Isolation And Biological Function Of The New Subpopulation NCCC In Colorectal Cancer

Objectives: In living cells, we will find an accurate and effective system that coulddistinguish the ki67low/-subpopulation from ki67high, which will facilitate the research ofki67low/-subpopulation’s function.Methods: We will construct a lentivirus vector in which ki67promoter drives GFPexpression, and produce lentivirus for infecting the tumor cells. Then, we pick up thepositive clones and give subcutaneous injection of null mice. After4-6weeks, we will killthe mice and get the xenograft tumors, which then are digested into single cell suspensionby collagenase II. We sorted the GFPlow/-subpopulation from GFPhighby FACS, and thendetected the endogenous ki67expresion by double parameter.Results: We successfully constructed the lentivirus system that ki67promoter drive theGFP expression. Using the lentivirus system, GFPhighcells have higher expression of ki67and cyclins, while GFPlow/-no or lower expression of ki67and cyclinsConclusions: the ki67-driving GFP expression lentivirus system could distinguish theki67low/-subpopulation from ki67higheffectively. Objective: we will compare the properties of cancer stem cell between ki67low/-and ki67highsubpopulation, such as tumorigenicity, self-renew, metastasis and chemoresistance.Methods: In the part one, we could sort the ki67low/-（GFPlow/-）and ki67high（GFPhigh）, andthen compared its tumorigenicity by clone formation and self-renew by serial sphereformation assay. In addition, we will inject the single cell suspension into the subcapsularregion of spleen, after4weeks, mice were killed and observed. For chemoresistance,five-week-old BALB/C nude mice were injected subcutanously in both flanks with twoindependent sorted cells, and one million cells suspended in50μl PBS were injected ineach flank, meanwhile we also inject the5-Fu into abdominal cavity. After4weeks, wewill kill the mice and observe the number and size of tumors.Results: the subpopulation of ki67low/（-GFPlow/-）has a more remarkable tumorigeneity andself-renew than ki67high（GFPhigh）. in addition,the subpopulation of ki67low/-（GFPlow/-）iseasier to colonize in liver. Finally, the subpopulation of ki67low/-（GFPlow/-）is difficult to bekilled by chemotherapeutic drugs.Conclusions: the subpopulation of ki67low/-（GFPlow/-）has a more significant cancer stemcell related features than ki67high（GFPhigh）. Objective: we will confirm the existence of the subpopulation of ki67low/-in cliniccolorectal cancer, and disclose the relationship among the expression of ki67, tumorpathology and patients survival.Methods: we collected the clinic samples of stage III in colorectal caners in2005, andinvestigated its clinic information and5-years survival time. Meanwhile, we detected theki67expression by immunohistochemistry (IHC), and found the relationship between ki67expression and tumor pathology and5-years survival time.Results: In clinic colorectal cancer, there is the subpopulation of ki67low/-in deed. Incolorectal cancer of stage III, the expression of ki67is not related with sex, age andcharacter of tumor (mass or invasive), while the tumors with the lower expression of ki67often exhibited the poor differentiation and easy metastasis. At the same time, the patientswith the lower expression of ki67have a shorter survival time.Conclusion: the subpopulation of ki67low/-exists in clinic colorectal cancer; the lowerexpression of ki67indicates a poor prognosis.