# US 10,865,388 B2 · Method for identifying anti-cancer agents using an in vitro cell culture system that maintains cancer cell stemness > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,865,388 B2 - Document ID: US10865388B2 - Status: Granted - Kind: B2 - Application number: 15/513,804 - Filed: 2014-09-24 - Published: 2018-08-16 - Granted: 2020-12-15 - Earliest claimed: 2014-09-24 - Assignees: National Taiwan University - Applicants: National Taiwan University - Inventors: Pan-Chyr Yang; Huei-Wen Chen; Wan-Jiun Chen - Web page: https://patentgrep.com/patent/US10865388B2 ## CPC - C12N 5/0695: {Stem cells; Progenitor cells; Precursor cells} - A61P 11/00: Drugs for disorders of the respiratory system - A61P 35/00: Antineoplastic agents - C12N 5/0693: {Tumour cells; Cancer cells} - G01N 33/5011: {for testing antineoplastic activity} - G01N 33/5017: {for testing neoplastic activity} - G01N 33/575: for cancer - G01N 33/5759: {involving compounds localised on the membrane of tumour or cancer cells} - G01N 33/57595: {involving intracellular compounds} - C12N 2501/605: Nanog - C12N 2502/30: tumour cells - G01N 2333/4706: stimulating, promoting or activating activity - G01N 2333/475: Assays involving growth factors - G01N 2333/4753: Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II - G01N 2333/52: Assays involving cytokines - G01N 2333/71: for growth factors; for growth regulators - G01N 2333/715: for cytokines; for lymphokines; for interferons - G01N 2333/7158: for chemokines - G01N 2800/52: Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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 An in vitro co-culture system comprising cancer-associated fibroblasts (CAFs) and cancer cells for producing and maintaining cancer stem cells and uses thereof for identifying agents capable of reducing cancer cell stemness. Also disclosed herein are a paracrine network through which CAFs facilitate production and/or maintenance of cancer stem cells and the use of components of such a paracrine network for prognosis purposes and for identifying cancer patients who are likely to respond to certain treatment. ## Claims ### Claim 1 A method for identifying an anti-cancer drug, comprising: providing an in vitro co-culture system comprising cancer associated fibroblasts (CAFs), a population of cancer cells, and a drug candidate; wherein the CAFs are CD90+, wherein in the in vitro co-culture system, the ratio between the cancer cells and the CAFs ranges from 1:100 to 1:10; and wherein the CAFs maintain or induce stemness of the cancer cells in the co-culture system; determining the level of cancer cell stemness in the co-culture, and identifying the drug candidate as an anti-cancer drug, if the level of cancer cell stemness in the co-culture is decreased as compared to that of a co-culture of cancer cells and CAFs of the same type as those in the in vitro co-culture system in the absence of the drug candidate. ### Claim 2 (depends on 1) The method of claim 1, wherein the population of cancer cells in the in vitro co-culture, system are from an established cancer cell line, or wherein the cancer cells are primary cancer cells obtained from a cancer patient. ### Claim 3 (depends on 1) The method of claim 1, wherein the population of cancer cells are lung cancer cells, breast cancer cells, kidney cancer cells, prostate cancer cells, ovary cancer cells, skin cancer cells, cervical cancer cells, colon cancer cells, liver cancer cells, melanoma cells, oral cancer cells, or pancreatic cancer cells. ### Claim 4 (depends on 1) The method of claim 1, wherein the CAFs in the in vitro co-culture system are obtained from a cancer patient. ### Claim 5 (depends on 1) The method of claim 1, wherein the level of cancer cell stemness is determined by: measuring the level of Oct3/4+ and Nanog+ cells in the co-culture; measuring the drug resistance of the cancer cells in the co-culture, measuring the level of one or more of IGF-II, HGF, LIF, and SDF1 expressed in the CAFs, the level of one or more of IGF1R, IGF2R, LIFR, CXCR4, and Nanog expressed in the cancer cells, or both, or measuring the ratio of cancer stem cells (CSCs) to total cells in a cancer cell colony formed in the co-culture. ### Claim 6 (depends on 1) The method of claim 1, wherein the determining step comprises: (i) staining the in vitro co-culture system comprising the CAFs and the cancer cells with a first agent for detecting Nanog, wherein the first agent is conjugated with a first label directly or indirectly; and a second agent for detecting CD90, wherein the second agent is conjugated with a second label directly or indirectly, the second label being different from the first label; (ii) imaging the CAFs and the cancer cells stained with the first and second agents; and (iii) determining stemness of the cancer cells in the in vitro co-culture system based on the signals released from the first and second labels. ### Claim 7 (depends on 1) The method of claim 1, wherein the population of cancer cells are non-small cell lung cancer cells. ### Claim 8 (depends on 7) The method of claim 7, wherein the CAFs are obtained from a non-small cell lung cancer patient. ### Claim 9 (depends on 6) The method of claim 6, wherein the first label, the second label, or both are fluorescent dyes. ### Claim 10 (depends on 6) The method of claim 6, further comprising staining the in vitro co-culture system with a third agent for detecting nuclei.