Cell Cycle and Proliferative Activity of Human Colon Cancer Initiating Cells

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, it has previously been 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 primary recipient mice. In order to investigate the cell cycle and proliferative activity of human colon TIC in vitro and in vivo, human colon cancer patient samples were dissociated and cultured under serum free conditions favoring the outgrowth of tumor spheres enriched for TIC. The CFSE label-retaining assay was used to analyze the proliferative activity of human colon TIC in vitro. It allowed discrimination of fast (F), slow (S) and rarely dividing (R) cell fractions suggesting that a rarely dividing population of human colon TIC might exist in vivo as well. Cell surface markers previously associated with tumor initiating potential (CD133, CD44, EpCAM and CD166) were equally expressed in all proliferative subfractions. A limiting dilution assay confirmed the self-renewal potential of spheroid cells. Furthermore, it revealed that the frequency of sphere forming cells (SFC) was similar in the fast, slow and rarely dividing fraction within individual sphere lines, demonstrating that the vast majority of all SFC were rapidly cycling in vitro. To assess the in vivo tumor initiating potential and self-renewal ability, equal cell numbers of sorted R, S and F cells were transplanted into immunodeficient mice. All sorted cell fractions of three patients formed tumors, irrespective of their proliferative kinetics in vitro. Moreover, the majority of cells within serially transplanted tumors originating from ...