Preliminary Study On Adenosine A_2aR Affecting NK92 Cell Tumor Killing Function And Its Potential Mechanism

NK92 is the only NK cell line that has entered clinical studies,which proves that NK92 cells whose irradiated NK92 cells and their derived CAR-NK cells are safe and tolerated.However,due to the particularity of the tumor microenvironment,the anti-tumor immunity of immune cells（such as NK cells）is severely inhibited.The study found that there was a high concentration adenosine（ADO）in the tumor microenvironment.Adenosine inhibits NK cell function of tumor infiltration by binding with adenosine A_2aR expressed in the cell surface.Therefore,knockout adenosine A_2aR NK92 cell（named as NK92-2AK）was constructed by CRISPR/Cas9 technology in previous study.The evaluation of the recovery of anti-tumor ability of NK92 cells after knocking out adenosine A_2aR was verified from in vitro and in vivo experiments,and the mechanism by which adenosine affects the killing function of NK cells（such as the release of exosomes）was studied.The main results are as follows:1.Effect of knockout adenosine A2a R on NK92 cell function in vitroThe morphology of NK92-2AK and wild-type NK92 cells were observed in optical microscopy and transmission electron microscopy.NK92-2AK cells have a similar morphological characteristic with the wild-type NK92 cells,which grew in suspended clumps with similar size,round or quasi-round cells and smooth surface.Detecting the effect of adenosine on the proliferation level of NK92 cells,it was found that the increase of the concentration of adenosine,the more obvious the inhibitory effect.Since the concentration of adenosine in the solid tumor environment in vivo is tens of micromolar,the subsequent experiments used adenosine concentration of 50μM as the work concentration.There was no significant difference in cell viability after NK92-2AK cells were treated with adenosine for 12 h.The effect of adenosine on the long-term proliferation of NK92cells and NK92-2AK cells was studied by counting method,and it was found that there was no significant difference in the number of cells between the two groups after 4 days of normal culture.However,the proliferation ability of NK92 group was inhibited in adenosine environment,and adenosine A2a R knockout could alleviate the proliferation inhibition of NK92 cells by adenosine.Adenosine can also inhibit the killing effect of NK92 cells on melanoma cells（A375 cells）and human lung adenocarcinoma cells（A549 cells）,and the inhibitory effect increases with the increase of adenosine concentration.Knockout of adenosine A_2aR improved the suppression of cytotoxicity in NK92 cells in a high-adenosine environment.After adenosine A_2aR was knocked out in NK92 cells,their cell morphology and proliferation were not significantly affected,but they could effectively restore cell activity and anti-tumor ability in a high adenosine environment.2.The effect of adenosine A2a R knockout on NK92 cell function was evaluated in vivoOn the sixth day of subcutaneous injection,the tumor cells in the NK92-2AK group completely disappeared,which was significantly different from that in the NK92 group,and the tumor cells in the NK92 group did not disappear until the 9th day.In subsequent experiments of intratumoral injection of cells,it was found that at41 days,the tumor volume of mice in the NK92-2AK group was significantly different from that of the NK92 group and the control group.Tumor tissue was harvested and weighed and photographed.The tumor inhibition rate was calculated according to the tumor weight,and it was found that the tumor weight and tumor inhibition rate of the NK92-2AK group and the NK92 group were significantly different.During the animal in vivo experiment,the body weight of the mice was weighed every two days,and it was found that the body weight of the mice in each group fluctuated and increased,and the mice had no obvious symptoms of slow response,loss of appetite,and hair loss,indicating that NK92 treatment has an effect on the growth of mice.Development was not significantly affected.In conclusion,A_2aR knockout NK92 cells still showed stronger anti-tumor ability in vivo.3.Effect of adenosine A2a R knockout on expression of key killer gene in NK92 cellsThe expression patterns of some NK cytotoxicity related genes were studied using GAPDH gene as reference gene.It was found that FASLG,TNF,GZMA and GZMB genes in NK92 cells were significantly down-regulated 12h after adenosine stimulation.This is consistent with the previous conclusion that the anti-tumor ability of NK92 cells could be significantly inhibited under adenosine environment.In NK92-2AK cells,these genes did not differ significantly with or without adenosine stimulation,confirming the previous conclusion.Knockout of adenosine A_2aR can improve adenosine antitumor inhibition of NK92.The immunosuppressive mechanism of adenosine on NK92 may be to inhibit FASL expression,granase A and B formation,and TNF-αand INF-γproduction.4.Biological characteristics of exosomes in NK92 cells after adenosine A2a R knockoutThe exosomes of NK92 cells were successfully isolated and extracted by ultracentrifugation.Western Blot,electron microscope and NTA were used to identify the characteristic enrichment proteins,morphological size and particle size distribution of exosomes.Further experiments on exosomes extracted showed that adenosine might inhibit secretion of exosomes by NK92 cells.Adenosine A_2aR knockout could significantly improve the inhibition of exosome secretion of NK92cells in high adenosine environment.Conclusively,the present study investigated the effects of adenosine on the activity and killing ability of NK92 cells before and after adenosine A_2aR knockout.And to verify the anti-tumor ability of NK92 cells before and after adenosine A_2agene knockout in living animals.The results preliminarily confirmed our hypothesis that adenosine immunosuppressed NK92 cells through adenosine A_2aR.Adenosine A_2aR knockout NK92 cells restored cellular activity and immunity in adenosine environment.The immunosuppressive mechanism of adenosine on NK92 may be to inhibit FASL expression,granase A and B formation,and TNF-αand INF-γproduction.Knockout of adenosine A_2aR can restore this function.In addition,exosomes were isolated and identified,and found adenosine inhibited secretion of exosomes by NK92 cells and inhibited antitumor activity of exosomes.