, operably connected to a constitutively active
promoter that drives the expression of the fluorescent protein and
a nucleotide sequence encoding a selection marker. The disclosed
fluorescent cell lines stably effect high level expression of the
fluorescent protein in the absence of a selection agent and
maintain high level expression of the fluorescent protein when the
fluorescent cell lines proliferate through multiple passages, for
example 10 passages, 20 passages, 30 passages, 40 passages, 50
passages, 100 passages, 150 passages, 200 passages, 250 passages,
300 passages, 400, or even greater than 500 passages of the cell
line.
[0190] The disclosed cell lines can be derived from any mammalian
species, for example humans, apes, monkeys, swine, bovine, and the
like. In some embodiments, the fluorescent cell line is an
endothelial cell line, for example the porcine aortic endothelial
cell line PAE (see for example FIG. 1A-1D), the human lymphatic
endothelial cell line HMEC-1, or the rhesus macaque choroidal
endothelial cell line RF/6A (ATCC CRL-1780). In some embodiments,
the fluorescent cell line is an epithelial cell line, for example
the human adenocarcinoma cell line A549. In some embodiments, the
fluorescent cell line is an adenocarcinoma cell line, for example
the human breast adenocarcinoma cell line MCF7. In some
embodiments, the fluorescent cell line is a mast cell line, such as
the human mast cell line HMC-1.
[0191] The fluorescent cell lines can be transfected with vectors
expressing different fluorescent proteins, such as green, yellow,
red and cyan among others, such that a cell line can be constructed
that stably and constitutively expresses each of the fluorescent
proteins. In other words, a particular parental cell line can be
divided into sub cell lines, in which each of the sub cell lines
expresses a different fluorescent protein. The nucleic acids
encoding fluorescent proteins can be expressed in mammalian cell
lines. Transfection of mammalian cell lines with recombinant DNA
may be carried out by conventional techniques as are well known to
those skilled in the art, for example as calcium phosphate
coprecipitates, the use of conventional mechanical procedures such
as microinjection, electroporation (for example using a
NUCLEOFECTOR.TM. II available from AMAXA.RTM.) and insertion of a
plasmid encased in liposomes.
[0192] Polynucleotide sequences encoding the fluorescent proteins
can be operatively connected to expression control sequences. An
expression control sequence operatively connected to a coding
sequence is ligated such that expression of the coding sequence is
achieved under conditions compatible with the expression control
sequences. The expression control sequences include, but are not
limited to appropriate promoters, enhancers, transcription
terminators, a start codon (i.e., ATG) in front of a
protein-encoding gene, splicing signal for introns, maintenance of
the correct reading frame of that gene to permit proper translation
of mRNA, and stop codons.
[0193] The polynucleotide sequences encoding the fluorescent
proteins can be inserted into an expression vector including, but
not limited to a plasmid, to allow insertion or incorporation of
sequences into mammalian cell lines. Biologically functional
plasmid DNA vectors capable of expression and replication in a
mammalian cell line are known in the art. Examples of vectors that
can be used in constructing the disclosed fluorescent cell lines
include those vectors available from AMAXA.RTM., such as
pmaxFP-Green-C, pmaxFP-Green-N, pmaxFP-Yellow-C, pmaxFP-Yellow-N,
pmaxFP-Yellow-PRL, pmaxFP-Red-C, and pmaxFP-Red-N, and vectors
available from Clontech, such as pAcGFP1-Hyg-N1, pAcGFP1-N1,
pAcGFP1-N2, pAcGFP1-N3, pAmCyan1-N1, pAsRed2-N1, pDsRed2-N1,
pDsRed-Express-N1, pD sRed-Monomer-Hyg-N1, pDsRed-Monomer-N1,
pHcRedl-N1/1, pZsGreenl-N1, pZsYellowl-N1 and the like.
[0194] Those of skill in the art will also recognize that the
selection marker component of the vector need not be restricted to
an antibiotic resistance gene. By "selection marker" it is meant a
gene encoding a protein wherein an activity of the expressed
protein is suitable for exerting selection pressure on the cell in
which it is expressed. Many selection markers are known to those of
skill in the art, including but not limited to resistance markers
for antibiotics such as ampicillin, streptomycin, kanamycin,
neomycin and the like. Any suitable selection marker may be
utilized to construct the vectors of the present disclosure for
transfection of the mammalian cell lines, so long as the selection
marker provides appropriate selection pressure on the cells within
which it is contained.
[0195] The disclosed cell lines can be further sorted by fluoresce
activated cell sorting (FACS) to select for cells from a particular
cell line that have the greatest fluorescence intensity for a
particular fluorescent protein, for example, by gating on the
brightest population of cells and sorting these cells for further
propagation. In certain embodiments, the disclosed fluorescent cell
lines have been sorted by FACS to select for cells that stably and
constitutively express fluorescent proteins. In some situations it
is advantageous to FACS sort a florescent cell line multiple times,
such as 2, 3, 4, 5, 6, 7, 8, 9, or even greater than 9 times to
enrich for a population of cells that stably and constitutively
expresses a fluorescent protein.
[0196] The ability to create multiple different cell lines
expressing different fluorescent proteins enhances the ability to
study cells in co-cultures where different cell lines are included
in the same assay. Different combinations of fluorescent cells
enable the study of the interaction between different cell types at
the qualitative (morphological) and quantitative level. The use of
these cells will also ease the development of new in vitro
multicellular angiogenesis models, such as those disclosed
herein.
[0197] In particular, the disclosed cell lines are useful in the
estimation of the angiogenic potential of patient serum samples and
assessment of physiologically active angiogenic/antiangiogenic drug
levels in patient samples. The disclosed cell lines also are well
suited for integration into existing angiogenesis assays, such as
growth assays, migration/invasion assays, tubule formation assays,
cell viability assay, cell to cell interaction assays, cell to
matrix interaction assays, apoptosis assays, and are particularly
amenable to study by fluorescent/confocal microscopy, for example
to determine on a cell by cell basis the effects co-culture has on
different cell lines, such as cell lines from different anatomical
origins.
[0198] The disclosed cell lines can be integrated into existing
kits to replace standard reagents. Tubule formation assays are an
example of an assay that would substantially benefit from inclusion
of one or more of the disclosed cell lines. Typical tubule
formation assays require the staining of the cells with calcein AM
prior to the assay. Multiple problems are associated with this is
approach, including interaction of the staining chemical with the
cellular objects of study (calcein AM is a known inhibitor of
certain cell types), inter-experimental variability of the staining
protocol, and the inability to use different cell types on the same
assay. The use of the disclosed stably transfected fluorescent cell
lines in tubule formation assays would eliminate the staining of
cells with calcein, eliminate the variability in emitted
fluorescence and greatly expand the capabilities of the assay
introducing the possibility of the use of multiple cell types in
the same assay.
[0199] Another advantage of the use of stable fluorescent cellular
in vitro assays is that at any time during the assay the cells can
be observed under a fluorescent microscope, which allows for
morphological analysis and comparison of mono-cultures versus
co-cultures. For example, tubule formation and morphological
analysis is assessed as a function of time.
C. AngioApplicaton.TM.
[0200] Angiogenesis in vitro assays (such as the tubule formation
assay disclosed herein) and ex vivo assays (such as chicken
chorioallantoic assay) are fundamental tools to the angiogenesis
field. One of the hurdles in determining the effects of exogenous
agents in such assays, for example, the tubule formation assays
disclosed herein, is determining and quantifying the effects of
such exogenous agents on the tubule formation potential of the cell
line or cell lines under study, for example the fluorescent cell
lines disclosed herein. Automated technologies assist with the
precise morphological quantification of a vasculature formation
assay, such as the tubule formation assay disclosed herein, for
example to determine the total area of the tubules, the total
number of tubules, number of nodes, number of branch points, the
number of tubes per node, and/or node area in a tubule formation
assay. In one example, AngioApplication.TM. is a computer
implemented automated analysis used to analyze the morphology of
the cell lines. AngioApplication.TM. is an image-analysis software
that automatically quantifies morphological parameters in assays
involving formation of vasculature. The program reports the total
area of the tubules, the total number of tubules, number of nodes,
number of branch points, the number of tubes per node, or node
among other parameters. This software utilizes the freely available
NIH ImageJ library (Rasband, W. S., ImageJ, U.S. National
Institutes of Health, Bethesda, Md., USA, available on the world
wide web at rsb.info.nih.gov/ij; Abramoff et al., Image Processing
with ImageJ, Biophotonics International 11:7, 36-42, 2004). As the
program is coded in Java it can potentially be used in any computer
platform (for example Windows, Macintosh, Unix, Linux, etc.). This
program allows for the rapid/automated quantification of
angiogenesis assays thus enhancing the capabilities of existing
technologies for robust drug screening, patient diagnosis, and
assessment of biologically active angiogenic or antiangiogenic
drugs in patient samples.
