The Study Of Functions Of NK Cells In The Lung Cancer Microenvironment

NK cells are effector lymphocytes with pivotal roles in the resistance against various tumors.NK cell-based cancer immunotherapy is currently receiving great interest,as NK cells can facilitate the adaptive immune response by secreting cytokines and directly kill tumor cells without prior antigen encounter or sensitization.However,tumor cells can induce NK cell dysfunction by various mechanisms,thereby evading NK cell-mediated surveillance.Tumors develop in three stages comprising initiation,promotion and progression,but little is known about the interrelationships between NK cells and tumor cells at different stages of tumor development.Based on above-mentioned points,our study launched and we got the following conclusions.1.NK cells prevent lung cancer initiationTo investigate the role of NK cells throughout all stages of lung cancer development,the Cre-inducible Kras lung cancer mouse model was used,in which tumor development could be evaluated using a three-stage system.These three stages(Stage 1,Stage 2 and Stage 3)closely recapitulated three main carcinogenesis phases,namely initiation,promotion and progression,respectively.Using PK136 depletion mouse model,we found that depletion of NK cells during tumor initiation significantly accelerated tumor development,while depletion of NK cells during tumor promotion and tumor progression did not affect tumor development.In addition,using the NK cell deficient mouse model,we found that Kras;Nfil3-/-(which are devoid of NK cells)mice developed more severe lung cancer compared with Kras mice.Taken together,these findings show that NK cells potently prevent tumor initiation,but fail to control the promotion and progression of lung cancer.2.NK cells clearly decrease in number during lung cancer progressionWe hypothesized that loss of the antitumor effect by the NK cells could be caused by their decreased number and effector functions during lung cancer promotion and progression.Accordingly,we quantified and localized NK cells during lung cancer development.FCM was used to analyze leukocytes isolated from the lungs and spleens of WT and Kras mice at different stages of lung cancer.We found that the frequency and number of NK cells,T cells,B cells and MDSCs in the lungs showed a progressive decline,while macrophages showed the opposite trend.Conversely,the composition of spleen leukocytes remained constant.Notably,the loss of NK cells in the lungs was the most pronounced,with a particularly sharp decline at Stage 2.Using immunofluorescence,we found that NK cells were mainly located adjacent to the tumor,with rare infiltration into the tumor lesions.These results show that the lung cancer microenvironment profoundly excludes NK cells,with a critical time point being during Stage 2.3.Attenuated effector functions of NK cells in the lung cancer microenvironmentWhen co-cultured with LLC or YAC-1 target cells,Stage 2 and Stage 3 lung NK cells showed significantly attenuated cytotoxicity compared with WT lung NK cells,while the cytotoxicity of spleen NK cells were unchanged during lung cancer development.We then characterized the markers associated with degranulation,cytokine production and activation of NK cells.The data showed that lung NK cells displayed an impaired degranulation ability,reduced cytokine production and a hypo-responsive state during lung cancer promotion and progression,while spleen NK cells did not change.Together,these imply diminished effector functions of NK cells in the lung cancer microenvironment.4.Impaired viability of NK cells in the lung cancer microenvironmentBy examining Ki67 expression,intracellular ROS production,and in vitro survival of NK cells,we found that Stage 3 lung NK cells exhibited significantly impaired viability and proliferation capacity.This was also reflected in transcriptional profiles of genes analyzed with Stage 3 lung NK cells enriched for genes associated with positive regulation of apoptotic process and negative regulation of cell growth compared with WT lung NK cells.While hardly any alterations were found in the spleen NK cells.Collectively,the diminished effector functions and the impaired viability signifies NK cell dysfunction in the lung cancer microenvironment.5.Increased FBP1 expression is concomitant with decreased glycolysis of NK cellsSince glucose metabolism is essential for human and mouse NK cell function,dysregulation of glucose metabolism may lead to NK cell dysfunction.To identify previously unknown metabolic mechanisms involved in NK cell dysfunction,we analyzed the genes associated with glucose metabolic process,focusing on Fbpl(which encodes fructose-1,6-bisphosphatase),which was robustly upregulated in Stage 3 lung NK cells.This finding was further confirmed by using immunofluorescence,western blotting and PCR.Further,we found that upregulation of FBP1 in NK cells was caused by TGF-P derived from lung cancer microenvironments.In addition,the extracellular acidification rate(ECAR)of NK cells was measured for evaluating glycolysis,and the results showed that Stage 3 lung NK cells had considerably reduced glycolysis relative to WT lung NK cells.While the glycolysis and the expression of Fbpl in spleen NK cells was remained unchanged.6.Inhibition of FBP1 restores NK cell functionTo verify whether FBP1 and glycolysis play an important role in inducing the dysfunction of NK cells,MB05032(an FBP1 inhibitor)and 2DG(a glycolytic inhibitor)were used.The results showed that inhibition of FBP1 could improve the glycolysis,effector functions and viability of Stage 2 lung NK cells to some extent,but not Stage 3 lung NK cells,indicating that FBP1 inhibits glycolysis,effector functions and viability of NK cells.Moreover,we found that inhibition of FBP1 failed to restore the cytotoxicity of Stage 3 lung NK cells after addition of 2DG,whereas inhibition of FBP1 consistently improved the viability of Stage 2 lung NK cells with or without 2DG.Thus,FBP1 weakens the cytotoxicity of NK cells by inhibition of glycolysis,but FBP1 directly impairs the viability of NK cells independent of glycolysis.In conclusion,we demonstrated that NK cells potently prevented tumor initiation but failed to prevent tumor promotion or progression in Kras-driven lung cancer.Moreover,loss of the antitumor effect in NK cells was closely associated with their dysfunctional state during tumor promotion and progression.Mechanistically,we found that aberrant FBP1 expression in NK cells elicited their dysfunction by inhibiting glycolysis and impairing cell viability.Notably,FBP1 inhibition significantly restored NK cell function.Taken together,our results show the dynamic alterations of NK cells during tumor development,and uncover a novel mechanism involved in NK cell dysfunction,providing valuable information for NK cell-based cancer immunotherapy.