Heterogenicity Of Metastatic Potentials Of CD133 Positive Single-cell Derived Clonal Sublines From Colorectal Cancer Cell Line SW480

BACKGROUND & OBJECTIVEThe occurrence and development of tumor is a complex process which refers to more than one factor involved,multiple genetic changes,and the development of multi-step.In the process of evolution of tumor cells,genetic instability of tumor cells and the pressure to choose the host environment resulted in constant cell mutation which leads to divisive and phenotypic diversity.Therefore,in the same tumor mass, there are subcloned cells which contain not exactly the same biological characteristics and metastatic abilities.Metastasis of tumor cells is the result of the interaction with the host which is a highly selective process where only the cell sublines with metastatic potential can metastasis and these tumor cells have a certain organizational tendencies,which is the result of intersection between tumor cells and organ specific microenvironment.Therefore,it is necessary to isolate and identify the subcloned tumor cells with different direction of metastasis,and compare and analyze the differences in biological characteristics in a targeted manner,which has important theoretical and practical significance,is helpful to investigate the mechanism of metastasis and to find an effective way of prevention and treatment of tumors.Studies have shown that in tumor tissues exist a small part of unlimited proliferation and tumor-forming ability of tumor cells,because of its many features and very similar to stem cells have been recognized as a tumor stem cell disease.In 2001,Reya,etc.proposed tumor stem cell theory,and then related research for tumor stem cell developed step by step.From AML(acute myeloid leukemia) to the tumor stem cell model of solid tumor and cell separation and purification technologies,all in future will have an important significance to cancer treatment.CD133,also known as prominin-1,is a cell surface antigen,contains five transmembrane regions,an extracellular N terminal domain,two extracellular macrocyclic structures,and two small roop structure in the cytoplasm,which its C-terminal domain is in the cytoplasm.It is confirmed that as a stem cell marker,CD133 has been expressed in human leukemia,brain tumors,lung and prostate cancer and B16 melanoma cells. Singh,etc.,isolated CD133⁺ tumor stem cells of the brain(brain tumor stem cell, BTSC) in a series of brain tumor including Medulloblastoma,Pilocytic astrocytoma, ependymoma and Ganglioglioma.in 2007,Lucia Ricci-Vitianil etc.and Catherine AO'Brien,etc.,separately conducted a study of colon cancer cells with stem cell markers CD133 to prove that CD133⁺ cells exist in colorectal cancer,that is,the cell subsets in colorectal cancer have the characteristics of stem cells.The aim of our study is to monoclonal cultivate colorectal cancer cell line SW480 with the limiting dilution method,and to screen single-cell derived sublines of the same genetic background.We labeled them with stem cell markers CD133,and compared the biological characteristics among these single-cell derived CD133⁺ sublines.All of these provide a high metastatic and low metastatic potential cell subline model for further studying colorectal cancer metastasis mechanism.Our conclusion is benefit to further study the high and low metastatic potential cell subline.Methods1.Construction of CD133⁺ single-cell-derived from SW480 cell clonal sublines Using the limited dilution method,we cultivated single-cell-derived monoclonal cell subline from the colon cancer cell line SW480,designed CD133^-specific primers, extracted the sublines of total RNA,identified their expression of CD133 mRNA using RT-PCR,immunocytochemistry.CD133⁺ single cell line-derived progeny cell subline was constructed.2.Screening different metastatic potential cell clonal sublines through BALB/c-nu nude mice animal model1×10⁶ single cell derived subline cells which were in a logarithmic phase were injected into a subcutaneous nude mice.When the tumor diameter was up to about 8 mm,removed the tumor in aseptic conditions into ice-cold serum-free RPMI-1640 medium,cut it into a small piece of 1 mm³ standby,anesthetized five of the 6-week-old BALB/c-nu nude mice with 1%pentobarbital(0.1 ml/kg),discissioed the left lower quadrant,took the cecum out,curettaged some serosa,sutured with 7/0 suture needle belt line 1 mm³ volume of the tumor in the injury,then sent back the cecum into the abdominal cavity and sutured the abdominal incision.3.Analysis of high and low metastatic potential of cell clonal sublines