Correlation Analysis Between Fibroblasts Activation In The Tumor Microenvironment And Immunotherapy Efficacy Of Non-Small Cell Lung Cancer And Its Mechanism

Lung cancer is the second most common malignancy and the leading cause of cancer mortality worldwide.Immunotherapy represented by immune checkpoint inhibitors(ICIs)has become a crucial part of the comprehensive treatment system of non-small cell lung cancer(NSCLC).However,only 20%-40%of NSCLC patients benefit from programmed death-1(PD-1)antibodies.Exploring predictive biomarkers from the tumor microenvironment is considered an important approach to realize the precision of immunotherapy.The purpose of this study is to focus on the correlation between fibroblast activation in the tumor microenvironment and NSCLC immunotherapy efficacy,and further explore its regulatory mechanism.Part 1 Correlation analysis between fibroblasts activation in the tumor microenvironment and immunotherapy efficacy of non-small cell lung cancerObjective:Fibroblast activation protein(FAP)is one of the specific markers of cancer associated fibroblasts(CAFs).We aim to investigate on the expression of FAP and the correlation between FAP expression and immune infiltration,and clinical outcomes of PD-1 antibodies in advanced NSCLC,so as to identify if FAP could be a robust predictive biomarker that would preselect advanced NSCLC patients who would derive benefit from PD-1 antibodies.Methods:1.The study cohort was selected from advanced NSCLC patients who received PD-1 blockade at the Fourth Hospital of Hebei Medical University from June 2018 to November 2020.135 NSCLC patients were included(57cases of squamous cell lung cancer,78 cases of non-squamous NSCLC).2.The expression levels of FAP,CD3 and CD8 in tumor tissues were detected by Immunohistochemistry(IHC).3.The associations between FAP expression and the clinicopathological characteristics were evaluated by the chi-square test.4.The correlation of FAP with programmed death-ligand 1(PD-L1)and the density of T cells was estimated by the Spearman correlation test.5.Bioinformatics analysis was used to explore the association between FAP expression and the degree of immune cell infiltrates and the expression of immune cell marker genes.6.Kaplan-Meier method was utilized to analyze the correlation between FAP expression level and progression free survival(PFS)and overall survival(OS)of NSCLC patients.Cox proportional hazard model was applied to analyze the clinical significance of FAP as a prognostic factor for PD-1 antibodies treatment in NSCLC.Results:1.FAP was highly expressed in NSCLC stroma.FAP expression positively correlated with the presence of distant metastasis(P<0.001).2.FAP was correlated with decreased density of CD8~+T cells(Spearman’s rho=-0.32,P<0.001).No correlations were detected between the density of CD3~+T cells and PD-L1.3.Bioinformatics analysis showed that FAP was inversely correlated with the infiltration of CD8~+T cells,activated DCs and activated NK cells(all P<0.01),and FAP was positively correlated with the abundance of Tregs,M2macrophages and neutrophils(all P<0.001).FAP was positively related to the upregulation of immune checkpoints and immunosuppressive genes,indicating that FAP was relevant to the features of the immunosuppressive tumor microenvironment in NSCLC.4.Kaplan-Meier survival analysis revealed that patients with higher expression of FAP conferred worse PFS(HR=2.56,95%CI:1.69-3.87,P<0.001).Patients with low FAP expression exhibited a trend with longer OS than that of patients with high FAP expression(HR=1.57,95%CI:0.99-2.48,P=0.053).FAP contributed to shortened OS in subgroups of the patients with squamous cell lung cancer(P=0.020),PD-1 antibody monotherapy(P=0.017)and first-line therapy(P=0.028).5.Univariate analysis of the Cox proportional hazards model revealed that the factors significantly associated with PFS were TNM stage,distant metastasis,line of systematic therapy,FAP expression,and CD8~+T cell density.Multivariate analysis of the Cox proportional hazards model revealed that FAP expression(HR=2.33,95%CI:1.50-3.64,P<0.001)and line of systematic therapy(HR=1.67,95%CI:1.05-2.65,P=0.031)were independent