Alarmin S100A8 imparts chemoresistance of esophageal cancer by reprogramming cancer-associated fibroblasts

Abstract

Chemotherapy remains the first-line treatment for advanced esophageal cancer. However, durable benefits are achieved by only a limited subset of individuals due to the elusive chemoresistance. Here, we utilize patient-derived xenografts (PDXs) from esophageal squamous-cell carcinoma to investigate chemoresistance mechanisms in preclinical settings. We observe that activated cancer-associated fibroblasts (CAFs) are enriched in the tumor microenvironment of PDXs resistant to chemotherapy. Mechanistically, we reveal that cancer-cell-derived S100A8 triggers the intracellular RhoA-ROCK-MLC2-MRTF-A pathway by binding to the CD147 receptor of CAFs, inducing CAF polarization and leading to chemoresistance. Therapeutically, we demonstrate that blocking the S100A8-CD147 pathway can improve chemotherapy efficiency. Prognostically, we found the S100A8 levels in peripheral blood can serve as an indicator of chemotherapy responsiveness. Collectively, our study offers a comprehensive understanding of the molecular mechanisms underlying chemoresistance in esophageal cancer and highlights the potential value of S100A8 in the clinical management of esophageal cancer.

Keywords: S100A8; cancer-associated fibroblasts; chemotherapy; esophageal squamous-cell carcinoma; patient-derived xenografts; the tumor microenvironment.

Graphical abstract

Figure 1

Generation of ESCC PDXs chemotherapy cohort and identification of myCAF-associated gene signatures in non-responsive PDXs (A) Graphical overview of the study. (B) RTV curves of tumor xenografts from 20 distinct ESCC PDXs that were treated with chemotherapeutics or vehicle (n = 3–5 mice per group). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (C) Boxplot exhibiting the percentage of tumor growth inhibition (TGI) of PDXs between the R (n = 8) and NR (n = 12) groups. Boxes represent the interquartile range, and whiskers represent the minimum and maximum values. p value is determined using two-tailed Student’s t test. (D) Principal-component analysis (PCA) plot showing overall patterns of gene expression of 20 PDX donors’ tumor tissues. Circles indicate two separate clusters (R cluster [n = 8], blue; NR cluster [n = 12], red). (E) Gene set enrichment analysis (GSEA) of pathways enriched in the NR group, using RNA-seq data of PDX donors’ tumor tissues. (F) Spearman correlation between the TGI (%) and the collagen-associated genes score. The gray area represents 95% CI (n = 20). (G) Representative images of H&E staining (top) and MTS (bottom) in the PDX donors’ biopsies of the R (n = 8) and NR (n = 12) groups. Scale bar, 100 μm. (H) Quantification of (G). Data are presented as mean ± SEM. ∗p < 0.05 of two-tailed Student’s t test. (I) Spearman correlation between the TGI (%) and the MTS-positive area (%). The gray area represents 95% CI (n = 20). See also Figure S1 and Table S1.

Figure 2

Cancer-cell-derived S100A8 imparts chemoresistance in a CAF-dependent manner (A) Volcano plots exhibiting differentially expressed genes between the R and NR groups, using RNA-seq data of the PDX mice’s tumor tissues. (B) Representative images of H&E staining (top) and S100A8 IHC staining (bottom) in the PDX tumor tissues of the R (n = 8) and NR (n = 12) groups. Scale bar, 100 μm. (C) Quantification of (B). Data are presented as mean ± SEM. p value is determined using two-tailed Student’s t test. (D) Spearman correlation between the TGI (%) and the S100A8 RNA level (left) and the S100A8 IHC intensity (right). The gray areas represent 95% CI (n = 20). (E) ELISA analysis of S100A8 in the CM (top) and western blot analysis of S100A8 in cell lysate (bottom) of KYSE510 and KYSE180 cells stably transfected with S100A8 shRNA or control nontargeting shRNA (n = 3 biological replicates). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (F) The left panel shows the schematic of co-culture system and cell viability assay. The right panel shows the quantification of cell viability in KYSE510 and KYSE180 cell lines (n = 3 biological replicates). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (G) Tumor growth curves of control and S100A8-knockdown xenografts treated with chemotherapeutics or vehicle reagents (n = 4 per group). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (H) Representative images of H&E, S100A8, MTS, αSMA, and CD31 staining of tumor tissues in (G). Scale bar, 100 μm. (I) Quantification of (H). Data are presented as mean ± SEM. p values are determined using two-tailed Student’s t test. (J) Representative images of αSMA and CD31 staining in the PDX tumors of the R (n = 8) and NR (n = 12) groups. Scale bar, 100 μm. (K) Quantification of (J). Data are presented as mean ± SEM. p values are determined using two-tailed Student’s t test. For all panels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and n.s., not significant. Each assay for western blot had three biological repeats. See also Figure S2.

