# US 2014/0155292 A1 · Methods of monitoring angiogenesis and metastasis in three dimensional co-cultures

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- Number: US 2014/0155292 A1
- Document ID: US20140155292A1
- Status: Published application
- Kind: A1
- Application number: 14/174,610
- Filed: 2014-02-06
- Published: 2014-06-05
- Earliest claimed: 2007-10-01
- Assignees: The United States of America, as represented by the Secretary, Department of Health and Human Services
- Applicants: The United States of America,as represented by the Secretary,Department of Health and Human Services
- Inventors: Changge Fang; Enrique Zudaire; Frank Cuttitta
- Web page: https://patentgrep.com/patent/US20140155292A1

## CPC

- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/5005: {involving human or animal cells (immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025)}
- G01N 33/5017: {for testing neoplastic activity}
- G01N 33/5044: {involving specific cell types}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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.

## Claims

### Claim 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.

### Claim 2 (depends on 1)

The method of claim 1, wherein the neutral polysaccharide polymer gel comprises agarose.

### Claim 3 (depends on 1)

The method of claim 1, wherein the endothelial cells stably and constitutively express a fluorescent protein.

### Claim 4 (depends on 3)

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.

### Claim 5 (depends on 3)

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.

### Claim 6 (depends on 4)

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.

### Claim 7 (depends on 1)

The method of claim 1, wherein the second layer further comprises at least one additional mammalian cell type dispersed in the solidified gel matrix.

### Claim 8 (depends on 7)

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.

### Claim 9 (depends on 1)

The method of claim 1, wherein the first, second, or third layer further comprises at least one test agent.

### Claim 10 (depends on 9)

The method of claim 9, wherein the test agent is a known or potential inhibitor of angiogenesis or metastasis.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 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.

### Claim 14 (depends on 13)

The method of claim 13, wherein the neutral polysaccharide polymer gel comprises agarose.

### Claim 15 (depends on 13)

The method of claim 13, wherein the endothelial cells stably and constitutively express a fluorescent protein.

### Claim 16 (depends on 15)

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.

### Claim 17 (depends on 15)

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.

### Claim 18 (depends on 16)

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.

### Claim 19 (depends on 13)

The method of claim 13, wherein the second layer further comprises at least one additional mammalian cell type dispersed in the solidified gel matrix.

### Claim 20 (depends on 19)

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.

### Claim 21 (depends on 1)

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.