biological characteristicsUsing MTT assay,flat colony formation experiment,Ki-67 marking the nuclear antigen to detect the proliferation ability of high and low metastasis sublines; trans-well chamber assay to detect their mobility in vitro;trans-well with matrigel chamber assay to detect their invasion ability in vitro;immunocytochemistry to detect metastasis-associated gene expression(nm23) and EMT(CK,VIMENTIN); immunocytochemistry to detect expression of E-cadherin and EGFR in nude mice subcutaneous tumor;nude mice subcutaneous tumor forming assay to detect tumorigenicity;nude mice inoculated tissue in situ assay to detect metastasis potentiality,flow cytometry and Western Blot to detect high and low metastasis cell sublines stem cell markers CD133,CD44 and CXCR4 expression.Results1.Construction of CD133⁺ single-cell-derived from SW480 cell clonal sublines We selected 48 single cell line-derived progeny cell sublines from human colon cancer cell line SW480 by limiting dilution method,and separately named L_1～48.. After detected a total of 33 cell sublines,there are 30 CD133⁺ cell sublines and 3 CD133^-cell sublines respectively.2.Screening different metastatic potential cell sublines through BALB/c-nu nude mice animal model(1).A total of 33 cell sublines were carried out to subcutaneous tumor forming experiment(30 CD133⁺ cell sublines and three CD133^-sublines),in which 31 sublines succeeded in formation of tumor mass but two sublines failed.All of CD133 ⁺ cell subsets have tumor formation while 2 of of the 3 CD133^- cell sublines can not develop tumor mass but only one of them produces a minimal cancerous mass.(2).A total of 13 cell sublines subcutaneous tumor tissue were inoculated into caecum of BALB/c-nu nude mice in situ metastatic experiments,in which there are 2 cell sublines with higher metastasis capacity(liver metastasis) and they are L₁₇(5/5) and L₂₆(3/5),2 sublines with low metastatic capacity(only in situ tumor development without liver metastasis) and they are L₂₄(0/5) and L₄₀(0/5).3.Analysis of biological characteristics of high and low metastatic potential of cell clonal sublinesBy MTT assay(F=65.484,P＜0.001),flat colony formation experiments(F = 17.937, P = 0.001),Ki-67 nuclear antigen marker(F = 201.628,P＜0.001),we detected proliferative capacity in vitro between high metastatic sublines and low metastatic sublines.The proliterative index has statistical significance(p＜0.001).Trans-well chamber assay showed that highly metastatic cell subline L₁₇ and L₂₆have an more enhanced mobility than the low metastatic cell subline L₂₄ and L₄₀,the mobility difference of them was significant(F=11.380,P＜0.001).Trans-well with matrigel chamber assay showed that highly metastatic cell subline L₁₇ and L₂₆ have stronger invasion than the low metastatic cell subline L₂₄ and L₄₀,the difference was significant(F= 38.719,P＜0.001).Immunocytochemical markers of metastasis-associated proteins(nm23),EMT(CK, Vimentin),results showed that highly metastatic cell sublines expressed a low level of nm23 to resistant tumor metastasis.Both of highly and low metastatic cell subclones showed high expression of CK for EMT,but only highly metastatic cell subline Vimentin expression can be seen and without expressed any single cells in low metastatic cell sublines.Subcutaneous tumor formation experiment and orthotopic tumor inoculation experiments metastasis showed that highly metastatic cell sublines played more meatstatic potentials than low metastatic cell sublines(p＜0.001).Flow cytometry and Western Blot detection showed that there are more CD133, CD44 and CXCR4 all positive cells in highly metastatic cell sublines than low metastatic cell sublines.CONCLUSION1.Construction of 48 single-cell derived sublines from human colon cancer SW480 cell line,and in which there were 30 CD133⁺ cell sublines and 3 CD133^- cell sublines;CD133⁺ cell subline has a more powerful ability to tumorigenicity.2.Obtaining 2 highly metastatic(liver metastasis)cell subline L₁₇ and L₂₆,2 low metastatic cell subline L₂₄ and L₄₀.Highly metastatic cell sublines possess proliferation,mobility,invasion ability and epithelial-mesenchymal transformation more than low metastatic cell sublines.3.With the cancer stem cell marker CD133,CD44,CXCR4 expression in colorectal cancer line SW480 clone sublines have different characteristics of infiltration and metastasis,suggesting that colorectal cancer cells in the stem cell stage of the heterogeneity of biological behavior,tumor metastasis may be the existence of stem cells for transfer mechanisms in colorectal cancer research provide new clues.