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 CFSE+ fractions lost fluorescence intensity indicating that they actively cycled after transplantation. Hoechst/Pyronin-staining of dissociated sphere cells allowed investigation of their cell cycle status. Equal numbers of G1-, S/G2/M- and G0-cells were transplanted under the kidney capsule of immunodeficient mice. Each cell fraction comprised self-renewing, human colon TIC as shown by a serial transplantation assay. In order to investigate the proliferative activity of human colon TIC within an established tumor in vivo, intra-tumoral cell divisions were tracked using a genetic high resolution label-retaining assay. A tetracycline-regulated H2B-GFP expression system was implemented into spheroid cells by lentiviral transduction prior to transplantation. H2B-GFP-expression was suppressed after establishment of the tumor microenvironment. Further cell divisions dilute the GFP-label and thereby enable Summary II analysis of the cell’s proliferative activity. FACS analysis of formed tumors revealed fast, slow and rarely dividing cell fractions in vivo. All cell fractions harbored selfrenewing, human colon TIC as shown by serial transplantation. Interestingly, only quiescent TIC showed a polyclonal contribution to tumor formation in mice. A proportion of quiescent TIC might have been activated to proliferate upon chemotherapeutic treatment. This study demonstrates that human colon cancer harbours tumor initiating cells with differing cell cycle status and proliferative activity. Self-renewing colon TIC were present in all cell cycle phases demonstrating that the tumor initiating potential is not restricted to a dormant cell cycle status. A rarely dividing population of human colon TIC derived from different patient samples exists in vitro and in vivo. However, the majority of colon TIC rapidly divided in sphere cultures as well as in vivo. Colon TIC were found to be enriched in the quiescent population and were recruited to tumor formation upon chemotherapeutic treatment. Our results provide basis for a better understanding of quiescence and proliferation of human colon TIC. This will hopefully lead to the development of innovative treatment strategies directed against colon cancer initiating cells.
|Supervisor:||Glimm, Prof. Dr. Hanno|
|Date of thesis defense:||20 September 2013|
|Date Deposited:||17 Oct 2013 11:44|
|Faculties / Institutes:||The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences|
|Subjects:||500 Natural sciences and mathematics|