Abstract 3379: Adaptation of soft agar colony formation assays to identify agents which block proliferation of putative colon cancer stem cells

Abstract

Abstract Cancer stem cells have been the focus of renewed interest in recent years. In theory these cells have the potential to limit the success of therapy, serving as therapy-resistant reservoirs of disease. In a previous study (Proc AACR 2011 #5202), each of seven human colon cancer cell lines was found to contain a subset of single cells, each of which possesses putative cancer stem cell properties: the capacity (a) to initiate and sustain highly prolific clonal growth in vitro as well as (b) to form tumor xenografts which are readily transplantable in vivo. Despite the extensive in vitro propagation of each human colon cancer cell line established from patient tumors many years ago, they have retained the functional capacity to initiate and sustain cancer cell growth from single cells both in vitro and in vivo. Because soft agar cultures require the clonal growth of single cells and permit the detection of stem-cell derived colonies on the basis of cumulative colony masses, we explored the utility of low cell inoculation density cultures and the stratification of colony data into multiple size categories as a means to identify agents which modulate cancer stem cell function. In the current study, HCT-116 and HT-29 parent cell lines were employed to evaluate the activity of selected differentiating agents, a stem-cell inducing agent, cytotoxic agents, and a battery of clinical and experimental therapeutic agents postulated to modulate cancer stem cell function in a three-step strategy: (1) testing each agent in 96-well plate colony formation assays; (2) a comparison of the indices of drug activity in colony formation assays with indices of drug activity measured in the NCI60 screen and identification of agents which are more active in blocking colony formation than inhibiting monolayer culture growth; (3) an evaluation of the highly active agents in a conventional 35-mm dish colony formation assay in order to quantify putative anti-cancer stem cell activity. Of 41 agents tested, 11 agents were observed to exhibit prominent effects upon colony formation at concentrations which were ½ - 1 log less than that required for the inhibition of growth in monolayer cultures. In contrast to cytotoxic agents which indiscriminately impact the growth of all colony categories, these 11 agents were differentially active against stem-cell derived colonies. Moreover, it is noteworthy that several clinical agents were highly active following long exposure to fractions of concentrations achievable in human patients. These results suggest that the soft agar colony formation assay of established human cancer cell lines in which cell inoculation and analysis parameters are optimized for the measurement of long-term growth in low cell inoculation density cultures provides a sensitive method for detecting agents which impact putative cancer stem cell function. (Supported in part by NCI Contract No. HHSN261200800001E) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3379. doi:1538-7445.AM2012-3379