Selection And Identification Of Liver-metastasis Subpopulation From Colorectal Cancer Cell Line And Its Protein

The occurrence and development of tumor is a complex process which refers tomore than one factor involved, multiple genetic changes, the development ofmulti-step. In the process of evolution of tumor cells, genetic instability of tumor cellsand the pressure to choose the host environment resulted in constant cell mutationwhich leads to divisive and phenotypic diversity. Therefore, in the same tumor mass, there are subcloned cells which contains not exactly the same biological character-istics and metastatic abilities. Metastasis of tumor cells is the result of the interactionwith the host which is a highly selective process where only the cell sublines withmetastatic potential can metastasis and these tumor cells have a certain organizationaltendencies, which is the result of intersection between tumor cells and organ specificmicro- environment. Therefore, It is necessary to isolate and identify the subclonedtumor cells with different direction of metastasis, and compare and analyze thedifferences in biological characteristics in a targeted manner, which has importanttheoretical and practical significance, is helpful to investigate the mechanism ofmetastasis and to find an effective way of prevention and treatment of tumors.Human cancer metastasis model of orthotopic transplantation strategy is animportant theoretical basis for the study of invasion and metastasis of human cancer. Research shows that the ability of human tumor cell invasion in vivo needs not onlythe right environment of transplantion, but also the interaction between the tumorcells, stromal cells and tumor cells, tumor cells and the host. Therefore, in situ tumortransplant model, a complete tumor tissue have a high incidence of metastasiscompared with the tumor cell suspension. Tissue surgical orthotopic implantationmodel for the transfer has high credibility and the incidence of metastasis haveclinical similar time-dependent manner, instead of transplanting process of celldisseminated. Nude mice can eliminate the effect of the host immune surveillance onthe tumor cells, the tumor microenvironment and orthotopic transplantation is similarto that of the primary organs. Therefore, the nude mice with transplantation of tumourtissue in situ can make tumor cells compatible with micro-environment of the host, sothat the biological behavior of tumors and natural attributes can be fully expressed.According to the characteristics of the tumor metastasis, this study found the cellsublines of liver metastases SW480/M5 from colon cancer cell line SW480, whichhas many metastatic potential through nude mice with many successive generations, and identified the biological characteristics of SW480/M5 in the control of SW480and SW620 which have homology with SW480. First we tested metastasis of thethree types of cells in vivo by surgical orthotopic implantation methods, the resultsshowed: SW480 have the potential of multiple-organ transfer, liver, lung, lymph nodemetastasis all occurred. Particularly peritoneal metastasis was stronger than the othertwo cells; SW480/M5 only have the ability of liver metastasis, and SW620 only havethe ability of lymph node metastasis, which showed the three different cells haddifferent tissu tendencies, SW480 from the original tumor, contained a number ofdifferent subcloned cells; SW480/M5 and SW620 were the cell line from metastaseslesions. They have a tendency liver and lymph node metastasis respectively.Therefore, We can think SW480/M5 and SW620 cells were two subgroups of SW480 cells. Although the SW480/M5 and SW620 mice occured distant metastasis, it had abetter mental state due to the absence of peritoneal metastasis, the relatively smalldistant metastases and not very serious exertion. Although we killed these nude micesat the same time, we believe that the node mice transplanted SW480/M5 and SW620tummoue tissue keep a long survival period than SW480, the model of colon cancerestablished with these two cells should be ideal model of clinical treatment on lymphnode and liver metastasis of colon cancer.The most basic steps of tumor metastasis is that tumor cells movement aroundthe organization and vascular system. Genetic mutation makes some cells have strongmovement ability, and in the early stages of the disease only a small part of the cellsubcolones had this capacity. The test compared the movement capacity of the threecells using the Transwell method, Transwell into two grooves from up to down, andseparated with 8μm microporous membrane, in the above one we added incompletemedium without serum, and in the lower we added the complete medium containingserum. The cell must possess the ability of deformation and movements, which canpass through the porous membrane. The comparison of cells number through the cellmembranes can show the different movement abilities. Our results showed themovement ability of SW480/M5 cells is stronger than of SW480 and SW 620 cells.However, in vivo tumor cells can meet a series of natural barriers, such as connectivetissue matrix, basement membrane, and tumor cells can not pass through thesebarriers without secretion of the enzyme which can degradate matrix and basementmembrane. In the test we compared invasion capacity of three cells by Cell InvasionAssay Kit, Cell Invasion Assay Kit is the same with Transwell in the structure, theonly difference is that there is a thin layer the artificial basement membrane in thesurface of microporous membrane, when cells pass through, it must degrade matrixfirst. The invasion ability of SW480/M5 is stronger than that of SW480 and SW 620 cells.After tumor cells through the vascular wall, most of which were killed orinhibited by host defense system, only the cells with strong proliferation and invasioncan form metastases. Therefore, we performed a series of tests on proliferation ofSW480/M5 cells. First, by MTT and colony formation we tested the proliferationindex in vitro of the cells: in 96-hole plate, the number of SW480/M5 and SW620cells exceeded the SW480 cells in the second day after inoculation; Colony formationrate test showed the proliferation of mononuclear cells of SW480/M5 and SW620were 1.64 and 1.52 times as great as SW480 cells. Secondly, using Subcutaneouslytumorigenicity in nude mice we tested proliferation in vivo of the three cells: sincethe ninth day after the injection cells, the volume of subcutaneous tumor ofSW480/M5 and SW620 cells exceeded that of SW480 cells. These show thatSW480/M5 and SW620 cells have stronger proliferation ability in vivo, which is thesame with test results in vitro.A series of experiments showed liver metastases subline SW480/M5 screenedfrom SW480 cells by successive generations had obviously organism tendency, andhad a high capability of campaigns, invasion, proliferation. Proteomics research cando quantitative and dynamic analysis of the protein expression, structure,post-translational modification, protein-protein interactions and intracellular locationin the overall and mass level, thereby can compensate the deficiency for genomeresearch can not provide all the functions of protein. The current study aboutmolecular mechanisms of tumor metastasis chiefly refered to the same pro-source celllines with different metastatic potential, the difference between the primary lesion andmetastases and finding the positive or negative correlation with the transfer of controlfactors. SW480/M5 is a good material for comparative proteomic analysis, which isselected from SW480 cells and has a definite metastatic direction and has the same genetic background as SW480 cells.In this study, proteomic strategy, combined with two-dimensional electrophor-esis (2-DE) separation and mass spectrometry (MS) identification was used tocompared differences between SWSW480 and SW480/M5 cells. Nine hepaticmetastasis-associated proteins were separated and identified by comparative proteometechnique. The use of fluorescence quantitative PCR and Western Blottingtechnology on three proteins (AnnexinⅠ, HMGB1 and FSCN 1) in the two cellmRNA and protein expression levels, the results showed that the results are reliable.With establishment of liver metastastatic cell subline SW480/M5, and analysisof differences in protein expression profile between SW480/M5 and its matrilinealSW480, it is of great significance for study hepatic metastases of colorectal cancer ina whole animal and the molecular level, and for early diagnosis and effectivetreatment of colon cancer liver metastases.