The Relationship Of Vascular Endothelial Growth Factor Receptor 1 Positive Hematopoietic Progenitor Cells And Human Colorectal Carcinoma Metastasis

BACKGROUND & OBJECTIVEColorectal cancer(CRC) is a common malignancy in the world.In recent years the incidence and mortality of colorectal cancer rates are rising year after year in China,the invasion and metastasis is the major cause of death.The occurrence and development of tumor is a complex process which refers to more than one factor involved,multiple genetic changes,the development of multi-step.In the process of evolution of tumor cells,intersection between the tumor cells and the host resulted in the formation and development of tumor.Therefore,the tumor cells have a certain organizational tendencies,which is the result of intersection between tumor cells and organ specific micro-environment.The metastatic predisposition is believed to reflect inherent molecular differences in tumour cells themselves and the potential influence by surrounding stromal cells, which include the vasculature,connective tissue and immune cells.Regulation mechanisms of specific tumor microenvironment affact the occurrence,development and invasion and metastasis of tumor.Therefore,tumor metastasis is decived by the interaction of tumor and host rather than tumor their biology peculiarity.Progenitor/stem cell is primitive cell of the ability of self-duplication and multi-differentiation.CD34 has been consided the important antigen of hematopoietic progenitor cell,and gradually weakened with the differentiation.To study surface antigen of stem cell,CD133-positive cell has stranger abilitity of chemotatics and metastasis than CD34-positive cell.It is possible that CD133-positive cell is the more primitive cell.In 2005,Kaplan RN firstly found that vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cell has important contribution in tumor metastasis,which was labled CD133 and VEGFR1,and not endothelial progenitor cells(VEGFR2- positive hematopoietic progenitor cell).Anti-VEGFR2 antibody did not prevent the formation of VEGFR1~+ clusters but limited metastatic progression. The VEGFR1~+ HPCs initiating the pre-metastatic cluster can attract tumour cells.β-galactosidase-positive BMDCs implantation following intradermal Lewis lung carcinoma(LLC) cells,B16 melanoma tumour cells injection in mice,or lymphoma cells injection in a spontaneous tumour model using c-Myc transgenic mice,showed VEGFR1~+ HPC were increased cellular clusters in common sites of metastasis before tumour spread,suggesting the potential of this tissue as a future site for metastasis. The precise cellular and molecular mechanisms that dictate metastasis of a specific tumour to a predetermined metastatic location are not known.Many tumours have a predilection for metastasis to specific organs.Cellular bookmarking- vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cell form metastasis microenvironment in specific organs,which promote tumor invasion and metastasis.But the current research all adopts to selectively transplant bone marrow progenitor cells into irradiated mice.That the radiative injury of each organ may result in the mobilizationg of the non-particularity bone marrow cells or bone marrow derivative cells is not confirmed in hunman cancer confirmation.In this study,we aim to approach the influence of hematopoietic progenitor cell in tumor metastasis.Firstly,VEGFR1-positive cell clusters was detected in metastasic and no metastasic lymphoid nodes by immumohischemistry.Then,human colorectal cancer cells were implanted into BALB/c nude mice,and the tumor tissue blocks were re-implantated into the colon of nude mice,and observe the quantity and the percentage of vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cells and human colorectal cancer cells in the pre-metastatic location by FCM,and observe the expression of metastasis relative factors by Western Blotting. Last,we study the influence of hematopoietic progenitor cell in cell proliferation, adhesion and invasion in colorectal cancer cell.CONTENTExpression VEGFR1-positive cell clusters or CD133-positive cell clusters were detected in metastasic and no metastasic lymphoid nodes by immumohischemistry.Human colorectal cancer cells SW480/M5 and SW480/EGFP~+ were implanted into BALB/c nude mice,and the tumor tissue blocks were re-implantated into the colon of nude mice,and observe the quantity and the percentage of vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cells and human colorectal cancer cells in the pre-metastatic location by FCM,and observe the expression of metastasis relative factors by Western Blot.Sort CD133~+HPC though magnetic activated cell sorting,then co-culture CD133~+HPC within colorectal cancer cells to detect the influence of CD133 in cell proliferation,adhesion and invasion in colorectal cancer.METHODS & RESULTS1 VEGFR1-positive cell clusters or CD133-positive cell clusters were detected in metastasic and no metastasic lymphoid nodes by immumohischemistry. The results indicated that VEGFR1-positive cell clusters were detected in metastasic lymphoid nodes and no metastasic lymphoid nodes.Furthermore, CD133-positive cell clusters were detected in metastasic lymphoid nodes and no metastasic lymphoid nodes.Hematopoietic progenitor cell was mainly detected in lymphatic capsule,lymphatic sinus,vassular adjacent and tumor location.2 Establishing the