Figure 3

S100A8 polarizes CAFs toward myCAFs (A) Spearman correlations between the S100A8 RNA level of epithelial cells and the myCAF (left) and iCAF (right) signature scores of fibroblasts. The gray areas represent 95% CIs (n = 60). (B) Representative multiple immunofluorescence images of KRT6A, S100A8, and αSMA in the PDX donors’ primary tumor tissues of the R (n = 8) and NR (n = 12) groups. Scale bar, 100 μm. (C) Quantification of S100A8 and αSMA fluorescence intensity in (B). Data are presented as mean ± SEM. p values are determined using two-tailed Student’s t test. (D) Spearman correlation between the S100A8 and the αSMA fluorescence intensity. The gray area represents 95% CI (n = 20). (E) Western blot analysis of the myCAF-related marker proteins collagen type 1 and αSMA, the iCAF-related marker CXCL1 in CAFs cocultured with KYSE510 and KYSE180 cells with or without S100A8 knockdown. (F) Representative images of CAF migration induced by CM derived of KYSE510 and KYSE180 cells with or without S100A8 knockdown. Scale bar, 100 μm. (G) Quantification of (F) (n = 3 biological replicates). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (H) Western blot analysis of collagen type 1, αSMA, and CXCL1 in CAFs treated with indicated concentration of rS100A8 proteins. (I) Representative images of CAF migration induced by indicated concentration of rS100A8 proteins. Scale bar, 100 μm. (J) Quantification of (I) (n = 3 biological replicates). Data are presented as mean ± SD. p value is determined using two-tailed Student’s t test. For all panels, ∗∗p < 0.01, ∗∗∗p < 0.001. Each assay for western blot had three biological repeats. See also Figure S3.

Figure 4

myCAF activation induced by S100A8 is triggered via CD147-RhoA-ROCK-MLC2-MRTF-A pathway (A) KEGG pathway enrichment analysis of distinct CAF subtypes. (B) Spearman correlation between the S100A8 RNA level and the pathway signature score. The gray area represents 95% CI (n = 60). (C) Western blot analysis of RhoA-associated protein markers in CAFs treated with indicated concentration of rS100A8 proteins. (D) Western blot analysis of RhoA-associated and myCAF-related protein markers in CAFs transfected with siRNAs targeting RhoA, ROCK1, MLC2, or negative control and treated with rS100A8 proteins or PBS. (E) Representative immunofluorescence images of MRTF-A nuclear translocation in CAFs treated with rS100A8 proteins or PBS. White arrows indicate MRTF-A nuclear translocation in cells. Scale bar, 100 μm. (F) Quantification of the percentage of MRTF-A nuclear translocation in (E) (n = 6 biological replicates). Data are presented as mean ± SD. ∗∗∗∗p < 0.0001 of two-tailed Student’s t test. (G) Western blot analysis of the indicated proteins in CAFs transfected with MRTF-A or control siRNA and treated with indicated concentration of rS100A8 proteins. (H) Representative immunofluorescence images of CD147 and αSMA in control and CD147-knockout CAFs. Scale bar, 100 μm. (I) Western blot analysis of the indicated proteins in control and CD147-knockout CAFs treated with indicated concentration of rS100A8 proteins. (J) Western blot analysis of the indicated proteins in control and CD147-knockout CAFs cocultured with control or S100A8-overexpression KYSE450 cells. Each assay for western blot had three biological repeats. See also Figures S4 and S5.

