Cancer-associated Fibroblasts Promotes The Progression And PD-L1 Expression Of Lung Adenocarcinoma Via Phd3 Signaling

Objective: Lung adenocarcinoma(LUAD)is the most common histologic type of nonsmall cell lung cancer(NSCLC).Recent years have witnessed increasing studies on stromal cells in the tumor microenvironment(TME),with more and more attention paid to the regulation of tumor-associated fibroblasts(CAFs)on NSCLCs.Prolyl hydroxylase domain protein 3(PHD3,encoded by EGLN3)is a class of hypoxiasensitive protein that can bind to hypoxia-induced factors(HIFs)in order to induce the ubiquitination and subsequent degradation of HIFs.However,it remains undetermined regarding the role of PHD3 in the regulation of LUAD progression mediated by CAFs.In this dissertation,we aimed to investigate the effect and mechanism of PHD3 in mediating CAFs-regulated LUAD progression.We also attempted to clarify the prognostic impact of programmed death factor ligand-1(PD-L1),a potential downstream molecule of PHD3,on LUAD receiving surgical resection.Methods: 1.The tissue sections of 109 patients with p-stage I-III LUAD or preinvasive lesions were obtained,and the expression levels of PHD3,PD-L1 and α-SMA were detected by immunohistochemical staining.The TIMER database was used to analyze the correlations between the expression levels of PHD3 as well as PD-L1 and the infiltration of CAFs in LUADs.2.CAFs were obtained via the isolation and culture of primary cells and identified by surface biomarkers(α-SMA,Vimentin).CAFs and A549 cell lines were inoculated into nude mice subcutaneously in different proportions to construct xenograft models.The tumor size and tumor formation rate were monitored regularly,and tumor tissues were obtained for paraffin section and immunohistochemical staining to detect the expression levels of PHD3,PD-L1 and α-SMA.3.Primary adenocarcinoma cells were obtained and identified by CK7 and TTF-1.The CAFs were co-cultured with A549,H1650 cell lines and primary adenocarcinoma cells in a transwell chamber,respectively.The m RNA and protein expression of PHD3,EGFR,p-EGFR,PD-L1 and other molecules were detected by q PCR and western blot.Formation assay,transwell migration and invasion assay were used to reflect cell proliferation,migration and invasion abilities.Meanwhile,the fold changes in the m RNA levels of 11 candidate paracrine molecules including TGF-α,TGF-β1,IL-6,and EGF in CAFs were detected by q PCR.Moreover,western blot was also employed to assess changes in the protein levels of PHD3,EGFR,p-EGFR,PDL1 and other molecules after administration of the screened factors,of which the m RNA levels increased significantly,to A549 and H1650 cell lines,respectively.Additionally,PHD3-overexpressed A549 and H1650 cell lines(A549-PHD3 OE and H1650-PHD3OE)were constructed by lentiviral transfection,and CAFs were co-cultured with A549-PHD3 OE and H1650-PHD3 OE in a transwell chamber.The effect of PHD3 overexpression on A549 and H1650 cells was then determined by CCK8 and transwell migration and invasion assays.STRING database was used to predict the potential interactions between PHD3 and PD-L1 which was further validated using coimmunoprecipitation and immunofluorescence co-localization in LUAD cells cocultured with CAFs.The protein levels of STAT-3/HIF-1α were detected in A549-PHD3 OE and H1650-PHD3 OE cell lines,revealing the molecular mechanism of CAFs regulating PD-L1 expression via PHD3 signalings.4.We performed a comprehensive online search to explore the association between PD-L1 expression(protein and m RNA)and overall survival(OS)or disease-free survival(DFS).Outcomes also included pooled rates of high PD-L1 protein expression in different cell types,per threshold used and per antibody used.A pooled gene expression analysis was also performed on three transcriptomic datasets that were obtained from The Cancer Genome Atlas(TCGA)database and the Gene Expression Omnibus(GEO)database.Results: 1.In terms of the tissue sections,immunohistochemical staining showed that the infiltration of α-SMA+CAFs in TME gradually increased during the evolution of pre-invasive lesions(AAH and AIS)to invasive lesions(MIA,I-III stages).The expression of PHD3 gradually increased from pre-invasive lesions(AAH and AIS)to MIA,and gradually decreased in invasive lesions(I-III stage)as the stages advanced;the expression of PD-L1 exhibited no significant changes from pre-invasive lesions(AAH and AIS)to MIA,but gradually increased in invasive lesions(stages I-III).According to the TIMER database,it was shown that PHD3 negatively correlated with CAFs infiltration in LUADs,while PD-L1 positively correlated with CAFs infiltration.2.Regarding the experiment in vivo,A549 cell line alone or together with CAFs were inoculated into nude mice in different