| Objective:Colorectal cancer is one of the most prevalent malignancies.Liver metastasis is the most common form of distant metastasis of colorectal cancer.The aim of this study was to analyze the effects of bone marrow-derived hematopoietic progenitor cells(HPCs)on the biological characteristics of colon cancer stem cells(CCSCs),and to investigate the molecular mechanisms underlying liver metastasis of colorectal cancer at the cellular level.Methods:In this study,we firstly isolated and identified the cell subpopulations of the colon cancer cell line HCT116 using the culturing method of serum-free medium(SFM),and explored the feasibility of this method to isolate the subgroups of the colon cancer stem cells(CCSCs).Then we co-cultured the subgroups of CCSCs with CD133 positive hematopoietic progenitor cells(HPCs)in vitro to study the change of their biological characteristics(transmigration,invasion,and clone proliferation).To observe the formation of "tumor cell spheres",the colon cancer cell line HCT116 was cultured using the method of SFM.The ratio of CD133 and CD24 positive cells in SSM and tumor cells in SFM,respectively,was detected by flow cytometry.The different of protein expression of CD24 positive cells and CD133 positive cells were detected using immunofluorescence.CCSCs subpopulation was co-cultured with CD133+ HPCs in vitro at a ratio of 20:1 for 48 h.The effect of on the proliferation changes of CCSCs subpopulation was observed using the methods of CCK-8 and plate colony.The alters of the cell migration and invasion were tested using the method of Transwell chamber.And the expression of genes and proteins related to tumor invasion and metastasis(MMP-9,VEGF)was detected by qPCR assays and Western blots.Results:The colon cancer cell line HCT116 was found to be suspended and growed in spherical status cultured in the method of SFM,and be passaged stably.Flow cytometry showed that the ratios of both CD133(P=0.000)and CD24(P=0.001)positive cells in tumor cell spheres were significantly higher than that in the monolayer cells.Immunofluorescence showed that the expression of CD133 and CD24 protein in tumor cell spheres was also stronger than that of monolayer cells.The effect of HPCs on the various changes of CCSCs subpopulation under co-culture conditions was observed as the following.CCK-8 and platelet cloning assays showed that the cell proliferation and colony formation of CCSCs subpopulation was induced and enforced(P=0.031).Migration experiments and invasion experiments by Transwell chamber showed that both migration ability(P=0.011)and invasive capacity(P=0.001)were enhenced.qPCR and Western blot results showed that the expression levels of genes(P=0.000)and proteins(P=0.005)in certain markers(MMP-9,VEGF)were both significantly increased.Conclusion:The culturing method of SFM can be used to form and isolate CCSCs subpopulations in colon cancer cell line HCT116.Cell migration,invasion,and proliferation of the CCSCs subpopulation can be induced and enhanced by CD133+ HPCs. |
PDF Full Text Request