In human colon cancer only a small subfraction of all tumor cells is able to rebuild the tumor in immunodeficient mice. It has been hypothesized that the proliferative activity of these tumor initiating cells (TIC) may differ from the bulk of the tumor cells and that mitotic quiescence of TIC may contribute to chemotherapy resistance or relapse after treatment. By genetic marking, we have previously shown that a variable proportion of all human TIC contributed to tumor xenograft formation only late after serial transplantation suggesting that these delayed contributing TIC indeed might have been quiescent in the primary recipient mice. We now investigated the proliferative activity of transplantable TIC in sphere cultures derived from primary human colon cancer samples. Samples from human colon cancer tumors were dissociated and cultured under serum free conditions that favor the outgrowth of tumor spheres that are enriched with TIC. Dissociated sphere cells from two individual patients (TSC01, TSC03) were stained using the fluorescent dye CFSE. Precise halving of the fluorescence intensity during each cell division allows us to define subfractions by FACS according to their proliferative activity. In both patients, fractions of fast (F), slow (S) and non-dividing (N) cells were distinguishable at day 8 after CFSE staining. Cell surface markers previously associated with tumor-initiating potential (CD133, CD44 and CD166) were equally expressed in all cell fractions. Sphere forming capacity was quantified in vitro. The frequency of sphere forming cells (SFC) was similar in the fast, slow and non-dividing fractions (TSC03: 1 SFC in 23 to 1 in 61 and TSC01: 1 SFC in 1,2 to 1 in 2,1), demonstrating that the vast majority of all SFC were rapidly cycling in vitro. SFC frequency as well as the kinetics of sphere formation remained stable after serial replating. To assess the in vivo tumor initiating potential and self-renewal ability, equal cell numbers of sorted N, S and F cells were transplanted into immunodeficient mice. All sorted cell fractions of both patients formed tumors, irrespective of their proliferative kinetics in vitro. Moreover, the majority of cells within serially transplanted tumors originating from CFSE+ fractions lost fluorescence intensity indicating that they actively cycled after transplantation. Our results demonstrate that functional assessment of CFSE stained primary human colon cancer spheres allows discriminating TIC subfractions with differing proliferative activity. The majority of all colon cancer TIC from two patient samples rapidly divided in sphere cultures suggesting that the tumor initiating potential is not tightly linked to mitotic quiescence.

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 5341. doi:1538-7445.AM2012-5341