prognostic factors for predicting PFS.6.Univariate analysis of the Cox proportional hazards model revealed that the factors significantly associated with OS were TNM stage(HR=2.49,95%CI:1.31-4.74,P=0.005)and distant metastasis(HR=2.76,95%CI:1.49-5.09,P=0.001).Multivariate analysis of the Cox proportional hazards model indicated that none of the variables were independent factors for predicting OS of advanced NSCLC patients.Summary:1.FAP is widely expressed in advanced NSCLC tissue.FAP is correlated with decreased density of CD8~+T cells and the formation of immunosuppressive tumor microenvironment.2.The expression level of FAP is negatively correlated with PFS of patients treated with PD-1 antibodies.FAP is a potential predictive biomarker of resistance to PD-1 antibodies.Part 2 In vitro study of the effect of cancer-associated fibroblasts on tumor biological behavior in non-small cell lung cancerObjective:To clarify the regulatory effects of cancer associated fibroblasts(CAFs)on tumor microenviroment,isolate and culture CAFs and normal fibroblasts(NFs)from human NSCLC tissues.To investigate the characteristics of CAFs and their effects on the invasion and metastasis of NSCLC cells.Methods:1.CAFs and paired NFs were isolated from tumor tissues and adjacent non-tumor tissues from NSCLC patients who underwent surgical resection(n=3).Tissue was digested enzymatically and fibroblasts were cultured.2.Immunofluorescence was used to identify fibroblasts.3.Western blotting was used to detect the expression of FAP andα-SMA in paired CAFs and NFs.4.The contraction capacity of CAFs and NFs was detected by collagen contraction assay.5.The conditioned medium(CM)of CAFs and NFs were collected.The changes of migration and invasion capacity of A549 and PC9 cells induced by different CMs were observed by wound healing assay and Transwell assay.Results:1.CAFs and NFs of human primary NSCLC were successfully isolated and cultured.The cells showed long spindle shape and cord-like growth,which was consistent with the morphological characteristics of fibroblasts.2.Immunofluorescence results showed thatα-SMA and FAP were positively expressed in CAFs,while Epithelial cell adhesion molecule(EPCAM)expression was negative.3.Western blotting results indicated that the expression level of FAP protein in CAFs was higher than that of NFs.4.The results of collagen contraction assay showed that CAFs had stronger contraction ability than NFs(P<0.05).5.The results of scratch healing assay and Transwell assay showed that compared with the NF-CM,CAF-CM significantly promoted the migration and invasion of A549 cells and PC9 cells(P<0.001).Summary:CAFs and NFs of human primary NSCLC are successfully isolated and cultured.Compared with NFs,CAFs have stronger promoting effect on the migration and invasion capacity of NSCLC cells.Part 3 The mechanism exploration of circ NOX4 mediated cancer-associated fibroblasts activation through upregulating FAPObjective:Based on the theory that fibroblast activation promotes tumor invasion,metastasis and immunotherapy resistance,we aim to screen the key circ RNA that mediate CAFs activation and to explore the epigenetic regulatory mechanism of FAP promoting NSCLC progression.Methods:1.The ce RNA chip technology was conducted to screen out differentially represented circ RNAs in CAFs and NFs.Reverse transcription-polymerase chain reaction(RT-PCR)was used to detect the expression of candidate circ RNAs in CAFs;Sanger sequencing identified the circularized splice junction sequence of circ NOX4.RNase R experiment was utilized to verify the characteristics of circ NOX4 in fibroblasts.Quantitative real-time polymerase chain reaction(q RT-PCR)was applied to determine the expression level of circ NOX4 in CAFs,NFs and NSCLC cell lines.2.Tumor tissues from 54 NSCLC patients(27 cases of lung adenocarcinoma,19 cases of squamous cell carcinoma,6 cases of large cell carcinoma,2 cases of sarcomatoid carcinoma)in the Fourth Hospital of Hebei Medical University from June 2018 to February 2019 were collected.Fluorescence in situ hybridization(FISH)was used to evaluate the expression of circ NOX4 in NSCLC tissues.Chi-square test was applied to