[0201] As shown in FIG. 15, the AngioApplication.TM. graphical user
interface (GUI) which allows the user to choose the image (or batch
of images) to be analyzed. The settings window of
AngioApplication.TM. (see FIG. 16) allows the user to dynamically
adjust several parameters to allow for a more precise analysis of
the morphological features of the image, for example total length
of the tubules, the total area of the tubules, the total number of
tubules, number of nodes, number of branch points, the number of
tubes per node, and/or node area. The program has been designed to
find tubes and nodes in the original image and produce an "overlay"
which shows both structures colored differently (see for example
FIG. 17A and FIG. 17B). As shown in FIG. 17A and FIG. 17B both
fluorescent images (FIG. 17A) and bright field images (FIG. 17B)
can be analyzed. Images can be stored for later analysis (see FIG.
18) and the data generated by AngioApplication.TM. are stored
directly into an Excel file (see FIG. 19).
D. Exemplary Test Agents
[0202] The methods disclosed herein are of use for identifying test
agents that are modulators of angiogenesis. A "test agent" is any
substance or any combination of substances that is useful for
achieving an end or result. The test agents identified using the
methods disclosed herein can be of use for affecting the normal
angiogenic potential of a fluorescent cell line. Any test agent
that has potential (whether or not ultimately realized) to affect
the angiogenic potential of the fluorescent cell lines disclosed
herein can be tested using the methods of this disclosure.
[0203] Exemplary test agents include, but are not limited to,
peptides such as, soluble peptides, including but not limited to
members of random peptide libraries (see for example, Lam et al.,
Nature, 354:82-84, 1991; Houghten et al., Nature, 354:84-86, 1991),
and combinatorial chemistry-derived molecular library made of D-
and/or L-configuration amino acids, phosphopeptides (including, but
not limited to, members of random or partially degenerate, directed
phosphopeptide libraries; for example, Songyang et al., Cell,
72:767-778, 1993), antibodies (including, but not limited to,
polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or
single chain antibodies, and Fab, F(ab').sub.2 and Fab expression
library fragments, and epitope-binding fragments thereof), small
organic or inorganic molecules (such as, so-called natural products
or members of chemical combinatorial libraries), molecular
complexes (such as protein complexes), or nucleic acids.
[0204] Appropriate tests agents can be contained in libraries, for
example, synthetic or natural compounds in a combinatorial library.
Numerous libraries are commercially available or can be readily
produced; means for random and directed synthesis of a wide variety
of organic compounds and biomolecules, including expression of
randomized oligonucleotides, such as antisense oligonucleotides and
oligopeptides, also are known. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant and animal
extracts are available or can be readily produced. Additionally,
natural or synthetically produced libraries and compounds are
readily modified through conventional chemical, physical and
biochemical means, and may be used to produce combinatorial
libraries. Such libraries are useful for the screening of a large
number of different compounds.
[0205] Libraries (such as combinatorial chemical libraries) useful
in the disclosed methods include, but are not limited to, peptide
libraries (for example see U.S. Pat. No. 5,010,175; Furka, Int. J.
Pept. Prot. Res., 37:487-493, 1991; Houghton et al., Nature,
354:84-88, 1991; and PCT Publication No. WO 91/19735), encoded
peptides (see for example PCT Publication WO 93/20242), random
bio-oligomers (see for example PCT Publication No. WO 92/00091),
benzodiazepines (see for example U.S. Pat. No. 5,288,514),
diversomers such as hydantoins, benzodiazepines and dipeptides (see
for example Hobbs et al., Proc. Natl. Acad. Sci. USA, 90:6909-6913,
1993), vinylogous polypeptides (see for example Hagihara et al., J.
Am. Chem. Soc., 114:6568, 1992), nonpeptidal peptidomimetics with
glucose scaffolding (see for example Hirschmann et al., J. Am.
Chem. Soc., 114:9217-9218, 1992), analogous organic syntheses of
small compound libraries (see for example Chen et al., J. Am. Chem.
Soc., 116:2661, 1994), oligocarbamates (see for example Cho et al.,
Science, 261:1303, 1003), and/or peptidyl phosphonates (see for
example Campbell et al., J. Org. Chem., 59:658, 1994), nucleic acid
libraries (see Sambrook et al. Molecular Cloning, A Laboratory
Manual, Cold Springs Harbor Press, N.Y., 1989; Ausubel et al.,
Current Protocols in Molecular Biology, Green Publishing Associates
and Wiley Interscience, N.Y., 1989), peptide nucleic acid libraries
(see for example U.S. Pat. No. 5,539,083), antibody libraries (see
for example Vaughn et al., Nat. Biotechnol., 14:309-314, 1996; PCT
App. No. PCT/US96/10287), carbohydrate libraries (see for example
Liang et al., Science, 274:1520-1522, 1996; U.S. Pat. No.
5,593,853), small organic molecule libraries (see for example
benzodiazepines, Baum, C&EN, Jan 18, page 33, 1993;
isoprenoids, U.S. Pat. No. 5,569,588; thiazolidionones and
methathiazones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat.
Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No.
5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514) and the
like.
[0206] Libraries useful for the disclosed screening methods can be
produced in a variety of manners including, but not limited to,
spatially arrayed multipin peptide synthesis (see for example
Geysen, et al., Proc. Natl. Acad. Sci., 81(13):3998 4002, 1984),
"tea bag" peptide synthesis (see for example Houghten, Proc. Natl.
Acad. Sci., 82(15):51315135, 1985), phage display (see for example
Scott and Smith, Science, 249:386-390, 1990), spot or disc
synthesis (see for example Dittrich et al., Bioorg. Med. Chem.
Lett., 8(17):23512356, 1998), or split and mix solid phase
synthesis on beads (see for example Furka et al., Int. J. Pept.
Protein Res., 37(6):487 493, 1991; Lam et al., Chem. Rev.,
97(2):411-448, 1997). Libraries may include a varying number of
compositions (members), such as up to about 100 members, such as up
to about 1000 members, such as up to about 5000 members, such as up
to about 10,000 members, such as up to about 100,000 members, such
as up to about 500,000 members, or even more than 500,000
members.
[0207] In one convenient embodiment, high throughput screening
methods involve providing a combinatorial chemical or peptide
library containing a large number of potential therapeutic
compounds. Such combinatorial libraries are then screened in one or
more assays as described herein to identify those library members
(particularly chemical species or subclasses) that display a
desired characteristic activity (such as in an increase or decrease
in tubule formation). In one example a test agent of use is
identified that increases the number of tubules formed. In another
example a test agent of use is identified that inhibits tubule
formation, for example by decreasing the relative number of tubules
formed.
[0208] The compounds identified using the methods disclosed herein
can serve as conventional "lead compounds" or can themselves be
used as potential or actual therapeutics. In some instances, pools
of candidate agents may be identify and further screened to
determine which individual or subpools of agents in the collective
have a desired activity.
E. Therapeutic Compounds, Formulations and Treatments
[0209] This disclosure further relates to methods for modulating
angiogenesis in a subject. The disclosed methods can identify
compounds that modulate angiogenesis. The compounds and derivatives
thereof are particularly useful for modulating angiogenesis in a
subject, such as a subject suffering from a disease or condition
accompanied by deregulated angiogenesis, for example cancer. The
methods of modulating angiogenesis include administering to a
subject a therapeutically effective amount of a test agent
identified as one that modulates angiogenesis. Thus in some
embodiments, the pharmaceutical compositions containing a test
agent that decreases angiogenesis is administered to a subject,
such as a subject with cancer. In some embodiments, the subject is
a human subject. It is also contemplated that the compositions can
be administered with conventional treatments for cancer, such as in
conjunction with a therapeutically effective amount
chemotherapeutic agent.
[0210] In some examples, a subject is selected for treatment with
an angiogenesis modulator that increases angiogenesis. Such a
subject can be treated with a test agent identified by the methods
disclosed herein that increase angiogenesis. In some embodiments,
the subject is a human subject.
[0211] Therapeutic compound(s) can be administered directly to a
subject for example a human subject. Administration is by any of
the routes normally used for introducing a compound into ultimate
contact with the tissue to be treated. The compounds are
administered in any suitable manner, optionally with
pharmaceutically acceptable carrier(s). Suitable methods of
administering therapeutic compounds are available and well known to
those of skill in the art, and although more than one route can be
used to administer a particular composition, a particular route can
often provide a more immediate and more effective reaction than
another route.
[0212] When the test agent is to be used as a pharmaceutical, the
test agent is placed in a form suitable for therapeutic
administration. The test agent may, for example, be included in a
pharmaceutically acceptable carrier such as excipients and
additives or auxiliaries, and administered to a subject. Frequently
used carriers or auxiliaries include magnesium carbonate, titanium
dioxide, lactose, mannitol and other sugars, talc, milk protein,
gelatin, starch, vitamins, cellulose and its derivatives, animal
and vegetable oils, polyethylene glycols and solvents, such as
sterile water, alcohols, glycerol and polyhydric alcohols.
Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial, anti-oxidants, chelating
agents and inert gases. Other pharmaceutically acceptable carriers
include aqueous solutions, nontoxic excipients, including salts,
preservatives, buffers and the like, as described, for instance, in
Remington's Pharmaceutical Sciences, 15th ed., Easton: Mack
Publishing Co., 1405-1412, 1461-1487, 1975, and The National
Formulary XIV., 14th ed., Washington: American Pharmaceutical
Association, 1975). The pH and exact concentration of the various
components of the pharmaceutical composition are adjusted according
to routine skills in the art. See Goodman and Gilman The
Pharmacological Basis for Therapeutics, 7th ed.
[0213] The pharmaceutical compositions are in general administered
topically, intravenously, orally or parenterally or as implants.
Suitable solid or liquid pharmaceutical preparation forms are, for
example, granules, powders, tablets, coated tablets,
(micro)capsules, suppositories, syrups, emulsions, suspensions,
creams, aerosols, drops or injectable solution in ampoule form and
also preparations with protracted release of active compounds, in
whose preparation excipients and additives and/or auxiliaries such
as disintegrants, binders, coating agents, swelling agents,
lubricants, flavorings, sweeteners or solubilizers are customarily
used as described above. The pharmaceutical compositions are
suitable for use in a variety of drug delivery systems. For a brief
review of methods for drug delivery, see Langer, Science,
249:1527-1533, 1990, which is incorporated herein by reference.
[0214] For treatment of a patient, depending on activity of the
compound, manner of administration, nature and severity of the
disorder, age and body weight of the patient, different daily doses
are necessary. Under certain circumstances, however, higher or
lower daily doses may be appropriate. The administration of the
daily dose can be carried out both by single administration in the
form of an individual dose unit or else several smaller dose units,
and also by multiple administrations of subdivided doses at
specific intervals.
[0215] A therapeutically effective dose is the quantity of a
compound according to the disclosure necessary to prevent, to cure
or at least partially ameliorate the symptoms of a disease and its
complications. Amounts effective for this use will, of course,
depend on the severity of the disease and the weight and general
state of the patient. Typically, dosages used in vitro may provide
useful guidance in the amounts useful for in situ administration of
the pharmaceutical composition, and animal models may be used to
determine effective dosages for treatment of particular disorders.
Various considerations are described, e.g., in Gilman et al., eds.,
Goodman and Gilman: the Pharmacological Bases of Therapeutics, 8th
ed., Pergamon Press, 1990; and Remington's Pharmaceutical Sciences,
17th ed., Mack Publishing Co., Easton, Pa., 1990. Effectiveness of
the dosage can be monitored by any method.
F. Detection
[0216] The disclosed fluorescent cell lines can be detected by
detecting the presence of the emission spectrum of the fluorescent
proteins expressed by the fluorescent cell lines, for example using
appropriate filters and/or monochrometers, (for excitation,
emission, or both) the different fluorescent proteins can be
detected using fluorescence microscopy and by FACS. However, it
will be readily understood by those of skill in the art that other
means for detecting the presence of fluorescent proteins and thus
the cells expressing such protein may also be used.
[0217] Separate populations of fluorescent proteins with different
emission spectra can be used to identify the cells containing such
proteins, such as the disclosed fluorescent cell lines. For
example, the characteristic emissions from the different
fluorescent proteins can be observed as colors or can be decoded to
provide information about the particular wavelength at which the
emission is observed, for example to identify the number of cells
of a particular kind or the location of such cell. Methods and
devices for eliciting and detecting emissions from fluorescent
proteins are well known in the art. In brief, a light source
typically has a range that emits light at a wavelength shorter than
the wavelength to be detected is used to elicit an emission by the
fluorescent proteins. Numerous such light sources (and devices
incorporating such light sources) are known in the art, including
without limitation: deuterium lamps and xenon lamps equipped with
filters, continuous or tunable gas lasers, such as argon ion, HeCd
lasers, solid state diode lasers (for example, GaN, GaAs lasers),
YAG and YLF lasers and pulsed lasers. The emissions of fluorescent
proteins can similarly be detected using known devices and methods,
including without limitation, spectral imaging systems. Optionally,
the emissions are passed through one or more filters or prisms
prior to detection. The simultaneous multicolor wavelength, such as
multicolor, identification of fluorescent proteins permits rapid
identification of cell without requiring fixation of the cells.
G. Kits and High Throughput Systems
[0218] This disclosure also provides kits including one or more of
the fluorescent cell lines disclosed herein. Such kits can be used
for the study of angiogenesis, for example to identify test agents
that modulate angiogenesis, or the impact of mixed populations of
cell types on angiogenic potential. The kits include at least one
of the fluorescent cell lines disclosed herein. The kits may
further include additional components such as instructional
materials and additional reagents (for example serum, growth media
and the like). The kits may also include additional components to
facilitate the particular application for which the kit is designed
(for example microtiter plates, optical filters and the like). Such
kits and appropriate contents are well known to those of skill in
the art. The instructional materials may be written, in an
electronic form (such as a computer diskette or compact disk) or
may be visual (such as video files). It is contemplated that the
kits can contain reagents for carrying out the assays described
herein, for example reagents for migration, proliferation, and/or
tubule formation assays. In some examples, the kit also includes
the AngioApplication.TM. program, for example supplied on a digital
medium (such as a computer diskette or compact disk).
[0219] This disclosure also provides integrated systems for
high-throughput screening of test agents for modulation of
angiogenesis. The systems typically include a robotic armature that
transfers fluid from a source to a destination, a controller that
controls the robotic armature, a tag detector, a data storage unit
that records tag detection, and an assay component such as a
microtiter dish comprising a well having a cell culture, for
example a cell culture containing one or more of the fluorescent
cell lines disclosed herein.
[0220] A number of robotic fluid transfer systems are available, or
can easily be made from existing components. For example, a Zymate
XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a
Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used
to transfer parallel samples to 96 well microtiter plates to set up
several parallel simultaneous assays of fluorescent cell lines, for
example the assay the effect of one or more test agents on
angiogenesis.
[0221] Optical images can viewed (and, if desired, recorded for
future analysis) by a camera or other recording device (for
example, a photodiode and data storage device) and are optionally
further processed such as by digitalizing, storing, and analyzing
the image on a computer. A variety of commercially available
peripheral equipment and software is available for digitizing,
storing and analyzing a digitized video or digitized optical image,
for example, using PC (Intelx86 or Pentium chip-compatible DOS.TM.,
OS2.TM. WINDOWS.TM., WINDOWS NT.TM. or WINDOWS95.TM. based
computers), MACINTOSH.TM., or UNIX based (for example, a SUN.TM., a
SGI.TM., or other work station) computers.
H. Three-Dimensional Co-Cultures
[0222] The stably-transfected fluorescent cells provided herein can
be used to monitor the response of endothelial cells in a
co-culture to one or more test agents, such as pharmaceutical
agents. The detection of endothelial cell proliferation, motility
and tubule formation in response to one or more test agents
indicates the angiogenic effect of the test agent and can provide
crucial information related to development of patient therapies.
However, as described herein, endothelial cell responses to tumor
cells co-cultured in two-dimensions (for example, separated by a
layer of gel matrix such as a BME matrix) do not correlate with the
in vivo angiogenic tumor activity.
[0223] To overcome deficiencies of 2D in vitro co-cultures,
described herein are three-dimensional (3D) in vitro co-cultures
that provide a mimetic of in vivo tumor activity. The co-cultures
can be prepared in any suitable culture vessel or chamber,
including multi-well or multi-chamber culture plates.
[0224] The 3D co-cultures described herein are prepared in three
layers, which can be of any volume or thickness. The first layer,
which is in contact with the bottom of the culture vessel, is any
solidified polymer of neutral charge that is known to the art, and
which does not alter the biological activity of the cells in
culture. In particular examples, the polymer is a polysaccharide of
neutral charge. In particular examples, the first layer comprises
solidified agarose. In other examples, this layer comprises a
neutral polysaccharide polymer of cellulose, curdlan, cellulose,
starch, glycogen, chitin, and the like.
