Analysis Of Cancer Cell Populations By Isolated Single Colonies For The Identification Of Drugtolerance Inducing Factors

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC [Background] Functional heterogeneity within cancer cell populations plays an important role in responses to genotoxic stresses caused by anticancer agents. We have characterized 2,400 colonies derived in the presence of anticancer drugs using human cancer cell lines by newly introduced colony lysate array technique (CoLA). In the present study, we attempted to identify molecular components that could induce anticancer drug-tolerance phenotypes. [Materials and Methods] Using five cancer cell lines and four anticancer agents, CoLA was produced using 2,400 drug-tolerant colonies, followed by protein expression analysis employing specific immunodetection and using a panel of primary antibodies. Transcriptional products that may be important to the formation of colonies in the presence of anticancer drugs were identified by DNA microarray. Candidate genes were examined to determine whether they were associated with the suppression of colony formation, using gene knockdown by siRNA. [Results] This study revealed that CoLA is capable of analyzing lysates from individual colonies in a quantitative manner, by a panel of protein markers. Colonies that expressed pluripotent and CSC markers tended to express low levels of epithelial proteins. However, individual protein expression levels were not well-associated with drug concentrations, suggesting that the phenotypes of drug-tolerant colonies were not necessarily induced by drugs, and emerged spontaneously. Subsequent analysis identifying molecular fractions important to drug-tolerant phenotypes revealed that transcriptional products play an important role in developing these phenotypes. DNA microarray analysis allowed for the narrowing down of 12 genes that are potentially associated with these phenotypes. A gene knockdown by siRNA identified two out of 12 candidate genes as involved in the suppressed colony formation of MCF7 human breast cancer cell line in the presence of cisplatin. [Discussion] CoLA assay revealed that a number of CSC marker-negative colonies exist in the presence of anticancer drugs, suggesting that drug-tolerant cells may not always be associated with CSCs. In the context of drug-tolerant phenotype acquisition, protein expression may merely be a handy marker, whereas transcriptional control may be more critical. Further functional analysis of candidate genes for colony suppression in the presence of anticancer drugs is necessary. Together, de novo drug-tolerance inducing factors are possibly involved in the heterogeneity of cancer cell populations. Citation Format: Kohei Kume, Satoshi Nishizuka, Miyuki Ikeda, Sawako Miura, Yuriko Wada, Shuji Fujikawa, Chihaya Maesawa, Go Wakabayashi. Analysis of cancer cell populations by isolated single colonies for the identification of drug-tolerance inducing factors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4060. doi:10.1158/1538-7445.AM2013-4060