analyze the relationship between the circ NOX4 expression level and clinicopathological parameters of NSCLC patients.Kaplan-Meier method was applied to analyze the correlation between circ NOX4 expression level and OS of NSCLC patients.Cox proportional hazards models was applied to evaluate the effect of circ NOX4 expression level on the prognosis of NSCLC patients.3.Fibroblasts with circ NOX4 knockdown and circ NOX4 overexpression cell model were constructed.q RT-PCR was adopted to confirm the transfection efficiency.4.Cell counting kit-8(CCK-8)assay was adopted to analyze the effect of circ NOX4 knockdown and overexpression on the proliferation capacity of fibroblasts.Collagen contraction assay was adopted to analyze the effects of circ NOX4 knockdown and overexpression on the contraction ability of fibroblasts.Western blotting was applied to detect FAP protein expression in circ NOX4 knockdown CAFs and circ NOX4 overexpression NFs.5.The subcellular localization of circ NOX4 in CAFs and NFs was determined by FISH.6.Bioinformatics databases of miRanda and RNAhybrid were used to predict miRNAs that could interact with circ NOX4.Targetscan database was used to predict the potential miRNAs that target FAP,in order to screen the miRNAs that take effects in the regulation of fibroblast activation by circ NOX4.7.q RT-PCR was used to detect the expression of candidate miRNA(miR-329-5p and miR-624-5p)in CAFs and NFs,and detect the expression changes of miR-329-5p and miR-624-5p in CAFs after knocking down circ NOX4.8.Dual luciferase reporter assay as well as RNA-binding protein immunoprecipitation(RIP)assay were used to confirm the binding capacity of circ NOX4 with miR-329-5p and miR-624-5p.Dual luciferase reporter assay was used to confirm whether circ NOX4 could interact with FAP.9.Western blotting was applied to test the changes of the FAP protein expression after knocking down and overexpressing miR-329-5p.10.miRNA rescue experiments and western blotting was applied to verify the circ NOX4/miR-329-5p/FAP regulatory axis,and the effect of this regulatory axis on the contractile ability of fibroblasts was verified by collagen contraction experiments.11.Wound healing assay and Transwell assay was applied to explore the effects of knocking down circ NOX4 in CAFs and overexpressing circ NOX4 in NFs on the migration and invasion ability of NSCLC cells.12.Cytokine array was applied to detect the cytokines secreted by CAFs and analyze the effect of circ NOX4 on its secretion level,the result of cytokine array was verified by enzyme linked immunosorbent assay(ELISA).13.A549 cells or A549 cells mixed with CAFs were used to construct subcutaneous CAFs-tumor cell transplantation model of nude mice,which were divided into A549 group,A549+sh NC-CAFs group and A549+sh-circ NOX4-CAFs group.The tumor growth was observed and the tumor volume and weight were measured.The expression levels of FAP,MMP2 and MMP9 proteins were detected by IHC.Results:1.The results of ce RNA chip,RT-PCR and q RT-PCR showed that compared with NFs and NSCLC cell lines,circ NOX4 was highly expressed in CAFs(P<0.01).Sanger sequencing and Rnase R results verified that circ NOX4had a closed continuous loop structure,laying foundation for further study of epigenetic regulation mechanism.2.FISH indicated that circ NOX4 was mainly expressed in tumor stroma of NSCLC.Chi-square test showed that the expression level of circ NOX4 was correlated with distant metastasis(P<0.01).Kaplan-Meier survival analysis showed that the OS was significantly shorter in patients with high circ NOX4expression compared with those with low circ NOX4 expression(P<0.0001).Cox proportional hazards model analysis showed that distant metastasis(P=0.019)and circ NOX4 expression(P=0.002)were correlated with OS in NSCLC patients,indicating circ NOX4 expression could be considered as an independent prognostic factor for NSCLC patients.3.The results of q RT-PCR showed that after transfection of circ NOX4small interfering RNA(si RNA)in CAFs,the expression level of circ NOX4 was significantly decreased(P<0.05).The expression level of circ NOX4 in the sh-circ NOX4 group was significantly downregulated compared