[0225] The second layer of the 3D co-cultures comprises a mixture
of two or more types of cells embedded in an extracellular matrix
gel extract or any suitable synthetic gel product. Exemplary
extracellular matrix gels include BD MATRIGEL.TM. gel matrix (BD
Bioscience), BME (Trevigen), GELTREX.RTM. gel matrix (Invitrogen),
Collagen Type I/IV and the like. The gel matrix layer also
comprises a mixture of endothelial cells that are dispersed
(distributed) throughout the second layer and tumor cells. In
particular examples, the endothelial cells can be any immortalized
endothelial cell line, including the stably-transfected fluorescent
endothelial cells described herein. In other examples, the
endothelial cells are non-immortalized endothelial cell cultures
that are transiently transfected with a fluorescent protein. The
tumor cells can be derived from a cell line or from a tumor biopsy
extracted from a subject. In particular embodiments, the tumor
cells are in the form of a tumor spheroid colony. In other
embodiments, the tumor cells are a piece of a tumor biopsy. The
tumor cells can also be stably transfected to express a fluorescent
protein.
[0226] In other examples, the second layer includes one or more
additional mammalian cell types. Any cell type known to the art
that is or might be part of the tumor microenvironment can be
included in the gel matrix layer, for example, macrophages, mast
cells, fibroblasts, adipocytes, and pericytes can all
(independently or in combination) be included in the co-culture. In
particular examples, multiple cell lines included in the second
layer are transfected with constructs that express fluorescent
proteins of distinguishable emission spectra.
[0227] The third layer of the 3D co-cultures is comprised of any
suitable liquid culture medium, and is provided on top of the
second layer. Any mammalian tissue culture media known to the art
can be used; thus, a skilled artisan will understand the parameters
that will influence selection and adaptation of media for cell
growth.
[0228] In particular embodiments, one or more test agents, such as
those described herein, is added to any layer of the co-culture,
such as the first, second, or third layer of the co-culture. In
particular examples, the test agent is added to the third layer of
the co-culture. In particular examples, the test agent is one or
more anti-angiogenic and/or anti-metastatic compound. In other
examples, the test agent is a promoter of angiogenesis or
metastasis. In some examples, the test agent directly affects
angiogenesis, in the manner of drugs such as Avastin.RTM.. In other
examples the test agent is one or more indirect anti-angiogenic
compounds such as "non steroidal anti-inflammatory drugs" or
NSAIDs.
[0229] In particular embodiments, the 3D co-cultures described
herein are used to monitor the angiogenic or metastatic potential
of tumor cells. Such methods involve preparing a 3D co-culture (in
three layers, as described herein), incubating the co-culture for a
period of time, and detecting endothelial cell proliferation or
tubule formation, or angiotropism (for instance, using the assays
described herein). In particular examples, the tumor cells are
derived from a tumor removed from a subject and the 3D co-cultures
can be used to assess the angiogenic or metastatic potential of the
tumor. The co-culture can be incubated for any length of time
necessary to observe the angiogenic or metastatic activity
including incubation times from several hours (6, 7, 8, 9 or more
hours) to several days (1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days)
to one, two or more weeks. The detection of endothelial cell
proliferation, tubule formation, or angiotropism is achieved by
fluorescence or standard microscopy alone or confocal laser
scanning microscopy or in combination with the detection methods
described herein. In particular embodiments, the methods of
monitoring angiogenic or metastatic potential of tumor cells are
used to determine the effects of at least one test agent on the
angiogenic or metastatic potential of tumor cells derived from a
subject. In such examples, the test agent may be a candidate
therapeutic compound that may be used for treatment of the subject.
In other examples, it may be a compound that has already been
administered to a subject (either the same subject or a subject
different from the subject from whom the tumor cells originated) as
part of a cancer treatment regimen. In this way, the development of
drug-resistance of a tumor and the efficacy of a treatment in a
subject can be monitored by repeatedly using the 3D co-cultures
over a period of time.
[0230] The development of 3D co-cultures that provide a mimetic of
in vivo tumor activity also enables methods for selecting
personalized anti-angiogenic and anti-metastatic therapies (that
is, therapies that are selected in order to be specifically
effective in a specific subject). These methods involve assaying
the anti-angiogenic or anti-metastatic activity of a panel of
compounds or other treatment variables (e.g., dosage, timing,
etc.), alone or in combination, using a 3D co-culture that contains
tumor cells from the specific subject. Such assays optionally can
be done in a multi-well plate. 3D co-cultures are prepared as
described using the target subject's tumor cells, and at least one
test compound (test agent) is added to the third layer of each of
some but not all of the 3D co-cultures. The co-cultures are
incubated and angiogenic or metastatic activity is observed, for
instance as described herein. The test agent (or dosage, or other
variable regimen) that has the most beneficial (strongest)
anti-angiogenic and/or anti-metastatic effect, for instance in
comparison to the angiogenic or metastatic activity observed in the
compound-free co-culture, is then selected for personalized
treatment. The continued efficacy of such therapies can be
monitored after any desired length of time, such as three months or
longer if necessary.
[0231] In another embodiment, the 3D co-cultures can also be used
in the development of computer programs to quantitate angiogenesis
and vessel complexity in three-dimensional projections. Such
programs would ultimately be applied to analyze the vascular
network around tumors and determine a 3D vascular density profile
for a given tumor via CAT or MRI scans. Such images could be
retaken after initial anti-angiogenic drug therapy to quantitate
treatment regimen efficacy before actually seeing alterations in
tumor size.
EXAMPLES
Example 1
Generation of Stably Transfected Fluorescent Cells
[0232] This example describes the materials and methods used in the
generation of stably transfected fluorescent cells.
[0233] Cell Lines.
[0234] Cell lines A549 (lung adenocarcinoma) and MCF-7 (breast
cancer) were acquired though the DTP 60 cell line library at
NCI/Frederick. RF/6A (ATCC CRL-1780) was obtained from the American
Type Culture Collection as a Macaca mulatta (rhesus monkey) retina
endothelial cell line but shown to be a monkey pericyte cell line
via surface marker expression. The mast cell line HMC-1 was derived
from primary mast cells exposed to 5-azacytindine, spontaneously
immortalized and established by Dr. John Butterfield (Butterfield
et al., Leuk. Res. 12:345-355, 1988) and was obtained from the
Department of Internal Medicine, Division of Allergic Diseases,
Mayo Clinic, Rochester Minn. 55905. Endothelial cell line HMEC-1 is
a human dermal microvascular blood vessel endothelial cell line
originally developed by Dr. Thomas Lawley (Emory University School
of Medicine, Atlanta, Ga., USA) via SV40 large T transfection (Ades
et al., J. Invest. Dermatol. 99:683-699, 1992), and was obtained
from Dr. Hynda Kleinman (NIDCR). PAE is a porcine aortic
endothelial cell line from Dr. Carl-Henrik Heldin (Ludwig Institute
for Cancer Research, Uppsala, Sweden) that became spontaneously
immortalized with continuous passaging (Ronnstrand et al. EMBO J.
11:3911-3919, 1992).
[0235] Plasmids.
[0236] Plasmids pmaxFP-GFP-C, pmaxFP-Yellow-C and pmax-Red-N were
obtained from AMAXA.RTM. Inc. (Gaithersburg, Md.). Plasmids
pDsRed2-C1 and pAmCyan1-C1 were obtained from BD Bioscience.
pcDNA3.1-GFP was built in house inserting the GFP coding reading
frame into the pcDNA3.1-TOPO-TA (INVITROGEN.TM.) backbone. All
plasmids contain a GENETICIN.RTM. selectable marker.
[0237] Transfections and Generation of Stable Transfectants.
[0238] Cell lines A549, MCF7, SK-LMS-1, 92-1, PC-12, HMEC-1, RF/6A
(ATCC CRL-1780), PAE, and HMC-1 were stably transfected with the
plasmids described above using a NUCLEOFECTOR.TM. II (AMAXA.RTM.
Inc.). Different transfection solutions were used following
manufacturer's suggestions if available (A549, MCF7, SK-LMS-1,
92-1, PC-12). For HMEC-1 and RF/6A (ATCC CRL-1780), AMAXA.RTM.
HMVEC-L solution together with NUCLEOFECTOR.TM. II program T-023
were used. For HMC-1, AMAXA.RTM. solution V and NUCLEOFECTOR.TM. II
program T-030 were used.
[0239] After transfection, cells were seeded in 6 well plates using
the following media. A549, 92-1, and MCF7 cells were cultured in
RPMI (INVITROGEN.TM., Carlsbad, Calif.) supplemented with 10% fetal
bovine serum (FBS) (Hyclone, Logan, Utah), SK-LMS-1 cells were
cultured in DMEM (INVITROGEN.TM., Carlsbad, Calif.), supplemented
with 10% FBS (Hyclone, Logan, Utah), PC-12 cells were cultured in
F-12K medium (INVITROGEN.TM., Carlsbad, Calif.), HMC-1 cells were
cultured in Iscove's minimum medium (INVITROGEN.TM.) supplemented
with 10% FBS and 1.2 mmol/L of monothioglycerol (Sigma-Aldrich, St
Louis, Mo.). HMEC-1 cells were cultured in EMB-2 (CLONETICS.RTM.,
San Diego, Calif.) supplemented with EGM-2 MV SingleQuots.RTM.
