Modulation Of Antitumor Immune Response By Targeting USP7 To Reset Tumor-associated Macrophages In Lung Cancer

Objective:Tumor-associated macrophages(TAMs)play an important role in occurrence and development of lung cancer.Resetting TAMs from M2 to tumor-killing M1macrophages(MΦs)is a meaningful strategy to ameliorate the immunosuppressive microenvironment and enhance the antitumor immune response.Therefore,exploring new targets for resetting TAMs has important research value and clinical significance.In this study,RT-PCR was used to quantify the expression of common genes related to the deubiquitinating enzymes(DUB)in M1 and M2 MΦs induced from ANA-1.We found that the differential expression of USP7 was the most significant one between M1 and M2MΦs.USP7 is a cysteine protease,an important member of the family of DUB.It is also the first DUB that has been specifically inhibited by drugs with good therapeutic effect in various tumors.Related studies have shown that USP7 mainly plays the role of an oncoprotein by regulating the MDM2-P53 axis in various tumors.At the same time,USP7can also stabilize the expression of Foxp3 to maintain the function of regulatory T(Treg)cells.However,the role of USP7 in resetting TAMs has not been reported,to the best of our knowledge.Here,we investigated the role and underlying mechanism of USP7 in resetting TAMs.Methods:(1)RT-PCR was used to quantify differential expressions of common genes related to DUB between M1 and M2 MΦs induced from ANA-1 murine macrophage cell line.Differential expressions of USP7 were most significant among all genes quantified,which were further validated by RT-PCR and WB in M1 and M2 MΦs induced from bone marrow-derived macrophages(BMDMs).(2)We evaluated effects of targeted inhibition of USP7(inhibitor and si RNA)on phenotypes of M2 MΦs and the conditioned medium from treated MΦs on proliferation of CD8~+T cells.We further confirmed effects of targeted inhibition of USP7 on lung cancer growth and antitumor immune response in a Lewis lung cancer model.(3)Clearance of macrophages in vivo,flow cytometry sorting control group and treatment group of TAMs for RNA sequencing and WB were used to explore the underlying mechanism of resetting TAMs following targeted USP7 inhibition.(4)we explored the therapeutic effect of targeted inhibition of USP7 combined with PD-1monoclonal antibody in vivo,and investigated the role of USP7 in lung adenocarcinoma from TCGA database.Results:(1)To screen the m RNA expression of 51 common DUB genes in M1 and M2MΦs induced form ANA-1,we found that differential expressions of USP7 were most significant among all genes quantified,which were further validated by RT-PCR and WB in M1 and M2 MΦs induced from BMDMs.(2)Targeted inhibition of USP7 significantly inhibited the phenotype and function of M2 MΦs in vitro.In vivo application of USP7targeted inhibitor P5091 significantly inhibited the growth of mouse Lewis lung cancer,resetted TAMs from M2 to M1 MΦs,and activated the antitumor immune response mediated by cytotoxic T cells(CTLs).(3)Targeted inhibition of USP7 inhibited tumor growth and promoted the activation of CTLs by resetting TAMs,which could be through the activation of JNK/ERK/p38 MAPK pathway.(4)Targeted inhibition of UPS7significantly increased the expression of PD-L1 in tumor microenvironment,and further combined with PD-1 monoclonal antibody could exert synergistic antitumor effect.(5)The role of USP7 oncoprotein and negative immune regulation in lung adenocarcinoma were further confirmed in TCGA database.Conclusion:We found that USP7 plays an important role in resetting TAMs of lung cancer for the first time.Targeted inhibition of USP7 may reset TAMs to M1 MΦs by activating JNK/ERK/p38 MAPK pathway,and then activate the anti-tumor effect mediated by CTL,and finally inhibit tumor growth.However,targeted inhibition of USP7 can increase the expression of PD-L1 in tumor microenvironment,and further combined with PD-1monoclonal antibody can exert synergistic antitumor effect.Moreover,targeted inhibition of USP7 resetting TAMs combined with PD-1 monoclonal antibody in the treatment of lung cancer is worthy of further study.