Figure 5

S100A8-induced myCAFs endow ESCC cells to acquire chemoresistance by activating anti-apoptotic pathways (A) The left panel shows the schematic of co-culture system. The right panel shows western blot analysis of the indicated proteins in control and S100A8-knockdown KYSE510 and KYSE180 cells cocultured with or without CAFs and treated with chemotherapeutics or vehicle reagents. (B) Western blot analysis of the indicated proteins in control and S100A8-overexpression KYSE450 cells cocultured with control or CD147-knockout CAFs and treated with chemotherapeutics or vehicle reagents. (C) The quantification of cell viability of control and S100A8-overexpression KYSE450 cells cocultured with control or CD147-knockout CAFs and treated with chemotherapeutics or vehicle reagents (n = 3 biological replicates). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (D) Tumor growth curves and excised tumor images of xenografts derived from co-injection of control and S100A8-overexpression KYSE450 cells with CAFs and treated with chemotherapeutics or vehicle reagents (n = 5 per group). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (E) Representative images of H&E, S100A8, MTS, αSMA, and cleaved-Caspase 3 staining of tumor tissues in (D). Scale bar, 100 μm. (F) Quantification of (E). Data are presented as mean ± SEM. p values are determined using two-tailed Student’s t test. For all panels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S5. Each assay for western blot had three biological repeats. See also Figure S6.

Figure 6

Disruption of S100A8-CD147 pathway improves chemotherapy efficiency in vivo (A) Tumor growth curves of xenografts derived from co-transplantation of control and CD147-knockout CAFs with PCs and treated with chemotherapeutics or vehicle reagents (n = 4 per group). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (B) Representative images of H&E, MTS, αSMA, cleaved-Caspase 3, and S100A8 staining of tumor tissues in (A). Scale bar, 100 μm. (C) Quantification of (B). Data are presented as mean ± SEM. p values are determined using two-tailed Student’s t test. (D) Tumor growth curves of xenografts of PDX-19 treated with AC-73 and control solvent and with chemotherapeutics or vehicle reagents (n = 4 per group). Data are presented as mean ± SD. p values are determined using two-tailed Student’s t test. (E) Representative images of H&E, MTS, αSMA, and cleaved-Caspase 3 staining of tumor tissues in (D). Scale bar, 100 μm. (F) Quantification of (E). Data are presented as mean ± SEM. p values are determined using two-tailed Student’s t test. For all panels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, and n.s., not significant. Each assay for western blot had three biological repeats. See also Figure S7.

Figure 7

S100A8 serves as a prognostic biomarker for predicting chemotherapy responsiveness (A) Schematic overview of the experimental design of liquid biopsy. (B) Representative images showing computed tomography of patients’ esophageal tumors of the R and NR groups before and after neoadjuvant chemotherapy. Scale bar, 5 cm. (C) Histogram exhibiting plasma S100A8/S100A9 concentration of patients with ESCC before receiving neoadjuvant chemotherapy in the R (n = 100) and NR (n = 52) groups. Data are presented as mean ± SEM. p value is determined using two-tailed Student’s t test. (D) Forest plot exhibiting odds ratio (OR) with 95% CI and p value calculated by univariate and multivariate logistic regression analysis for age, gender, histologic grade, pathologic TNM stage, treatment cycles, and S100A8/A9 level. (E) Histogram exhibiting serum S100A8/S100A9 concentration of vehicle-treated PDX mice between the R (n = 8) and NR (n = 12) groups. Data are presented as mean ± SEM. p value is determined using two-tailed Student’s t test. (F) Spearman correlation between the TGI and S100A8/A9 concentration. The gray area represents 95% CI (n = 20). (G) Representative images of S100A8 IHC staining in clinical pre-treatment ESCC biopsies of the stable disease (n = 10) and partial response (n = 23) groups. Scale bar, 100 μm. (H) Quantification of (G). Data are presented as mean ± SEM. p value is determined using two-tailed Student’s t test. (I) Kaplan-Meier plot comparing the overall survival (OS) of patients with ESCC treated with chemotherapy with low or high S100A8 RNA level. Hazard ratio (HR) and 95% CI are calculated by Cox proportional hazards model with age, gender, and tumor stage as covariates. (J) Dot plot showing normalized score of HRs of high or low S100A8 and S100A9 RNA level in patients with different cancers receiving chemotherapy from The Cancer Genome Atlas (TCGA). HR and p values are determined by Cox proportional hazards model with age, gender, and tumor stage as covariates. For all panels, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001. See also Figure S8, Tables S2 and S3.