Targeting Inhibition Of CYP4F Improves Anti-PD-1 Therapy In Lung Cancer And Its Mechanism Of Action

Background and purpose:Anti-PD-1/PD-L1 therapy shows exciting therapeutic effects in non-small cell lung cancer（NSCLC）,however,its overall response rate is only20-30%.Therefore,studying the potential mechanism of resistance to anti-PD-1/PD-L1therapy and finding new targets are critical for the treatment of NSCLC.Metabolic dysregulation of cancer cells contributes to ineffective immune function and confers immunotherapy resistance.Cytochrome P450（CYP）4A/F mainly catalyzesω-hydroxylation of arachidonic acid（AA）to biologically active eicosanoids20-hydroxyeicosatetraenoic acid（20-HETE）,which contributes to tumor progression.Cancer-associated fibroblasts（CAFs）,the most abundant cell population in the tumor microenvironment,are recently emerging as immunosuppressive regulators.The goal of the study was to uncover the role of CYP4F-mediated immunosuppression of cancer-associated fibroblasts to promote resistance to anti-PD-1 therapy and its mechanism of action,attempting to provide new targets for improving the efficacy of anti-PD-1 therapy.Methods:（1）CYP4F2 expression in human non-small cell lung cancer using GEO database was analyzed;Kaplan-Meier survival analysis of relationship between CYP4F2expression and overall survival（OS）;the correlations of CYP4F2 expression with the infiltration and effector function（Gzm B and IFN-γ）of CD8⁺T cells were analyzed in tissue microarray and TCGA database;（2）The correlations of CYP4F2 expression with markers of tumor-associated fibroblasts（FAP）and angiogenesis（CD31）were analyzed;（3）Lewis-m Cherry-luciferase cells（2×10⁵）were injected subcutaneously into the right flank of C57BL/6 mice.7 days after the tumor reached size of about 100 mm³,the mice were treated with HET0016（5 mg/kg/five times weekly,i.p.）,DDMS（7.5 mg/kg/five times weekly,i.p.）or vehicle liposome.Tumor growth was detected by ex vivo luciferase-based bioluminescence imaging;the tumor weights were measured;the number of CD8⁺T and CD4⁺T cell in the LLC tumor tissues were measured by immunofluorescence and flow cytometry;the expression of IFN-γand granzyme B were measured by immunofluorescence;the level ofα-SMA and FAP was measured by q PCR and immunofluorescence;（4）GFP⁺L929 fibroblasts（2×10⁵）were mixed with LLC cells,negative control lentivirus or CYP4F13 lentiviral activation particle(CYP4F13^high)transfected LLC cells at a ratio of 1:1,and were subcutaneously injected into the right flank of C57BL/6 mice.LLC cells（2×10⁵）injected alone were as control.The tumor weights,CD8⁺T cell infiltration,IFN-γand granzyme B expression and the level ofα-SMA and FAP were measured as described above;（5）C57BL/6 mice were injected with parental or CYP4F13^highLLC cells（2×10⁵）in the right flank,and then treated with tranilast（200 mg/kg/daily,i.g.）or vehicle for 14 days.The tumor weights,CD8⁺T cell infiltration,IFN-γand granzyme B expression and the level ofα-SMA and FAP were measured as described above;（6）MRC-5 fibroblasts were treated with the conditioned medium（CM）from CYP4F2^high,DDMS（10μM）,HET0016（5μM）or vehicle-treated A549 cells.The expression ofα-SMA was measured by immunofluorescence;（7）WI38fibroblasts incubated with the CM from CYP4F2^high,DDMS（10μM）,HET0016（5μM）or vehicle-treated A549 cells were cultured with CD8⁺T cells for the indicated time.The proliferation of CD8⁺T cells was measured by carboxyfluorescein succinimidyl ester（CFSE）dilution;IFN-γin CD8⁺T cells were determined by q PCR and ELISA;（8）Immunosuppressive cytokines and coinhibitory molecule in MRC-5 fibroblasts incubated with the CM from CYP4F2^highor vehicle-treated A549 cells were measured by q PCR;IL-6and TGF-βprodution were measured by ELISA;The MRC-5 fibroblasts incubated with the CM from the