Effects Of Chemoradiotherapy On Tumor Microenvironment Of Esophageal Squamous Cell Carcinoma And The Involved Mechanisms

Part 1Objectives The tumor microenvironment plays an important role in the development and progression of tumors.This study aimed to explore the effect of chemoradiation on the tumor microenvironment of esophageal squamous cell carcinoma,and to evaluate the prognostic value of tumor infiltrating lymphocytes.Methods The study retrospectively enrolled 104 patients with ESCC who underwent surgery alone or preoperative neo-adjuvant chemoradiotherapy.The total TILs were assessed in hematoxylin-eosin staining tissue sections.Immunohistochemistry was used to detect the expression of Fox P3+ T cells,CD4+ T cells,CD8+ T cells,CD56+ NK cells and PD-L1 expression in tumors.Results 1.Compared with patients who received surgery only,the density of TILs,CD8+ TILs,CD4+ TILs and NK TILs in TME of patients who received preoperative n CRT was high(P<0.05),and no significant difference was found in Foxp3+ TILs counts(P>0.05),and the expression of PD-L1 was significantly increased after n CRT(P<0.05).2.The density of TILs,CD8+ TILs and CD4+ TILs was correlated with pathological T stage and N stage(P<0.05).There was no significant correlation between CD56+ NK TILs and Foxp3+ TILs with gender,age,stage,tumor location,soft tissue infiltration,etc.(P>0.05).The positive expression of PD-L1 was associated with pathological T stage and N stage(P< 0.05).3.Univariate analysis showed that T stage,N stage,CD8+ TILs,CD4+ TILs and Fox P3+ TILs were associated with DFS.Cox multivariate survival analysis showed that CD4+ TILs and Fox P3+ TILs were independent prognostic factors for DFS(P<0.05).In univariate analysis,T stage,N stage,CD8+ and CD4+ TILs were associated with OS.However,only T stage-9and CD8+ TILs were independent predictors of OS on multivariate analysis.4.In 48 patients who received preoperative n CRT,25 patients with significant tumor response(tumor regression grade,TRG 1-2)after n CRT had earlier yp T stage(P < 0.001)compared with tumor non-responders(TRG3-5),significant lymph node response(P = 0.004),and higher CD8+ TILs density(P = 0.027).Compared with non-lymph node responders,27 patients with lymph node downstaging(c N+-p N0)had earlier yp N stage(P <0.001).Conclusions 1.The density of TILs,CD8+ TILs,CD4+ TILs,CD56+ TILs and PD-L1 expression increased in TME after n CRT.2.TILs,CD8+ TILs,CD4+ TILs were associated with pathological T and N stage.Patients with poor disease stage had low tumor infiltrating lymphocyte density.3.Foxp3+ TILs,CD4+ and CD8+ T cells are reliable predictors of prognosis in ESCC patients,which provide a new basis for comprehensive anti-tumor strategies combining with CRT and immunotherapy.4.Chemoradiotherapy response was associated with the density of CD8+ TILs.The greater the density of CD8+ TILs,the more obvious the chemoradiotherapy response.Part 2Objectives Recent research findings support the role of CRT in activating an anti-tumor immune response.A critical aspect of tumor-targeted immune responses is mediated by circulating lymphocytes,a cell type that also exhibits significant radiation sensitivity.The aim of this study was to measure CRT-induced changes to lymphocyte subpopulations and to evaluate the prognostic value of lymphocyte alterations for patients with ESCC.Methods In total,this pilot study enrolled 64 patients with ESCC who received neo-adjuvant CRT or definitive CRT.Peripheral blood samples were collected before and during treatment and were analyzed by flow cytometry for CD19,CD3,CD4,CD8,CD56,and CD16.Relationships between lymphocyte subset alterations and overall survival(OS)and progression-free survival(PFS)were evaluated using the log-rank test and a Cox regression model.Results 1.Compared to pre-treatment specimens,post-treatment blood samples had decreased proportions of CD19+ B-cells and increased proportions of CD3+ and CD8+ T-cells(all P values <0.05).However,there were no changes in the proportions of CD4+ cells or NK cells(P >0.05).2.There was no significant correlation between CRT-induced CD8+ T cell and CD4+ T cell alterations and clinical pathological factors(P>0.05).CRT-induced CD19+ B cell alterations were associated with age(P<0.05),and no statistically significant differences were found regarding gender,smoking status or disease stage,etc.(P>0.05).CD56+ NK cells were associated with weight loss and tumor location(P<0.05),and no statistically significant differences were found regarding gender,age,smoking status or disease stage,etc.(P>0.05).3.In univariate analysis,TNM stage,tumor location,and increased CD4+ T-cell ratio were associated with PFS.However,only increased CD4+ T-cell ratio(P =0.042)and TNM stage(P =0.029)were independent predictors of PFS on multivariate analysis.Similarly,univariate analysis showed TNM stage,tumor location,and increased CD8+ T-cell ratio were associated with OS.More importantly,multivariate analysis showed that increased CD8+ T-cell ratio(P =0.040)was the only independent predictor of OS.When the 64 patients with ESCCs were separated into two groups,the 32 patients with both increased CD4+ and CD8+ ratios had a superior PFS and OS,compared to the 32 patients with an increased CD4+ ratio only or CD8+ ratio only or neither(1-year PFS rate 63% VS 25%,1-year OS rate 80% vs 62%,P =0.005 and 0.025,respectively).Conclusions 1.In ESCC patients who underwent CRT,proportions of CD19+ B-cells decreased and proportions of CD3+ and CD8+ T-cells increased(all P values < 0.05).2.CRT-induced increases in CD4+ and CD8+ T-cell ratios are reliable biomarker predictors of PFS and OS in patients with ESCC.Thus,we propose that immune therapy during CRT may promote anti-tumor immune response to confer a survival benefit to patients with esophageal squamous cell carcinoma.Part 3Objectives Inflammatory cytokines play an important role in the tumor microenvironment and are closely related to the development and progression of tumors.The aim of this study was to investigate cytokine profiles of epidermal growth factor(EGF)and urokinase plasminogen activator receptor(u PAR)in 68 ESCC patients,and to evaluate the clinical utility of these markers.Methods This pilot study enrolled patients who received neoadjuvant chemoradiotherapy followed by radical surgery or definitive chemoradiotherapy between 2015 and 2017.Informed consent was obtained from each patient to undergo blood and tissue samples collection before treatment and at the time of administration of total doses of 40 Gy.Cytokines expression analyses were performed in pretreated serum compared to that at the doses of 40 Gy from 8 ESCCs.7 differentially expressed cytokines identified by cytokine antibody arrays were validated by enzyme-linked immunosorbant assay.Of these,up-regulation of EGF and u PAR in serum at the doses of 40 Gy were associated with poor prognosis.The predictive value of EGF and u PAR were further assessed in a second set of 60 ESCCs.Results 1.Cytokine microarrays containing 120 human cytokines were performed to detect the expression of serum cytokines before and after CRT from 8 ESCC patients.7 differentially expressed cytokines were validated in 4 patients with favorable prognosis and 4 patients with adverse prognosis(EGF,u PAR,MIP-1?,MIF,IL-8,PDGF-BB and BDNF).Of these,up-regulation of EGF and u PAR in serum after CRT were associated with a poor response to CRT.2.In univariate analysis,age,TNM stage,and u PAR ratio were associated with PFS.However,u PAR ratio and TNM stage were independent predictors of PFS on multivariate analysis.Similarly,univariate analysis showed TNM stage,tumor location,and EGF ratio were associated with OS.More importantly,multivariate analysis showed that EGF ratio and TNM stage were independent predictors of OS.Patients with both high EGF and u PAR ratios had an inferior PFS and OS,compared to patients with an high EGF ratio only or u PAR ratio only or neither(1-year PFS rate 44.2% vs 61.4%,1-year OS rate 64.2% vs 83.4%,P =0.033 and 0.029,respectively).3.Of note,u PAR expression was significantly positive correlation with EGF expression in pre and post-treatment serum(P=0.0001,P=0.0038).Conclusions 1.The levels of EGF and u PAR in serum are a reliable and predictive biomarker for survival in ESCC patients.2.Serum u PAR expression was significantly positive correlation with EGF expression in pre and post-treatment serum.These data may provide the rationale for multipronged approaches to combine CRT with molecular targets in patients with ESCC,with the goal of overcoming CRT resistance to improve survival.Part 4Objective The PD-1/PD-L1 pathway plays an important role in tumor immune escape.It reported that epidermal growth factor receptor(EGFR)mutation status was associated with the PD-L1 expression in non-small cell lung cancer.The purpose of this study was to investigate the relationship between PD-L1 expression and EGFR signaling pathway and radiation in esophageal squamous carcinoma cells.Methods Flow cytometry and western blot methods were used to assess PD-L1 expression on ESCC cells when EGFR signaling pathway was activated by epidermal growth factor(EGF)with or without EGFR-specific inhibitor AG-1478,and then EGFR signaling array was applied to analyze the potential signaling pathways involved.Flow cytometry and western blot methods were used to assess PD-L1 expression when ESCC cells were exposed to radiation with or without EGFR-specific inhibitor AG-1478.Results 1.When cultured in complete culture media,ESCC cells had different levels of PD-L1 expression in a pattern similar to the level of EGFR expression 2.After EGF treatment,the expression of PD-L1 elevated highest in kyse30 cells and moderate in TE7,TE1,Eca109,and kyse140 cells(P<0.05),whereas no significant increase was found in kyse510 and Ca Es-17 cells(P>0.05).When kyse30 and TE-1 cells were pretreated with EGFR tyrosine kinase inhibitor AG1478,the up-regulated percentage of PD-L1 induced by EGF decreased significantly.3.To further assess the molecular pathways involved in PD-L1 expression in ESCC cells following EGFR activation,the EGFR downstream signaling pathways were examined in wild-type EGFR expression kyse30 and TE1 cells,and EGFR mutant TE7 cells using EGFR signaling antibody array kit.Similar results were found in all these three cell lines,which indicated that EGFR–PI3K–AKT,EGFR–Ras–Raf–Erk,and EGR–PLC-? signaling pathways may participate in the regulation of PD-L1 expression.4.After 24 h of radiation exposure,PD-L1 expression on the surface of the tumor cells increased significantly in a dose-dependent manner and corresponding with different levels of EGFR expression.Next,AG1478 inhibited radiation-induced PD-L1 expression dramatically.Conclusion1.The up-regulated expression of PD-L1 after EGF treatment may be affected by EGFR–PI3K–AKT,EGFR–Ras–Raf–Erk,and EGR–PLC-? signaling pathways.2.This finding suggested that radiation-induced EGFR signaling activation deregulated PD-L1 in ESCC,and these data may provide the rationale for multipronged approaches to combine CRT with molecular targets and immunotherapy in patients with ESCC.