A Study On The Effects Of Overexpression Of NK Cell Chemokine Receptors CCR7 And CXCR4 On Tumor Chemotaxis And Its Anti-human Colon Cancer Effect

Objectives: NK cells have become a cancer treatment method because of their unique anti-tumor effect,but NK cell deficiency in the solid tumor microenvironment is an important factor restricting its clinical efficacy.In this study,the NK cell chemokine receptor CCR7 was overexpressed by gene modification.And CXCR4 on the chemotaxis of NK cells to human colon cancer cells in vitro and in vivo and their anti-colon cancer effects.Methods: 1.ELISA method was used to detect the expression of chemokine CCL21(CCR7 ligand)and CXCL12(CXCR4 ligand)in serum of healthy people and colon cancer patients.2.The chemokine receptor CXCR4 and CCR7 lentiviral vectors were up-regulated,and Lipo2000 was transfected into NK cells to screen for NK cell lines stably overexpressing CXCR4 and CCR7.The expression of CXCR4 and CCR7 in stable cell lines was detected by RT-PCR and Western blot.4.MTT assay was used to detect NK cell killing activity,and tanswell was used to study the chemotaxis of NK cells to colon cancer cells in vitro.5.Firstly,the SCID mouse model of subcutaneous colon cancer was constructed,then injecting NK cells overexpressing CXCR4 and CCR7,and the distribution of NK cells in tumor-bearing mice was analyzed by IVIS,The tumor volume and survival cycle of tumor-bearing mice were observed to study the effect of NK cells against colon tumors.Result: 1.The expression of CXCL12 and CCL21 was confirmed by ELISA in this study: the serum level of colon cancer patients was higher than that of healthy people,and the difference was statistically significant(P<0.001).In this study,a stable NK cell line stably overexpressing the dual chemokine receptors CXCR4 and CCR7 was successfully constructed by lentiviral transfection technology.The CXCR4 and CCR7 m RNA levels were up-regulated and compared with wild-type NK cells.Significantly up-regulated,the difference was statistically significant(P <0.05).The MTT assay showed that there was no significant change in the killing activity of NK92 cells overexpressing the double chemokine receptor compared with the wild type NK cell killing activity,indicating that lentiviral transfection did not impair the killing activity of NK92 cells.3.Transwell experiments and small animal in vivo imaging techniques demonstrated that NK cells overexpressing the dual chemokine receptors CXCR4 and CCR7 showed stronger chemotaxis to human colon cancer cells in vivo and in vitro.The difference was statistically significant(P<0.05).4.Mean survival time of SCID-bearing mice treated with PBS(25d)and average survival time of SCID-bearing mice of LW238 NK92 cell group(33.5d),average survival time of tumor-bearing mice treated with LW750 NK92 group(45d)There was a significant prolongation(P<0.05);there was no significant difference in the survival cycle between the PBS group and the LW238 NK92 group.Conclusion: 1.The lentiviral transfection gene modification technology can make the NK cell surface chemokine receptors CXCR4 and CCR7 constantly up-regulated at the level of gene and protein molecules;lentiviral transfection gene modification itself does not affect the killing activity of NK cells.2.Upregulation of cell surface receptors CCR7 and CXCR4 can significantly promote the chemotaxis and aggregation process of NK cells themselves to human colon cancer cells or colon cancer tissues(chemokine sources),and increase the number of local NK cells in the tumor.3.NK cells with up-regulated expression of chemokine receptors CCR7 and CXCR4 enhanced the inhibition of human colon cancer growth by increasing the number and activity of NK cells in the tumor microenvironment,and significantly prolonged the survival of tumor-bearing mice.4.Modification of the chemokine receptor gene of NK cells is expected to be an important method for improving the immunotherapy of adoptive tumors.