CYP4F2^highA549 cells were treated with or without anti-PD-L1（20ng/ml）,anti-TGF-β（10 ng/ml）,or anti-IL-6（20 ng/ml）for 24 h.The proliferation of CD8⁺T cells co-cultured with the above treated MRC-5 fibroblasts was measured;IFN-γin CD8⁺T cells were determined by q PCR and ELISA;（9）GPR75 in MRC-5 and L929fibroblasts treated with 20-HETE（1μM）was determined by Western blot;MRC-5 and L929 fibroblasts pre-treated with or without GPR75-si RNA were incubated with 20-HETE（1μM）for 24 h.STAT3 and p-STAT3 were determined by Western blot;α-SMA and PD-L1 were determined by q PCR;TGF-βwas determined by ELISA;The proliferation of CD8⁺T cells was measured;（10）Lewis-m Cherry-luciferase cells（2×10⁵）were injected subcutaneously into the right flank of C57BL/6 mice.When tumors had reached a size of about 100 mm³,the mice were treated with anti-PD-1 antibody（200 mg/mouse every 3days,i.p.）,HET0016（5 mg/kg daily,i.p.）,anti-PD-1 antibody plus HET0016,or vehicle for 12 days.The tumor volume,tumor weights and survival time were measured.Results:（1）High CYP4F2 expression was correlated with poor overall survival（OS）in patients with NSCLC in the GEO database;CYP4F2 expression correlated with the infiltration and effector function of CD8⁺T cells;（2）GEO database analysis showed that CYP4F2 was significantly positively correlated with angiogenesis marker CD31 and fibroblast activation markerα-SMA and FAP;（3）CYP4F inhibition by HET0016 and DDMS significantly inhibited tumor growth as evidenced by decreases in luciferase intensity and tumor weight;the number of CD8⁺T cells was markedly increased in the HET0016-and DDMS-treated tumors,yet the number of CD4⁺T cells was little changed as compared with the control;HET0016 and DDMS treatment significantly increased the number of IFN-γ⁺CD8⁺T cells and GZMB⁺CD8⁺T cell;HET0016 and DDMS reduced microvessel density but enhanced vessel pericyte coverage;HET0016 and DDMS reduced the levels ofα-SMA and FAP in CAFs;（4）Co-implantation of CYP4F13^highLLC cells with GFP⁺L929 fibroblasts enhanced tumor growth as compared with co-injection of LLC-LV-NC cells with GFP⁺L929 fibroblasts;co-implantation of LLC-CYP4F13 with GFP⁺L929 fibroblasts increased the number ofα-SMA⁺GFP⁺cells,and reduced intratumoral number of CD8⁺T cells and their expression of Gzm B and IFN-γ;the pericyte coverage of tumor vessels was decreased by CYP4F13^highLLC cells plus GFP⁺L929 fibroblasts;（5）Treatment with tranilast partly reversed the effects of CYP4F13overexpression on tumor weight,vessel pericyte coverage,CD8⁺T cell number and its expression of Gzm B and IFN-γ;（6）Immunofluorescence analysis showed thatα-SMA expression was significantly increased in the MRC-5 fibroblasts treated with the CYP4F2^high-CM,but decreased in HET0016-and DDMS-CM;（7）The proliferation of CD8⁺T cells and its expression of effector cytokine IFN-γwere decreased in the CYP4F2^highgroup as compared with the LV-NC group;（8）Compared with the fibroblasts treated with LV-NC CM,CYP4F2^high-CM increased IL-6,TGF-β,and PD-L1 expression;neutralizing antibodies against PD-L1,IL-6,and TGF-βpartially abolished the effect of CYP4F2 overexpression on CD8⁺T cell proliferation,their expression of Gzm B and IFN-γand the migration of pericytes and endothelial cells;（9）The expression of GPR75was increased after treatment of MRC-5 and L929 fibroblasts with 20-HETE;knockdown of GPR75 significantly attenuated the effects of 20-HETE on p-STAT3,α-SMA and PD-L1 m RNA expression,TGF-βsecretion and CD8⁺T cell proliferation;（10）Combined HET0016 with anti-PD-1 treatment significantly inhibited tumor growth and delayed survival time.Conclusion:Targeting CYP4F-dependent arachidonic acid metabolism improves anti-PD-1 therapy through blocking tumor-stromal crosstalk,and may serve as a novel combination strategy for human lung cancer.