proportions((1)A549 1*10^6+CAFs 1*10^6,(2)A549 1*10^6,(3)A549 1*10^5+CAFs 1*10^6,(4)A549 1*10^5,(5)A5491*10^4+CAFs 1*10^6,(6)A549 1*10^4,(7)CAFs 1*10^6,5 mice in each group)and monitored for 6 weeks.The tumor formation rates were(1)5/5;(2)5/5;(3)3/5;(4)3/5;(5)2/5;(6)0/5;(7)0/5.We also monitored and compared the tumor volume of group(1)and group(2)synchronously,and observed that the volumes of subcutaneous tumors in group(1)were significantly larger than that in group(2).Immunohistochemical staining showed that compared with group(2),the expression levels of α-SMA in tumor tissues in group(1)was higher,while those of PHD3 were lower.Notably,the PD-L1 expression levels were found to be higher in group(1)compared with group(2).3.Aprops of the experiments in vitro,q PCR indicated that the expression of PHD3 m RNA in LUAD cells(A549,H1650 cell lines and primary adenocarcinoma cells)was significantly decreased after co-culture with CAFs,and that of PD-L1 m RNA was significantly increased,while that of EGFR was not altered significantly.Meanwhile,we found increased protein levels of p-EGFR and PD-L1 in LUAD cells,and decreased protein level of PHD3,but no significant change in EGFR by western blot.Besides,it was detected that the proliferation and invasion abilities of A549 cells and H1650 cells were significantly enhanced after co-culture with CAFs.It was confirmed by q PCR that the transcription levels of TGF-α,TGF-β1,IL-6,and EGF in CAFs co-cultured with A549 or H1650 cell lines or primary adenocarcinoma cells were significantly increased.Interestingly,administration of the aforementioned factors alone to A549 and H1650 cell lines can produce similar effects as co-culture with CAFs,resulting in the aforementioned changes in the protein levels of PHD3,p-EGFR and PD-L1.After the successful construction of A549-PHD3 OE and H1650-PHD3 OE cells,transwell migration and invasion assays confirmed that A549-PHD3 OE and H1650-PHD3 OE cells had impaired migration and invasion abilities than A549-NC and H1650-NC cells after PHD3 overexpression,which could be restored by co-culture with CAFs.CCK8 assay showed that the proliferation of A549-PHD3 OE and H1650-PHD3 OE cells was attenuated compared with A549-NC and H1650-NC cells after PHD3 overexpression,while co-culture with CAFs could promote the proliferation of A549-PHD3 OE and H1650-PHD3 OE.Furthermore,in A549-PHD3 OE and H1650-PHD3 OE cells,PHD3 inhibitors and CAFs synergistically reversed the diminished migration,invasion and proliferation abilities caused by PHD3 overexpression.By analyzing the STRING database,we identified a potential protein interaction network among EGLN3,EGFR,CD274(the gene encoding PD-L1),STAT-3 and HIF-1α.Co-immunoprecipitation and immunofluorescence co-localization further confirmed the interaction between PHD3 and PD-L1.Furthermore,we observed upregulated expression of PD-L1,and activation of STAT-3/HIF-1α in LUAD cell lines(A549-PHD3 OE,H1650-PHD3 OE,A549-NC and H1650-NC)after co-culture with CAFs by western blot,with downregulated PHD3 expression.In addition,both HIF-1α inhibitors and STAT-3 inhibitors could attenuate the elevated PD-L1 expression caused by the co-culture of these cells with CAFs.4.To provide robust evidence for clinical practice,a total of 6488 patients from 25 studies were included.The pooled results suggested that high PD-L1 expression was associated with shorter OS(HR[hazard ratio] = 1.57;P < 0.001)and DFS(HR = 1.341;P = 0.037)in the overall population.The overall pooled rate of high PD-L1 protein expression was29%(95% CIs[confidence interval]: 23–34%)in tumor cells.In subgroup analysis,high PD-L1 protein expression in tumor cells was a prognosticator for worse OS and DFS.A pooled analysis on of TCGA dataset and GEO datasets revealed that higher levels of PD-L1 m RNA predicted poorer OS in the entire population.Conclusions: The progression of LUAD is accompanied by increased infiltration of CAFs in TME and complicated changes in the expression of PHD3 and PD-L1 in tumor cells.CAFs can downregulate the expression of PHD3 in LUAD cells via paracrine of TGF-α,TGF-β1,IL-6,and EGF,thereby promoting the proliferation,migration and invasion of LUAD as well as PD-L1 expression.Overexpression of PHD3 can attenuate the promoting effect of CAFs on the proliferation,migration and invasion of LUAD cells,and can downregulate the expression of PD-L1 induced by CAFs through STAT-3/HIF-1α pathway.By pooling the clinical evidences available,our analyses on the subject shed light on the high expression rate of PD-L1 and the prognostic value of high PD-L1 expression in resected LUADs.PD-L1 gene expression is a promising prognostic factor for patients with surgically resected LUAD.Standardization of staining should be underscored prior to routine implementation.