with the sh-NC group(P<0.05).The expression level of circ NOX4 in the circ NOX4 group was significantly upregulated compared with the Vector group(P<0.001),suggesting that CAFs cell lines of knocking down circ NOX4 and NFs cell lines of overexpressing circ NOX4 were successfully constructed.4.The results of CCK-8 method and collagen contraction assay showed that circ NOX4 knockdown reduced the proliferation and contraction capacities of CAFs(P<0.01),circ NOX4 overexpression enhanced the proliferation and contraction capacities of NFs(P<0.01).Western blotting showed that the FAP protein expression in CAFs was downregulated after knocking down circ NOX4,and upregulated in NFs after overexpressing circ NOX4.5.The results of FISH assay indicated that circ NOX4 was primarily located in the cytoplasm of CAFs and NFs.6.The miRanda,RNAhybrid and Targetscan databases predicted the potential miRNAs that can both bind to circ NOX4 and FAP,and miR-329-5p and miR-624-5p was identified by overlapping the prediction results.7.q RT-PCR results showed that expression levels of miR-329-5p and miR-624-5p in CAFs were significantly downregulated compared with NFs.After knocking down circ NOX4,the expression levels of miR-329-5p and miR-624-5p were significantly upregulated(P<0.001).8.Dual luciferase reporter assay and RIP assay revealed that circ NOX4could interact with miR-329-5p as a miRNA sponge in CAFs(P<0.05),while circ NOX4 failed to bind miR-624-5p in CAFs.miR-329-5p could bind to FAP3’UTR(P<0.05).9.Western blotting results showed that overexpression of miR-329-5p reduced the expression of FAP in CAFs.10.The results of miRNA rescue experiments exhibited that knocking down circ NOX4 in CAFs reduced the expression level of FAP protein,while miR-329-5p inhibitors saved this phenomenon.Overexpression of circ NOX4 in NFs upregulated the expression level of FAP protein,which could be saved by miR-329-5p mimic.The results of collagen contraction experiments showed that knocking down of circ NOX4 in CAFs reduced the contractile ability,while miR-329-5p inhibitors partially restored the contractile ability(P<0.001).The results confirmed circ NOX4/miR-329-5p/FAP regulatory axis in CAFs.11.The wound healing assay and transwell assay suggested knocking down circ NOX4 in CAFs inhibited the migration and invasion capacity of A549and PC9 cells(P<0.05),Overexpressing circ NOX4 in NFs promoted the migration and invasion capacity of A549 and PC9 cells(P<0.01).12.Cytokine array and ELISA analysis confirmed that interleukin-6(IL-6)was the most downregulated cytokine in the supernatant of CAFs after knocking down circ NOX4(P<0.001).It is suggested that circ NOX4-mediated CAFs promote the progression of NSCLC,which may be related to the upregulation of IL-6 expression and the formation of immunosuppressive tumor microenvironment.13.In mixed xenograft tumor models of nude mice,CAFs promoted the growth of xenograft tumor in nude mice(P<0.001);sh-circ NOX4-CAFs significantly stunted tumor volume and tumor weight compared with A549 cells cotransplanting with sh NC-CAFs(P<0.001);IHC analysis confirmed that compared with the A549+sh NC-CAFs group,the expression of FAP,MMP2and MMP9 in the A549+sh-circ NOX4-CAFs group was downregulated,indicating that circ NOX4 knockdown in CAFs downregulated FAP and inhibited the tumorigenesis of xenograft tumor in vivo.Summary:1.A novel circ RNA circ NOX4 specific to CAFs is identified,which is highly expressed in CAFs of NSCLC microenvironment and correlates with distant metastasis and poor prognosis of NSCLC patients.2.circ NOX4 promotes FAP expression and CAFs activation through sponging miR-329-5p as a ce RNA,thus promoting the progression of NSCLC.Conclusions:1.FAP is correlated with decreased density of CD8~+T cells and the formation of immunosuppressive tumor microenvironment,and negatively correlated with the efficacy of PD-1 antibodies in advanced NSCLC.FAP could be considered as a predictive biomarker of resistance to immunotherapy.2.circ NOX4 bind to miR-329-5p as ce RNA to promote the upregulation of FAP expression and activation of CAFs,thus promoting the immunosuppressive tumor microenvironment and progression of NSCLC.