(CLONETICS.RTM.). PAE cells were cultured in DMEM/F12 1:1 medium
(Invitrogen). RF/6A (ATCC CRL-1780) cells were cultured in RPMI
(INVITROGEN.TM.) supplemented with 10% FBS.
[0240] After 18 hours transfection efficiency was assessed under a
fluorescent microscope and when considered appropriate (>40%
transfection efficiency) cells were exposed to 800 .mu.g/ml
GENETICIN.RTM. (INVITROGEN.TM.). For all cells types, antibiotic
resistant clones showed a high range of fluorescence intensities
including clones (high proportion in some cell types such as A549)
which were negative. In order to enrich for positive clones and to
obtain a more homogeneous population of fluorescent cells the cells
were sorted by FACS gated on the fluorescent signal of the cells.
In some cases, such as the A549 cells, several cycles of cell
sorting were used to obtain a population stably transfected and
with homogeneous fluorescence. Once cells were confirmed to be
stable transfectants with homogeneous fluorescence levels map
testing and mycoplasma testing were performed.
Example 2
Growth Assay
[0241] This example describes exemplary procedures for measuring
the growth of the cell lines disclosed herein as either
monocultures or co-cultures of two or more cell lines.
[0242] Co-cultures of different cell lines were performed, with
each cell line expressing a different fluorophore than the other
(for example MCF7-RFP expressing red fluorescent protein and
PAE-YFP expressing yellow fluorescent protein). The co-cultures
were grown in black, clear bottom Costar 96-well plates (Corning
Costar Corp., Cambridge, Mass.). A direct relationship between
fluorescence and number of cells in culture was established for all
different fluorescent cell lines tested (see for example FIG.
2).
[0243] Different densities of cells were used in different trials.
Fluorescence intensity was obtained using an INFINITE.TM. M200
(TECAN.RTM. Group Ltd. Switzerland) fluorometer. The spectra for
the different fluorophores used overlap at maximum
excitation/emission. In order to avoid spectral bleed through and
discriminate fluorescence from different cells types, two systems
were used. In the first system measurements were taken at
suboptimal emission/excitation wavelengths ensuring no overlap.
Precisely gated fluorescence emission and excitation wavelengths
allowed the complete discrimination of fluorescence emitted by
different cell types in a co-culture. As shown in FIG. 3A, when
fluorescence emission is measured in the yellow range, only the
cells emitting in the yellow range (i.e. PAE endothelial cells
expressing YFP) show a linear relationship with the number of
cells, while cell emitting in the red range (i.e. MCF7 cells
expressing RFP) do not show such a relationship. As shown in FIG.
3B, when fluorescence emission measurements are done in the red
range, only cells emitting in that range (i.e. MCF7 cells
expressing RFP) show a linear relationship with cell number.
[0244] In the second system measurements obtained at maximum
excitation/emission were corrected through spectral linear unmixing
using a modification of the ImageJ algorithm first implemented by
Dr. Joachim Walter (the program is available on the world wide web
at rsb.info.nih.gov/ij/plugins/spectral-unmixing.html); based on
the work by Timo Zimmermann (Zimmermann, "Spectral imaging and
linear unmixing in light microscopy" Adv Biochem Eng Biotechnol,
95: 245-265, 2005). The fluorescent cells were also examined under
a fluorescent microscope allowing for morphological analysis and
comparison of mono-cultures versus co-cultures, as shown in FIG. 5.
Continuous real time readings were carried out to assay the growth
of mono- and co-cultures of PAE and MCF7 cells (see for example
FIGS. 4A and 4B).
Example 3
Tubule Formation Assay
[0245] This example describes exemplary procedures for measuring
the ability of the cell lines disclosed herein for tubule formation
potential. 50 .mu.l of low growth factor BME (Basement Membrane
Extract, Trevigen, Inc. Gaithersburg, Md.) were laid down in each
well and the plate was incubated for 1 hour at 37.degree. C. The
extract gels at 37.degree. C. to form a reconstituted basement
membrane and stimulates tubule formation by endothelial cells. The
major components of the Basement Membrane Extract (BME) include
laminin I, collagen IV, entactin, and heparin sulfate proteoglycan.
15,000 PAE-GFP cells were then added on top of the gelled BME and
images were acquired with a fluorescent microscope after 3.5-24
hours. Cells were imaged on the BD Pathway.TM. Bioimager. FIG. 7A
shows the impact of increasing concentrations of suramin (an
established inhibitor of tubule formation) (from 0 to 26 .mu.M) on
tubule formation by PAE green cells (GFP transfected). FIGS. 7B and
7C shows the dose response of PAE green cells to suramin in a
tubule formation assay. As expected the suramin inhibited tubule
formation and the EC50 calculated (.about.26 .mu.M) is in agreement
with the value previously published. Tubule formation assays were
quantified using the Java based software AngioApplication.TM..
AngioApplication.TM. has been specifically designed for the
quantification of tubule formation assays. Several morphological
parameters were assessed in images including the total area of the
tubules, the total number of tubules, number of nodes, number of
branch points, the number of tubes per node, or node.
[0246] AngioApplication.TM. was validated by comparison of the
published EC50 value of suramin (.about.26 .mu.M) and the EC50
value of suramin as determined experimentally using the disclosed
fluorescent cell lines in a tubule formation assay. As shown in
FIG. 7A increasing concentrations of suramin causes disruption of
tubule formation. AngioApplication.TM. was used to automatically
assess tube length at several concentrations of suramin.
Traditionally quantification of tube formation was based in the
measurement of complete (long) tubes in the image.
AngioApplication.TM. determined that as the concentration of
suramin was increased the number of long tubules was diminished. As
shown in FIG. 7B, using AngioApplication.TM. the EC50 of suramin
was determined to by approximately 26 .mu.M, in close agreement
with the published EC50 of suramin. In addition, because
AngioApplication.TM. calculates the length for all tubes in the
image (including incomplete tubes) a more refined analysis was
performed using the measurement of short (or incomplete) tubules.
Interestingly, AngioApplication.TM. found that the number of small
tubes increased as a function of suramin concentration. This is in
agreement with the fact that Suramin interferes with the mechanism
of tube formation. As shown in FIG. 7C, the EC50 calculated for
suramin using this additional parameter was approximately 26 .mu.M.
This result demonstrates that both measurements (short and long
tubes) can be used to evaluate potency of antiangiogenic drugs and
potentially other proangiogenic factors found in patient serum
samples.
Example 4
Migration Assay
[0247] This example describes exemplary procedures for measuring
the ability of the cell lines disclosed herein to migrate in
response to a chemical stimulation. In this example migration is up
a chemical gradient, from a region of lower concentration to higher
concentration of a chemical attractant.
[0248] Migration assays were performed using the ChemoTx.RTM. 96
well cell migration system (Neuro Probes Inc.) following the
manufacturer's recommendations. Every plate contained an internal
negative (absence of stimulus) and a positive (presence of a known
chemotactic substance) control together with wells containing
different cell densities which allow for the construction of
standard curves which mathematically correlate number of cells and
fluorescence intensity. Different putative chemotactic factors were
assayed using this system including plasma obtained from patients.
Fluorescence measurements were obtained using an INFINITE.TM. M200
fluorescence plate reader to measure the accumulation of
fluorescent cells in the lower chamber of the ChemoTx.RTM. 96 plate
that had migrated through the membrane to the lower chamber in
response to chemoattractant in the lower chamber. Migration can be
quantitated and the relative migration determined by determining
the intensity of the fluorescent signal, for example at the
emission maxima, of the fluorescent cells in the lower chamber,
wherein greater fluoresce intensity in the lower chamber relative
to the control indicates that migration has occurred. The migration
potential of various samples can be correlated with characteristics
of the sample or the subject from which the sample was taken. In
several examples, the fluorescent cells disclosed herein were
tested for migratory potential in the presence of
enhancers/blockers of migration.
[0249] Using this migration assay the presence of biologically
active chemotactic factors in a sample can be tested. FIG. 8A shows
the effect of decreasing human normal serum on the motility of YFP
expressing PAE endothelial cells. PEA cells were placed on the
upper membrane of the ChemoTx.RTM. 96 well cell migration plate.
The migration of PAE cells to the lower chamber as a function of
concentration of human normal serum was then determined using an
INFINITE.TM. M200 fluorescence plate reader to measure the
accumulation of fluorescent cells in the lower chamber. As
expected, as the serum concentration increases (and therefore the
presence of chemotactic factors increases in the serum) higher
levels of fluorescence are detected, which are directly related to
the migratory capacity of the cells. This assay was also directly
applied to the assessment of the migratory potential of patients'
serum samples. FIG. 8 shows a study wherein the serum of different
patients with or without tumors and with or without treatments was
screened for induced migratory potential. As expected all samples
show higher induced migratory potential than the negative control
(un-stimulated cells). For example, greater or less migratory
potential can be correlated with the presence or absence of tumors
or tumor types.
