Nanocarriers Regulating Tumor Microenvironment For Sensitizing Anti-PD-1 Immunotherapy

Cancer,the second leading cause of human death,has always been a public health problem attracting worldwide attention.Immunotherapy,which remedies cancer by actively mobilizing or enhancing the body's own immune response,is considered to be one of the most promising approaches in the field of cancer treatment in recent years.However,tumors adopt various strategies to devitalize immunotherapy,among which the most prominent way of immune escape is the programmed cell death(PD)pathway.Programmed death receptor 1(PD-1)is expressed on the surface of activated T and B lymphocytes.After binding to the ligands PD-L1 or PD-L2 on the surface of tumor cells,it induces uncontrolled tumor progression by restricting the immune activity of tumorspecific effector T cells.Plenty of experiments have shown that blocking the PD-1 pathway has certain therapeutic effect in many types of tumors.To date,several anti-PD-1 antibodies have been approved by the FDA for the treatment of more than 40 cancers.However,clinical results show that only 20%-30% of patients are responsive to immunotherapy,and the clinical effect of immunotherapy is not optimistic.Therefore,how to enhance the efficacy of anti-PD-1 antibody is the top priority of current immunotherapy.Hypoxia,one of the most significant features of the tumor microenvironment(TME),impairs the anti-tumor effect of immunotherapy by activating multiple signaling pathways.Studies have shown that hypoxia is one of the important factors hampering the efficacy of immune checkpoint inhibitors(ICIs).To sensitize immunotherapy,this study first focused on improving the hypoxic TME.Three methods were adopted to ameliorate hypoxic TME,including direct oxygen delivery using two different carriers,liposomes loaded with perfluorooctane or hemoglobin(PFC@lipo or Hb@lipo)and an indirect way involving hypoxia inducible factor 1?(HIF-1?)inhibition with PX-478.Both in vivo and in vitro experiments showed that both liposomes could load and release oxygen,relieving tumor hypoxia.Compared with PX-478 and oxygenated PFC@lipo,oxygenated Hb@lipo had significantly better biosecurity and clinical applicability.Anti-tumor experiments showed that mouse 4T1 tumor had limited response to anti-PD-1 immunotherapy,while oxygenated Hb@lipo significantly enhanced the immunotherapy effect of anti-PD-1 antibody.However,in mouse colon cancer(CT26),neither immunotherapy alone nor combination therapy produced meaningful antitumor effects.In-depth study found that the proportion of tumor associated macrophages(TAMs)with different phenotypes(M1/M2)was completely different in the above two types of tumors.4T1 tumors had more immune-promoting M1 cells(M1/M2 > 1),whereas CT26 tumors had far more immunosuppressive M2 cells than M1 cells(M1/M2 < 1)even after treatment.In conclusion,we believed that the sensitizing effect of alleviating hypoxia on antiPD-1 immunotherapy was limited by the phenotype of TAMs,and the efficacy of immunotherapy alone was also restricted by the phenotype of TAMs.The polarization of TAMs in TME,that was,the ratio of TAMs with different phenotypes(M1/M2),was the key to sensitizing anti-PD-1 immunotherapy.Therefore,the follow-up research was devoted to sensitizing anti-PD-1 immunotherapy through the phenotypic regulation of macrophages.Macrophages are highly plastic cells,and inducing the repolarization of M2-TAMs into M1 phenotype has become a hot topic of enhancing the efficacy of ICIs.M2 macrophages are mostly concentrated in tumor hypoxic area.However,there is still a lack of targeted regulatory strategies for the repolarization of M2 macrophages under hypoxia.In-depth research found that the metabolism of these two types of macrophages is significantly different: M1 mainly relies on glycolysis for energy supply,while M2 relies on oxidative phosphorylation(OXPHOS)and fatty acid oxidation(FAO).And the metabolic regulation of macrophages is closely related to their endoplasmic reticulum stress(ERs).Therefore,we encapsulated the ERs-related inositol-requiring enzyme 1(IRE1)-XBP1 pathway inhibitor KIRA6 into a reductive nanoemulsion containing ?-tocopherol,the active ingredient of Vitamin E(VE),to obtain a nanoemulsion with dual inhibitory effects on ERs and oxidative stress(?-T-K).In vitro and in vivo experiments confirmed that ?-T-K effectively induced the repolarization of M2-TAMs into M1 phenotype under normoxia or hypoxia by regulating their metabolism.To maximize the effect of nanoemulsions,mouse Lewis lung cancer(LLC)with abundant macrophage infiltration in the TME was screened as a tumor model.Pharmacodynamic experiments suggested that ?-T-K significantly enhanced the therapeutic effect of anti-PD-1 antibody against LLC tumors.In summary,this study firstly improved the hypoxic TME to sensitize anti-PD-1 immunotherapy.The results showed that the polarization of TAMs was not only the key to determine the effectiveness of hypoxia relief in sensitizing PD-1 blockade therapy,but the core to evaluate the efficacy of anti-PD-1 immunotherapy.Therefore,the follow-up experiments of this study focused on sensitizing the immunotherapy effect of anti-PD-1 antibody by regulating the phenotype of TAMs,which provided a feasible new idea for clinical sensitizing immunotherapy.