visible spontaneous metastasis nude mice model by orthotopic implantation.SW480/M5 and SW480/EGFP~+ cell suspension(5×10~6/ml) the mice were executed euthanasia.The tumor tissues were taken down and stripped into 1mm~3 particles.The tissue particles were inoculated into cecum subserosa of nude mice respectively(6/group).Then these tumor growth,evolution and metastasis in the nude mice were observed by whole-body fluorescence imaging at intervals.We tested metastasis of the two types of cells in vivo by surgical orthotopic implantation methods,the results showed:SW480/EGFP~+ have the potential of multiple-organ transfer,liver,lung,spleen,renal,lymph node metastasis all occurred. Particularly peritoneal metastasis was stronger than SW480/M5 cells;SW480/M5 has the stronger ability of liver metastasis,no metastasis on 21 days,the metastasis rate was 50%and 66.7%on 35 and 49 days after orthotopic implantation.3 Detection the quantity and the percentage of vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cells and CD 133 positive cells in the pre -metastatic location by FCM,without or with orthotopic implantation of SW480/M5 and SW480/EGFP~+ tissue in nude mice.First,We deteced the the quantity and the percentage of vascular endothelial growth factor receptor 1 positive and CD133 positive hematopoietic progenitor cells and human colorectal cancer cells in the pre-metastatic location by FCM,without or with orthotopic implantation of SW480/M5 and SW480/EGFP~+ tissue in nude mice, the results showed:the quantity and the percentage of both positive cells increased with different stages(F=474.570,P=0.000),and arrived in the liver before tumour cells.Furthermore,at the same time,SW480/M5 group has more cells than SW480/ EGFP~+ group in nude mouse liver(t=11.619,P=0.000).SW480/M5,which is the cell sublines of liver metastases from colon cancer cell line SW480,have a certain stronger organizational tendencies;SW480/EGFP~+ have the potential of multiple-organ transfer.4 Detection by western blot of the MMP-9 and SDF-1 protein in the liver of the nude mouse.We analyzed the expression level of MMP-9 and SDF-1 protein in the pre-metastatic location by western blot,without or with orthotopic implantation of SW480/M5 tissue in nude mice,the results showed:The expression of MMP-9 protein was enhanced in different stages,the expression of SDF-1 protein was enhanced also.5 Sort CD133~+HPC though magnetic activated cell sorting,then co-culture CD133~+HPC with colorectal cancer cells to detect the influence of CD133 in cell proliferation,adhesion and invasion in colorectal cancer.Select healthy and fresh cord blood and sort CD133~+HPC by anti-human CD133 immunomagnetic beads though magnetic activated cell sorting.Culture CD133~+HPC in vitro in low sugar MDEM with 20%fetal bovine serum for 1～2 weeks,then co-culture CD133~+HPC with colorectal cancer cells for 24 hours.MTT and Transwell assay was engineered to detect effect of CD133~+HPC on cell proliferation,adhere and invasion.Results show that:Cells expressed CD133 sorted by magnetic activated cell sorting can reach up to 73%.Sorted cells will not induce cell differentiation in 1～2 weeks in vitro and still remain the characters of progenitor cell.Firstly,by adhesion experiment we found CD133~+ hematopoietic progenitor cell can enhangce the adherent abilitity of SW480/M5,SW480/EGFP~+ and SW620 cell.Secondly,the test compared the movement capacity of the three group cells using the Transwell method. Our results showed the movement ability of SW480/M5,SW480/EGFP~+ and SW620 cells with CD133~+ hematopoietic progenitor cell in the lower is stronger than the control group cells.Therefore,we performed a series of tests on proliferation of SW480/M5 SW480/EGFP~+,and SW620 cells with or without CD133~+ hematopoietic progenitor cell co-cultured.By MTT we tested the proliferation index in vitro of the cells:in 96-hole plate,the number of SW480/M5,SW480/EGFP~+and SW620 cells with CD133~+ hematopoietic progenitor cell exceeded the control group cells in the second day after inoculation.CONCLUSION:1 The results indicated that VEGFR1-positive cell clusters was detected in metastasic lymphoid nodes and no metastasic lymphoid nodes.Furthermore, CD133-positive cell clusters was detected in metastasic lymphoid nodes and no metastasic lymphoid nodes.VEGFR-1~+HPC was not detected in patients without cancer,such as reactive hyperplastic lymphoid node and chronic tonsillitis tissues.It is possible that VEGFR1-positive cell clusters home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells,providing a permissive niche for incoming tumour cells.2 Vascular endothelial growth factor receptor 1 positive hematopoietic progenitor cell may have important contribution in colon cancer metastasis.The percentage of VEGFR-1~+HPC increased in different stages,and arrived in the liver before colon cancer cells.3 CD133~+ hematopoietic progenitor cells can promote proliferation,adheration and invasion of colon cancer cells in vitro. 4 We analyzed the expression level of MMP-9 and SDF-1 protein in the pre-metastatic location by western blotting,without or with orthotopic implantation of SW480/M5 tissue in nude mice,the results showed:The expression of MMP-9 protein was enhanced in different stages,the expression of SDF-1 protein was enhanced also.VEGFR-1~+HPC promote tumor metastasis mainly by increase the expression of SDF-1 and MMP-9.