[0250] FIGS. 11A and 11B shows the applicability of the endothelial
fluorescent cells to assess the angiogenic status of clinical
samples. In this case the migratory potential of the sputum from
Idiopatic Pulmonary Fibrosis (IPF) patients was tested to determine
the effect of the sputum on the migratory potential of PAE cells
expressing fluorescent protein. This example assesses whether
angiogenic factors are present in this type of sample and can be
used in combination with the experimental procedures described in
this patent application as a diagnostic/prognostic end point.
Sputum obtained from the patients was placed in the lower chamber
of the ChemoTx.RTM. 96 well cell migration system. The migration of
PAE cells through the membrane to the lower chamber in response to
the sputum was then determined using an INFINITE.TM. M200
fluorescence plate reader to measure the accumulation of
fluorescent cells in the lower chamber. First, a standard curve was
generated with one of the samples from an IPF patient demonstrating
that it contains chemotactic factors for endothelial cells (see
FIG. 8A). Subsequently, sputum obtained from 13 normal subjects was
compared to sputum obtained from 13 IPF patients (see FIG. 8B). As
shown in FIG. 8B, sputum from normal subjects does not induce
migration above the phosphate buffered saline (PBS) control.
However, samples from IPF patients showed a strong migratory
potential.
[0251] This assay provides a fast and reliable system to detect the
presence of biologically active enhancers (for example
tumor-derived enhancers), or suppressors (for example test agents,
such as drugs) of cellular migration in patients' samples.
Example 5
Cell Viability Assay
[0252] This example describes exemplary procedures for measuring
the cytotoxicity of an agent on the cell lines disclosed
herein.
[0253] Increasing concentrations of Triton.RTM. X were added to
fluorescent cells and after 20 minutes supernatants were collected
and transferred to a different plate and measured at the
appropriate wavelength. As shown in FIG. 9, the relative
fluorescence as a function of the log of concentration produces a
sigmoidal curve correlating the amount of Triton.RTM. X to the
percentage of cytotoxicity. Using standard curve fitting software
(such as a KaleidaGraph.RTM. available from Synergy Software) the
EC50 and other parameters commonly used to assess cytotoxicity can
be calculated.
Example 6
Exemplary Screen of Small Molecules as Modulators of
Angiogenesis
[0254] This example describes exemplary procedures for screening of
test agents as modulators of angiogenesis. A flow-chart
representation of an exemplary implementation of a screening
procedure is shown in FIG. 10. As shown in FIG. 10 a primary screen
of a library of small molecules is done using growth and tube
formation assays (exemplified above in Examples 2 and 3). This
primary screen identifies antiangiogenic compounds which in some
cases are cytotoxic. A counterscreen using the disclosed cell
viability assay (as exemplified in Example 5) is performed to
determine those compounds that are cytotoxic. Antiangiogenic
compounds which show little or no cytotoxicity are considered
putative antiangiogenic candidates and move forward to in vivo
studies.
[0255] In some examples, the growth of a cell line of interest is
determined in the presence of a test agent. In some example, the
growth of a mixture of cell lines of interest is determined in the
presence of a test agent. In some examples, this is done in a
multiwell format, such as a 96 well plate or a 384 well plate. For
example (as exemplified in FIG. 11), the assays are performed in 96
well format which contains negative controls (column 1), positive
controls (column 12) and 80 wells containing the small molecules to
be tested. A sample containing a mixture of the fluorescent cell
lines disclosed herein is provided in the wells of the multiwell
plate. Test agents are added to the plate either at a single
concentration or at graded concentrations, for example from about 1
picomolar to about 100 millimolar. The growth of the cells in the
presence of the test agent is determined, for example by
determining the fluoresce signal attributable to the fluorescent
cell line of interest in the well as compared to a control, for
example a control well in which no test agent has been added. FIGS.
12A and 12B shows a montage of micrographs representing an example
of one growth assay plate. As shown in FIG. 12, column 1 contains
cells that have not been stimulated, such that the cells do not
proliferate (the background autofluorescence of the cells is
measured that way) and column 12 shows growth of the endothelial
cells upon exposure to a growth factor cocktail. Test wells will
show different levels of cell growth or growth inhibition, based on
the fluorescence quantified from each well. Wells which contain
growth inhibitors (hits are defined using the SASD: sum of the
average squared inside-cluster distances, Gagarin et al. J. Biomol.
Screen 11:1-12, 2006) are shown in white boxes. Agents that cause a
measurable decrease in the growth of the fluorescent cell line of
interest are potential inhibitors of angiogenesis. Potential
inhibitors of angiogenesis can then be tested for there effect on
migration, and tubule formation, and for cytotoxicity using the
disclosed assays.
[0256] In some examples, the effectiveness of the small molecules
to inhibit tubule formation is determined. Exemplary methods for
determining the effect of an agent on tube formation is given in
Example 4. In a some assays, 50 .mu.l of low growth factor BME is
laid down in each well of a multiwell plate (such as a 96 well
plate) and the plate is incubated for 1 hour at 37.degree. C. Test
agents are added to the plate either at a single concentration or
at graded concentrations, for example from about 1 picomolar to
about 100 millimolar (in some examples the test agents are added
directly to the BME prior to plating). A cell mixture containing
about 15,000 fluorescent PAE cells is then added on top of the
gelled BME. The ability of the test agent to block tubule formation
is determined, for example by comparing the number of tubes or
related structures formed in a sample contacted with a test agent
relative to a control, such as a sample not contacted with a test
agent. In some examples, this is done by eye, for example by visual
inspection of the cells with a fluorescent microscope after 3.5-24
hours. In some examples, images are acquired with a fluorescent
microscope after 3.5-24 hours and stored, for example digitally. In
some examples, quantitative evaluation of the effectiveness of the
small molecules to block tube formation is assessed using the
AngioApplication.TM. software. AngioApplication.TM. can compute
multiple parameters which including but are not restricted to:
single tube length, single tube area, total tube length, total tube
area, node area, total number of tubes, total number of nodes,
single node branching points, total number of branching points,
average node branching points average tube length, average tube
area, average node area, etc. Test agents identified as capable of
inhibiting tubule formation are identified as potential
angiogenesis inhibitors.
[0257] Potential angiogenesis inhibitors can be screened for
cytotoxicity using the disclosed cytotoxicity assays, such as
exemplified by Example 5. Increasing concentrations of potential
angiogenesis inhibitors (such as from about 1 picomolar to about
100 millimolar) are added to cell mixtures containing fluorescent
cell lines with distinguishable emission spectra. This method
permits the cytotoxicity of a potential angiogenesis inhibitor on
multiple cell lines to be determined simultaneously. After about 20
minutes supernatants are collected and transferred to a different
plate and the fluorescence of the supernatants is measured at the
appropriate wavelength corresponding to the emission spectra of the
distinguishable emission spectra of the fluorescent proteins. The
measured emission spectra from each of the distinguishable emission
spectra is then used to determine the cytotoxicity of the potential
angiogenesis inhibitor on the fluorescent cell lines in the
mixture, for example by determining the EC50 of the potential
angiogenesis inhibitor on the individual cell lines in the mixture.
Potential angiogenesis inhibitors which show no or little
cytotoxicity are considered putative antiangiogenic candidates and
can move forward to in vivo studies.
Example 7
Tumor Stimulated Angiogenesis in 2D Co-Cultures does not Correlate
with Xenograft Angiogenesis
[0258] The stably-transfected fluorescent endothelial cells
described herein enable the detection of angiogenic cell activities
such as migration and tubule formation. Examples 3 and 4
demonstrate the stimulation of angiogenic activities in endothelial
cultures incubated with various angiogenic stimuli. This example
illustrates that tubule formation is analogously induced by tumor
cells in 2D co-cultures, but that the angiogenic potential of
particular tumor cell types in the 2D co-cultures does not
correlate with the angiogenic behavior of xenografts of the same
cell types.
Methods
[0259] Unless specified, all methods are as described in the
previous examples.
[0260] 2D Cell Cultures.
[0261] Tumor cells were grown in a 96-well plate in the medium
previously described such as RPMI1640, DMEM, or F-12K, in
accordance with the cell lines used herein, +10% fetal calf serum,
to approximately 70% confluence and gently washed three times in
PBS. 50 .mu.l gel matrix were added and solidified at 37.degree. C.
A 100 .mu.l aliquot of endothelial basal medium-2 (EBM-2) without
serum supplementation and containing BEC or LEC at between
150,000-300,000 cell/ml were then added on top of the solidified
gel matrix. Cultures were incubated at 37.degree. C. and resulting
tube formation determined in 4-6 hours.
