Development Of Novel High-efficiency NK Cells With Increased Tumor Chemotaxis And Sustainable Proliferation And Their Anti-tumor Effects On Human Colon Cancer

Purpose: Natural killer(NK)cells have good application prospects in adoptive cellular immunotherapy against cancer.However,the therapeutic effect of infused NK cells has been limited in solid tumors,such as colorectal cancer.This study aimed to develop novel high-efficiency NK cells with increased tumor chemotaxis,sustainable proliferation,and better anti-tumor effects.Methods: 1.The number of NK cells in patients with advanced colon cancer and healthy people was collected to understand their basic cellular immune status.The treatment data of advanced colon cancer patients treated with autologous NK cells was collected retrospectively.Clinical effects of NK cell transfusion were analyzed.2.We up-regulated CXC chemokine receptor 2(CXCR2)and cytokine interleukin 2(IL2)of NK92 cells via CRISPR-Cas9 gene-editing technology and lentivirus transfection.3.The expression levels of CXCR2 and IL2 in NK92 cells were measured by qRT-PCR and ELISA.4.The chemotactic capacity,proliferation ability,and cell-killing activity were tested by Transwell assay and CCK8 assay in vitro.5.Fresh colon cancer tissue specimens were obtained and prepared into single cell suspension.The killing effect of NK cells on tumor cells in patients with colon cancer was also detected.6.Human colon cancer xenograft tumor model was established in SCID mice.NK cells were injected via the tail vein to study the distribution and anti-tumor effects in vivo.The number of tumor-infiltrating NK cells and the surface expression of CXCR2 were detected by immunohistochemistry.The tumor volume and life cycle of tumor-bearing mice in each group were observed.Results: 1.The number of NK cells in patients with advanced colon cancer significantly decreased(P<0.001).In colon cancer patients treated with autologous NK cell transfusion,the number of NK cells increased,the NLR decreased,and many tumor markers decreased in varying degrees,indicating clinical improvement,but the difference was not statistically significant(P>0.05).The PS score of patients reduced after treatment(P<0.05),indicating their physical condition was improving.2.CXCR2-IL2 sgRNA plasmid and d Cas9-VP64 plasmid were successfully transferred into NK92 cells,and activated NK92 cells to constantly up-regulate the expression of CXCR2 and IL2.qRT-PCR and ELISA confirmed the higher expression of CXCR2 and IL2 in CXCR2-IL2-NK92 cells(P<0.05).3.Through Transwell assay and animal in vivo imaging technology,it was confirmed that CXCR2-IL2-NK92 cells achieved stronger chemotactic ability towards human colon cancer cells in vitro and in vivo(P<0.001).4.Through CCK8 assay and animal in vivo imaging technology,it was proved that CXCR2-IL2-NK92 cells had stronger killing and proliferating ability in vitro and in vivo(P<0.01).5.The human colon cancer xenograft tumor model was successfully established in SCID mice.In CXCR2-IL2-NK92 treated group,we noted a considerable delay of tumor growth(P<0.01),and the average survival time of tumor-bearing mice was significantly prolonged(P<0.01).Through immunohistochemistry,it was found that CXCR2-IL2-NK92 cells increased infiltration into tumor sites,and the expression of CXCR2 on the cell surface was stronger than that of the control group.Conclusion: 1.The number of NK cells in patients with colon cancer decreased and the cellular immune function was impaired.Adoptive transfusion of NK cells is feasible in the treatment of colon cancer.2.The CRISPR-Cas9 gene-editing technology can activate NK cells to stably up-regulate the expression of chemokine receptor CXCR2 and cytokine IL2.3.The up-regulated expression of CXCR2 and IL2 can promote the infiltration and chemotaxis of NK cells into human colon cancer cells or tissues,and the killing and proliferation activity of NK cells are also enhanced.4.Overexpressing CXCR2 and IL2 can improve the inhibitory effects of NK cells on the growth of human colon cancer,reduce the tumor burden of tumor-bearing mice,and prolong their survival time.5.Gene-editing modification of chemokine receptors and cytokines on NK cells is expected to become a novel and promising tumor immunotherapy strategy.