The Roles And Mechanisms Of P2X7 Mediated Macrophage Regulation In Tumor Microenvironment

Tumor-associated macrophages(TAMs)are major components of leukocytic infiltrate of tumors,which are predominantly differentiated from monocytes in the blood,presenting a remarkable heterogeneity and plasticity.TAMs are mainly induced into M2 phenotype by a variety of cytokines and growth factors in tumor microenvironment and promote tumor progression and metastasis by accelerating tumor cell growth,angiogenesis and immune escape.Growing clinical observations have indicated that excessive TAMs infiltration are associated with the low survival rate or the poor prognosis of lung cancer patients,which implicated the essential roles of TAMs in maintenance of tumor microenvironment.Recent findings have found that the presence of abundant ATP was involved in the immune regulation of microenvironment by activating purine receptors in tumor microenvironment.Among them the function of P2X7 receptor is most pivotal,which plays an important role in the inflammatory cascade,the survival and proliferation of tumor cells.However,the roles and mechanisms of P2X7 receptor on TAMs are still unclear in tumor microenvironment.To detect the expression of P2X7 receptor on macrophages,we used Q-PCR,Western blot and flow cytometry and found that P2X7 receptor was up-regulated on TAMs or IL-4 induced macrophages,suggesting its crucial role on TAMs.Functional polarization and phagocytosis of macrophages have been reported to play a key role in tumor development.To clarify the function of P2X7 receptor in macrophages,BMDMs were induced to M1 phenotype using LPS or IFN-?,and M2phenotype using IL-4 or LLC tumor supernatants.Then,we examined the different markers of M1/M2 macrophages by Q-PCR and flow cytometry.Our data demonstrated that the expression of some prototypical M2 markers(such as Arg1,CD206 and others)were significantly reduced in P2X7 receptor knockout or inhibited macrophages,while M1 markers were little influenced,indicating that P2X7 receptor was involved in the polarization of macrophages.However,co-culture of macrophages with tumor cells showed that P2X7 receptor did not significantly affect the phagocytic function of macrophages.In order to explore whether P2X7 receptor participate in tumor progression via regulating macrophage polarization,we established a series of mouse tumor models,including subcutaneous transplantation of LLC,orthotopic tumor in lung and urethane induced lung cancer.The results showed that tumors were reduced and survival was improved in P2X7 receptor knockout or inhibited mice.Further analysis indicated that P2X7 receptor and TAMs showed a highly co-localized expression in tumor tissues and P2X7 receptor deficiency mainly inhibited tumor growth by impairing M2 phenotype of TAMs,which was consistent with that in vitro.To verify the specific regulation of P2X7 receptor on TAMs,we also subcutaneously co-injected WT or P2X7KO macrophages with tumor cells into WT or P2X7KO mice respectively and the results showed that WT macrophages significantly promote the tumor growth,while P2X7KO macrophages inhibit tumor growth both in WT and P2X7KO mice.After determining the immune regulation of P2X7 receptor on macrophages both in vitro and in vivo,we investigated the molecular mechanism of polarization by P2X7receptor and its function in tumors.Western blot analysis showed that P2X7 receptor deficiency impaired M2 phenotype of macrophages by decreasing the level of phosphorylation of Stat6 and the expression of IRF4 instead of Stat3,CREB,PPAR?and PI3K.In addition,functional experiments demonstrated that the P2X7 receptor deficiency attenuated the effect of TAMs on tumor cell proliferation,angiogenesis and inhibition of CD8~+T cell activity.Finally,to further explore the potential application of P2X7 receptor as a target for cancer immunotherapy,we combined P2X7 receptor inhibitor O-ATP with clinical chemotherapy drug Cisplatin to treat tumor-bearing mice,which showed synergistic action than monotherapies in controlling tumor progression.In conclusion,our data reveal the vital role of P2X7 receptor in the functional polarization of TAMs,which accelerates tumor progression.Therefore,targeting P2X7receptor is an attractive strategy for developing an immunotherapy for lung cancer.