[0262] Xenografts.
[0263] 1.times.10.sup.6 or 1.times.10.sup.7 tumor cells were
injected subcutaneously in the hind flank of a nude mouse. A
palpable mass is felt under the skin in 1-2 weeks having a tumor
volume of approximately 50-100 mm.sup.3. Mice were randomized into
groups of 10 mice/group having tumor volumes of 50-100 mm.sup.3,
and drug treatment was started at this time. Treatment was
continued for an additional 2-3 weeks or until tumors reached a
maximum volume of 2000 mm.sup.3. For biopsy studies, tumors were
excised at a volume <1000 mm.sup.3, gross morphology photographs
were taken and either sectioned in half for a second gross
morphology picture showing internal structure of tumor mass or a
core biopsy taken through the entire tumor nodule resulting in a
traversing "sausage" tissue sample having peripheral, mid section
and center anatomical regions that were ultimately sliced into
cross-sections and placed into the 3D drug sensitivity assay (see
Example 8).
Results
[0264] Induction of angiogenesis in the tumor microenvironment is a
multi-stage process, involving multiple factors and cell types
(FIG. 20). As shown in Examples 3 and 4, stably-transfected
fluorescent endothelial cells can be used to model angiogenic
activities in vitro in response to chemical stimuli provided in
culture medium. Tubule formation was monitored in Example 3 in 2D
cultures that were prepared with endothelial cells layered on top
of solidified gel matrix (FIG. 21 top right).
[0265] To determine the influence of tumor cells on endothelial
tubule formation, tubule formation was monitored in modified 2D
co-cultures of endothelial cells layered on top of gel matrix that
was solidified on top of a monolayer of a tumor cell line (FIG. 21,
top center). Using this modified 2D co-culture assay, tubule
formation in fluorescent PAE cells was stimulated by several
different tumor cell lines and observed after a six-hour incubation
(FIG. 22). Of the cell lines tested, ocular melanoma 92-1 cells
displayed the least tubule inductive potential, while lung
carcinoma A549 induced robust tubule formation.
[0266] The separate influences of three tumor cell lines (lung
carcinoma A549, pheochromocytoma PC-12 (CRL-1721), and ocular
melanoma 92-1) on tubule formation in three endothelial cell lines
(PAE, HMEC-1, and LEC-1) was similarly tested. As shown in FIG. 23,
A549 cells induced robust tubule formation in all endothelial cells
tested. In contrast, 92-1 cells did not stimulate tubule formation
in any of the cells tested.
[0267] One goal of the modified 2D co-culture assays was to develop
a mimetic of the in vivo effects of a tumor on angiogenesis. To
establish the correlation between the modified 2D co-culture
results and in vivo tumor activity, tumor xenografts were produced
in nude mice using lung carcinoma A549, pheochromocytoma PC-12, and
ocular melanoma 92-1 cells. Resultant tumors were excised and blood
vessel formation observed in the periphery (FIG. 24, top panels)
and interior (FIG. 24, bottom panels) of the tumors. PC-12 and 92-1
tumors had abundant vasculature both on the periphery as well as
the interior of the tumors. In contrast, the A549 tumor displayed
moderate vascularization on the periphery of the tumor, but little
vasculature in the tumor interior.
[0268] These xenograft results strongly diverge from the induction
of tubule formation observed in the modified 2D co-cultures. Thus,
the modified 2D co-cultures cannot serve as a mimetic of in vivo
tumor-induced angiogenesis.
Example 8
Recapitulation of In Vivo Tumor Activities in 3D Co-Cultures
[0269] As shown in Example 7, tumor cell-induced endothelial tubule
formation in modified 2D co-cultures does not correlate with
angiogenesis in a corresponding nude mouse xenograft model. This
example describes a 3D co-culture assay system that accurately
recapitulates the in vivo activity of tumor xenografts. Migration
of tumor cells along endothelial tubules, or angiotropism, was also
observed in the described 3D co-culture system. Thus, the 3D
co-cultures provide a model to monitor both angiogenic and
metastatic potential of a tumor.
Methods
[0270] Unless specified herein, methods were as described in the
preceding examples.
[0271] Tumor Spheroids.
[0272] Tumor spheroid colonies were prepared according to a
modified protocol based on Hamburger et al. (Science, 197:461-463,
1977). 2% SEAPLAQUE.RTM. Agarose (FMC BioProducts) was prepared in
deionized water and autoclaved. The agarose was cooled in a
40.degree. C. water bath. 10.times.RPMI 1640 medium
(Sigma-Aldrich), FBS, Antibiotic-Antimycotic (a.k.a. Anti-Anti),
and sterile deionized water (Invitrogen) were warmed in a
40.degree. C. water bath. Appropriate volumes of the deionized
water, 100.times. Antibiotic-Antimycotic, FBS, 10.times. RPMI1640
medium, and 2% agarose were mixed to make the final concentration
of 1% agarose with 20% FBS, 2.times. Antibiotic-Antimycotic, and
1.times.RPMI1640. 1.5 ml of the mixture was added to each well of
6-well plate (Corning) and set aside to solidify for 20 minutes in
the hood. In the meantime, tumor cells at approximately 70%
confluence were harvested and counted. Tumor cells were suspended
at 15,000 cells/ml in 0.2% of agarose, 2.times.
Antibiotic-Antimycotic, 20% FBS, and 1.times.RPMI1640. 3 ml of the
tumor cell suspension was added to the well with the first
solidified layer in the 6-well plate. This plate was left in the
hood for 10 minutes and then carefully transferred to an incubator
with 100% humidity for 20-30 days. The well-formed colonies were
harvested by adding 2 ml of 1.times.PBS to each well of a 6-well
plate and pipetted up and down for sufficient number of times until
the agarose was broken into tiny pieces. The colonies were washed
three times in PBS to get rid of the agarose residue. The colonies
were suspended in sterile PBS with 1% glucose, 0.3 mM EDTA, 0.5%
BSA and 1.times. Antibiotic-Antimycotic. The bright fluorescent
colonies were picked up using an Olympus inverted fluorescent
microscopy (Olympus, Japan) for 3D co-culture.
[0273] Xenograft Biopsy.
[0274] Xenografts were prepared as in Example 7. Tumor xenografts
were dissected when they reached about 1 cm in diameter and put
into the sterile 15 ml or 50 ml tubes (Corning) with RPMI1640
medium supplemented with 10% FBS, 1% Glucose (Sigma), and 4.times.
Antibiotic-Antimycotic (Invitrogen). The tumor xenografts were
placed on wet ice and shipped to the lab within 2-3 hours. The
Xenografts were rinsed 3 times in 70% ethanol and then 3 times in
PBS. In the hood, the core biopsy was performed by biopsy punch
(Miltex, Inc.) and was washed out into a 100 cm cell culture dish
(Corning) by using a pipette to blow the top opening of the biopsy
applicator. Using a disposable scalpel (Feather Safety Razor, Co.
Japan), the core biopsy was dissected in three stages. 1 mm of both
ends of the core biopsy was carefully cut off; they were
transferred into a new 100 cm cell culture dish labeled P
(peripheral section). Next, 1 mm of both ends of the remaining core
biopsy were removed and discarded. 1 mm of both ends of the core
biopsy were cut off and transferred into a new 100 cm cell culture
dish labeled M (middle section). Lastly, 1 mm of both ends of the
remaining biopsy tissue were removed and discarded. The rest of the
core biopsy was transferred into a new 100 cm cell culture dish
labeled C (center section). A drop of PBS was added to each section
P, M, and C to keep them moist. Using the disposable scalpel, each
section was cut into small pieces, approximately 10 pieces per 1 mm
section, under the dissection microscope (LeicaMZ125, Leica,
Germany). For the 3D co-cultures, each piece of the biopsy was
transferred to the center of the well of the second layer of the
gel matrix suspension comprising individual endothelial cells
and/or other component cells in a prepared 96-well plate kept on
wet ice. The plate was then placed in the incubator at 37.degree.
C. for 45 minutes to allow the gel matrix to solidify. The third
layer comprising the liquid medium was then added to the wells for
culturing.
[0275] 3D Co-Cultures.
[0276] 3D co-cultures were prepared as follows: 50 .mu.l 1%
SEAKEM.TM. LE agarose were added to individual wells of a 96-well
plate and allowed to solidify at room temperature for 20 minutes.
30 .mu.l GELTREX.RTM. gel matrix (Invitrogen) were combined with
PAE, LEC or HMEC-1 cells at a cell density of 560,000 cells/ml and
added to each well of the plate (atop the solidified agarose).
