Changes In The Cellular Structure Of CCCR-NK92 And Its Antitumor Effect

BackgroundIn recent years,chimeric antigen receptor T-cell immunotherapy(Chimeric Antigen Receptor T-Cell Immunotherapy,CAR-T)has emerged gradually and has played an important role in non-solid tumors(leukemia and lymphoma)treatment.However,the technical defects are obvious,for example,off-target effect,cytokine storm,long-term survival of CAR-T cells,insertion mutation,and so on.The no significant effect has been achieved in the treatment of solid tumors.And there was no significant effect on the treatment of solid tumors.Therefore,the modification of CAR in NK cells obtained much attention.Based on the design idea of classical CAR-T,we designed CAR-NK to re-innovate and develop the biological characteristics of NK cells.In human body,NK cells from different sources have different biological characteristics,such as NK cells derived from stem cell differentiation,isolated and cultured NK cell lines and peripheral blood NK cells.These biological characteristics have been widely used in the research and development of CAR-NK.In previous reports,both in vitro experiments and animal models have shown that CAR-NK is effective in targeted treatment of multiple hematological and solid tumors.CAR-NK can kill tumor cells by releasing cytokines and cytotoxic granules.However,the mechanism of CAR-NK killing without detection of cytokines and cytotoxic granules has not been reported,to reveal the pathway has great significance for the development of anti-tumor immunotherapy by CAR-NK.ObjectiveThe chimeric costimulatory transformation receptor was constructed by using the whole gene synthesis technique,and the NK92 cells(CCCR-NK92)were modified to improve the anti-tumor therapeutic effect of NK92 cells.To explore the anti-tumor mechanism of CCCR-NK92 in addition to releasing cytokines and cytotoxic granules from cellular structure,morphology and histology.MethodsThe recombinant vector PD1-NKG2D-41BB-pCDH plasmid was extracted,then the lentivirus was packaged and concentrated,after that the collected virus concentrate was transfected into NK92 cells.NK92 cells(CCCR-NK92 cells)with the chimeric costimulatory receptor were obtained.The transfection efficiency of CCCR-NK92 cells was analyzed by flow cytometry,the morphological changes of CCCR-NK92 cells were detected by H-E staining and Wright`s staining,and the structural changes of CCCR-NK92 cells were detected by transmission electron microscopy(TEM).Transwell assay was used to detect the killing mode of CCCR-NK92 cells and NK92 cells,CM assay was used to verify the release of cytokines in CCCR-NK92 and NK92 cell target assay,and CM-Dil staining solution and Hoechst 33342 staining solution were used to detect apoptosis.High-connotation imaging and TEM were used to detect target killing mode.The model of human lung cancer transplanted subcutaneously in NOG mice was established.The anti-tumor effect of CCCR-NK92 cells was observed by immunohistochemical staining,immunohistochemistry and immunofluorescence in frozen tissue sections.Results 1.Compared with NK92 cells,the HE,Wright`s staining and ransmission electron microscopy(TEM)proved that the lentivirus-transfected CCCR-NK92 cells has no change in nucleus,the cytoplasm of CCCR-NK92 cells was rich in organelles and formed protuberances on the cell membrane.2.CM-Dil,Hoechst 33342,Transwell assay,Conditioned medium experiments and High-connotation imaging analysis showed that Compared with NK92 group,the number of apoptotic cells in CCCR-NK92 group increased,and there was swelling and rupture and nuclear staining showed that the fluorescence intensity of CCCR-NK92 group was lower than that of apoptotic cells.3.TEM showed that there was no significant change in NK92 after exposure to H1299,and cytotoxicity particles were released from CCCR-NK92 after exposure to H1299.4.CD56 and PD1 positive cells were found in the tumor by immunohistochemistry and immunofluorescence assay.5.HE staining showed that the necrosis of CCCR-NK92 group was higher than that of NK92 group and PBS group.Conclusions The NK92 cells which was modified by The chimeric costimulatory transformation receptor have more abundant and protuberances on the cell membrane.The morphology and structure of CCCR-NK92 cells were changed,which may be related to the killing of target cells by apoptosis and suspected pyroptosis of CCCR-NK92 cells.