NLRP4-mediated Response Immune Checkpoint Inhibitor Therapy Benefits In Tumor Microenvironment

Objective:Immunotherapy has made a huge breakthrough in cancer care,successfully changing the traditional treatment based on surgery,radiotherapy,chemotherapy and targeted therapy.In particular,in the treatment of lung cancer,immune checkpoint inhibitors have been approved for first-line treatment.However,only 20%advanced NSCLC patients response to immune checkpoint inhibitors.As tumor progression involves complex interactions between tumor cells and immune cells,contributing to the formation of tumor microenvironment depended on tumor charicateristc.Specific gene mutations and its tumor-environment are associated with clinical outcomes after immuno-checkpoint therapy.Methods:In this study,tumor and matched blood DNA samples underwent whole-exome sequencing,and was used to analyze tumor biopsy samples to investigate the specific gene mutations that could reversed tumor resistance to immunotherapy.To further explore the immunotherapy benifical tumor microenvironment,we performed multiplex immunofluorescence analysis on pretreatment biopsy.And futher study focus on the gene regulated fuction and its induced tumor microenvironment using PCR,WB IHC and flowcytometre.Results:The NLRP4 mutation only happened in the response group in NSCLC patients received immnotherarpy,and the patients with this mutation had a durable response.After si NLRP4 in 293T cell lines,IFN-αand IFN-β’s upregulation was observed.To analyze the effect of NLRP4 mutations in tumors,sh RNA was done in LLC(tumor cell lines),WB and RT-PCRwere used for identificated the phenotypes and pathways.It was found that NLRP4 negative regulated of STING-TBK1-IRF3-IFN-Is pathway.Although NLRP4 did not affect cell growth rate in vitro,NLRP4 KD inhibited tumor growth in vivo.RNA-seq analysis of tumor tissues revealed NLRP4 KD tumor high expression of CD8~+T and CD3~+T.Immunohistochemical and flowcytometre showed more CD8~+T cells in NLRP4 KD tumor microenvironment and spleen.The M2macrophage was enriched in NLRP4 KD tumor,the co-culture experiment in vitro was also verified.Multi immunofluorescence of pre-treatment tumor biopsy were used to analyse the tumor microenvironment,expression of CD8~+T and its position are related to the prognosis of patients,and high expression of M2 macrophage and high expression of PD-L1 in M2 macrophage are also related to the response to ICIs.These fingings were similar to NLRP4 KD induced tumor environment.To further explore the mechanism by which NLRP4 KD induces CD8~+T cell migration,transwell experiments in vitro demonstrated that NLRP4 KD or IFN-Is can induce more CD8~+T cell metastasis.After GSVA analysis of the online WES database,there were significant differences in the type Ⅰ IFN among 33 NSCLC patients with different therapy outcomes.Luminex detected cytokines in peripheral blood of patients at the baseline of immunotherapy,and the increase of IFN-αcytokines in peripheral blood could prolong the PFS of patients undergoing immunotherapy.Subsequently,PD-L1 and NLPR4 KD were combined in animal experiments to further inhibit the tumor.The long PFS of patients with NLRP4 KD was further validated.Conclusions:NLRP4 reshapes tumor microenvironment with more recruiting of M2macrophage cells and CD8~+T cells by negatively regulating tumor local type Ⅰ interferon through STING/TBK1/IRF3 pathway,and finally enhance antitumor response of ICIs.Our results will provide direct evidence for IFN-I to improve the clinical efficacy of ICIs in the treatment of lung cancer,and provide new strategies for screening effective population and overcoming ICIs resistance.