Plates were maintained on wet ice (4.degree. C.) to prevent
solidification of the gel matrix/cell mixture (the second layer). A
single tumor cell spheroid colony or single ringlet of xenograft
core biopsy was added to the center of the well and the matrix gel
was solidified at 37.degree. C. On top of the tumor/endothelial
cell gel layer, 80 .mu.l EBM-2+1% FBS were added to a final
concentration of EMB-2+0.5% FBS in relation to the total volume of
the culture. Cultures were incubated at 37.degree. C. for 5-20 days
and endothelial vessel network formation was observed by confocal
laser scanning fluorescence microscopy.
Results
[0277] Modified 2D co-cultures (Example 7) enabled observation of
induction of endothelial tubule formation by a tumor cell line.
However, angiogenesis in the modified 2D co-cultures did not
correlate with in vivo tumor activity in nude mouse xenografts.
[0278] In order to more accurately reproduce the tumor
microenvironment illustrated in FIG. 20, 3D co-cultures were
developed (FIG. 21, bottom). In the 3D co-cultures, tumor and
endothelial cells were mixed together in gel matrix (second layer)
and layered on top of agarose (first layer) that had been
previously solidified in the wells of a 96-cell culture plate.
Culture media (third layer) was provided on top of the
tumor/endothelial cell gel layer (second layer).
[0279] Employing the 3D co-culture assay, endothelial tubule
formation was observed in the presence of tumor xenograft tissue
(FIG. 25) and single tumor spheroid colonies (FIGS. 26-29). Tubule
formation was not observed in co-cultures between endothelial cells
and dispersed tumor cells, though endothelial cell migration was
observed.
[0280] In contrast to the 2D co-cultures, tubule formation in the
3D co-cultures recapitulated in vivo angiogenesis in the nude mice
xenografts. Specifically, ocular melanoma 92-1, which produced a
highly vascularized xenograft tumor, induced robust tubule
formation after a nine-day incubation (FIG. 26) in the described 3D
co-culture system. Moreover, lung carcinoma A549 cells, which
produced a xenograft tumor that was only poorly and peripherally
vascularized, induced moderate peripheral tubule formation after a
twelve-day incubation (FIG. 28) in the described 3D co-culture
system. Similar results were observed for both tumor cell lines
after a twenty-day incubation (FIG. 29). Thus, the described 3D
co-cultures provide an in vitro model for angiogenesis that
directly correlate with in vivo tumor activity.
[0281] Evaluation of highly metastatic human tumor cell lines such
as pheochromocytoma or melanoma in the 3D in vitro co-culture model
system also demonstrated migration of individual cancer cells along
vascular highways (FIGS. 27 and 29). This cellular migration
extended far beyond cellular branch projections from the main tumor
spheroid seed colony. Vessel-mediated cancer cell migration is
known as angiotropism, and has been reported to occur in
pathological specimens of human glioma/glioblastoma and melanoma
(Lugassy et al., Am. J. Dermatopath., 24:473-478, 2002; and Lugassy
and Barnhill, Adv. Anat. Pathol., 14:195-201, 2007). Thus, in
addition to providing an in vitro model for tumor-induced
angiogenesis, the 3D co-cultures also provide a model for tumor
metastasis.
Example 9
Methods of Testing Anti-Angiogenic Tumor Therapies Using 3D
Co-Cultures
[0282] The 3D co-cultures described herein provide an in vitro
model that correlates with in vivo tumor induction of angiogenesis.
With the 3D co-cultures, it becomes possible to design
individualized anti-angiogenic tumor therapies that are tailored to
best inhibit induction of angiogenesis by a tumor in a subject.
This example shows the testing of anti-angiogenesis treatments
using the 3D co-culture assay of endothelial tubule formation.
Methods
[0283] Unless specified, all methods were as described in the
previous examples.
[0284] 3D Cell Cultures.
[0285] 3D cultures were prepared as in Example 8, except EBM-2
medium was provided on top of the solidified tumor/endothelium/gel
layer containing final concentrations of 0.5% FBS and 0.1% DMSO,
plus angiogenesis inhibitor. Final drug concentrations were as
follows: Avastin.RTM. at 100 .mu.g/ml; Thalidomide at 100 .mu.M;
Sunitinib at 6 .mu.g/ml; and Fumagilin at 1 .mu.M.
Results
[0286] Tumor cell-induced endothelial cell tubule formation in 3D
co-cultures correlated with angiogenesis in tumor xenografts. The
3D co-cultures can therefore be used to monitor the in vitro
angiogenic potential of tumor tissue isolated from a subject. In
particular, the efficacy of multiple angiogenesis inhibitors in
vitro can be tested and monitored with a high degree of predictive
correlation with the in vivo context.
[0287] To examine the utility of the 3D co-cultures to test the
effects of an angiogenesis inhibitor, 3D co-cultures were prepared
combining stably transfected fluorescent PAE cells and biopsy
tissue from peripheral or central tissue of a leiomyosarcoma HTB-88
xenograft. The prepared co-cultures were incubated for six days
with the FDA-approved angiogenesis inhibitor Avastin.RTM.. As shown
in FIG. 30, Avastin.RTM. treatment inhibited both PAE proliferation
as well as tubule formation. Moreover, regional differences of
biopsy material (periphery, midsection, core) in the tumor
angiogenic potential were observed, with outer geographic tumor
areas always demonstrating dramatically more vessel induction than
inner tissue regions (FIG. 30, compare top and bottom panels).
[0288] Identifying the most efficacious therapy for a particular
patient is a crucial goal for successful cancer treatment. To
demonstrate the use of the 3D co-culture assay for tailoring
anti-angiogenesis treatment to a particular tumor from a particular
patient, the effect of several different angiogenesis inhibitors on
tubule formation in 3D co-cultures was tested. Several FDA approved
anti-angiogenic compounds were assessed in this capacity, including
Avastin.RTM., Thalidomide, and Sunitinib as well as the non-FDA
approved drug Fumagillin. After a five-day incubation, Avastin.RTM.
demonstrated the greatest inhibition of proliferation and tubule
formation.
[0289] Together, these observations validate the use of 3D
co-cultures to personalize anti-angiogenesis cancer therapy and
monitor the efficacy of the chosen treatment. The existence of
tumor heterogeneity was also observed.
Example 10
Testing Anti-Metastatic Tumor Therapies Using 3D Co-Cultures
[0290] Example 9 demonstrated that the 3D co-cultures described
herein can be used to test the efficacy of a panel of
anti-angiogenic tumor therapies for given subject. The tumor cell
angiotropism observed in FIGS. 27 and 29 indicates that tumor
metastasis can also be detected using the 3D co-cultures. This
example provides a method for optimizing anti-metastatic therapy to
inhibit metastasis in a subject.
[0291] 3D co-cultures can be established as described herein. For
example, co-cultures can be prepared in several wells of a 96-well
culture plate. Tumor cells used in the co-culture can be a sample
of a tumor biopsy from a subject or a spheroid colony derived from
a tumor biopsy from a subject. As with the anti-angiogenic
compounds tested in Example 9, one or more anti-metastatic
compounds can be combined with culture medium and applied to some
but not all of the established 3D co-cultures. The 3D co-cultures
are incubated for any length of time sufficient to detect
angiotropism, such as nine days, and angiotropism observed under a
microscope. The relative efficacy of an anti-metastatic compound is
determined by the comparative inhibition of angiotropism in
relation, for instance, to the 3D co-culture that did not receive
any anti-metastatic compound. Those compounds with the strongest
inhibitory effect on angiotropic cell motility (for example, the
distance moved from core tumor cell colony) are most
efficacious.
Example 11
Personalized Anti-Angiogenic or Anti-Metastatic Cancer Therapy
[0292] This example provides representative methods of selecting
and monitoring the efficacy of an anti-angiogenic or
anti-metastatic cancer therapy for a specific subject.
[0293] Using the 3D co-cultures described herein, cancer therapy
can be tailored to a specific subject and the effectiveness of the
therapy monitored over time. A tumor biopsy or other cancer cell
sample from a patient (the target patient or target subject) can be
the source of cancerous tissue for incorporation into multiple 3D
co-cultures. As described Examples 9 and 10, the 3D co-cultures can
be used to select the most efficacious anti-angiogenic and/or
anti-metastatic compound for the target patient, from among a panel
of anti-angiogenic or anti-metastatic drugs. Additionally, the
effects of combining the selected anti-angiogenic and
anti-metastatic drugs can also be observed. The selected
compound(s) can then be administered to the target patient as part
of an anti-cancer therapy regimen.
[0294] The continued efficacy of the selected compound(s) can be
monitored by obtaining a tumor biopsy from the subject after a
given period of time, for example three months, and preparing 3D
co-cultures to observe the effect of the administered treatment on
angiogenesis and/or metastasis. If the development drug resistance
is observed, a new personalized treatment can then identified using
the foregoing methods.
[0295] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that the illustrated embodiment is only a preferred example of the
invention and should not be taken as a limitation on the scope of
the invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
* * * * *