Screening Molecular Markers Of Colorectal Cancer Metastatic Cancer Stem Cells By Gene Chips

BackgroundMetastasis has been the most important factor in the recurrence and cure of colorectal cancer patients.At present,the main mechanisms of tumor metastasis are tumor stem cell theory,epithelial-mesenchymal transition(EMT),tumor cell apoptosis and tumor organ-specific metastasis.Among them,the theory of cancer stem cell(CSC)is a hot spot for studying tumor metastasis recently.Accumulating evidences suggest that cancers may have a small population of cells that have the distinctive capability to start and maintain tumor's growth and heterogeneity in successive transplantation experiments.The most likely source of normal stem cells is cancer stem cells[1].Researchers believe that metastatic cancer stem cells are subpopulations that belong to cancer stem cells and play a crucial role in the metastasis of colorectal cancer[2].The most important difference between metastatic cancer stem cells and cancer stem cells is their potential to form metastases[3].At present,researchers have matured on markers of CSCs in colorectal cancer.However,the research on MCSC has just started,looking for the key markers of MCSC in order to more accurately decrypt the mechanism of tumor metastasis.Objects1.Select colorectal cancer cell lines for three-dimensional cultivation of cancer stem cell clones.2.Animal experiments to simulate the process of tumor metastasis in colorectal cancer by orthotopic implantation of cecum..3.Identify the key markers of colorectal cancer metastasis stem cells.MethodsWe adopted the method of three-dimensional culture of serum-free stem cells and in situ implantation of nude mice to obtain high-purity MCSC.This not only solves the problem of limited access to MCSC but also increases the purity of the obtained MCSC.This is the innovation of this experiment.Next,through gene chip technology,it was found that there was indeed a difference in gene expression between the primary cancer foci of metastatic nude mice and other organ metastases,and genes related to MCSC were screened out.The resulting differential gene was then verified by fresh human specimens,which was consistent with the results obtained from the nude mouse samples.Finally,the expression of differential genes at the protein level was obtained by immunohistochemistry.Results1.Enrichment of tumor stem cell sphere:The tumor sphere is formed when cultured for 5-6 days.Cells are mulberry-like with good refractivity and closely arranged cells,which can not accurately distinguish the boundaries between cells.2.Establishment of MCSC in vivo metastasis model:Approximately 500 tumor spheres were subcutaneously injected into nude mice,and all the nude mice developed subcutaneous tumors with a tumorigenic rate of 100%.Establishment of orthotopic implantation model in nude mice:4/6 nude mice in group H2 showed metastases,3/6 metastases in group C2,and 4/6 metastases in group S2.3.Differential Expressed Gene Analysis:Three differential expressed mRNAs:CRYAB,KCNMA1,SERPING1 were found by comparison.Since our group has previously studied this LncRNA involved in the regulation of colorectal cancer metastasis,the focus of the first three mRNA analysis.4.Real-time fluorescence quantitative PCR and immunohistochemistry results:We verified the expression of CRYAB,KCNMA1,and SERPING1 in metastases higher than those in primary tumors in both animal samples and fresh human samples using real-time fluorescence quantitative PCR.The difference was statistically significant(P<0.05).Subsequently,we confirmed by immunohistochemistry that the positive rates of CRYAB,KCNMA1,and SERPING1 in metastatic cancer tissues were:60%,80%,and 90%,respectively,which were higher than those in primary cancer tissues.The difference was statistically significant(P<0.05).Conclusions1.Successfully cultivated colorectal cancer stem cell cloned spheres.The tumorigenicity of the nude mice subcutaneous transplantation model established using it was 400 times stronger than that of ordinary cell lines.2.Successfully established a cecal in situ implanted cancer metastasis model in nude mice.3.Three differential expressed genes CRYAB,SERPING1 and KCNMA1 in primary cancer CSC and metastatic cancer tissue MCSC were screened by mRNA microarray and further verified that they are associated with the metastasis of colorectal cancer,but further functional and mechanism studies are still needed to identify whether they are colorectal cancer MCSC markers.