Esophageal Squamous Cell Carcinoma Microenvironment Research Articles

Gram-negative microflora dysbiosis facilitates tumor progression and immune evasion by activating CCL3/CCL5-CCR1-MAPK-PD-L1 pathway in esophageal squamous cell carcinoma.

Gram-negative micro-flora dysbiosis occurs in multiple digestive tumors and is found to be the dominant micro-flora in esophageal squamous cell carcinoma (ESCC) micro-environment. The continuous stimulation of G- bacterium metabolites may cause tumorigenesis and reshape the micro-immune environment in ESCC. However, the mechanism of G- bacilli causing immune evasion in ESCC remains underexplored. We identified CC Chemokine receptor 1 (CCR1) as a tumor-indicating gene in ESCC. Interestingly, expression levels of CCR1 and PD-L1 were mutually up regulated after G- bacilli metabolites lipopolysaccharide (LPS) stimulation. Firstly, we found CCR1 high expression level to be associated with poor overall survival in ESCC. Importantly, we found that high level expression of CCR1 up-regulated PD-L1 expression by activating MAPK phosphorylation in ESCC and induced tumor malignant behavior. Finally, we found that T cells exhaustion and cytotoxicity suppression were associated with CCR1 expression in ESCC, which were decreased after CCR1 inhibiting. Our work identifies CCR1 as a potential immune check point regulator of PD-L1 and may cause T cell exhaustion and cytotoxicity suppression in ESCC micro-environment and highlights the potential value of CCR1 as therapeutic target of immunotherapy. Implications: The esophageal microbial environment and its metabolites significantly affect the outcome of immunotherapy for ESCC.

Landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment

AimsWe sought to reveal the landscape of epithelial cell subpopulations in the human esophageal squamous cell carcinoma microenvironment and investigate their parts on esophageal squamous carcinoma (ESCC) development. BackgroundEpithelial cells play an important role in the occurrence and development of ESCC through multiple mechanisms. While the landscape of epithelial cell subpopulations in ESCC, remains unclear. ObjectiveExploring the role of epithelial cell subpopulations in ESCC progression. MethodsSeurat R package was used for single-cell RNA sequencing (scRNA-seq) data filtering, dimensionality reduction, clustering and differentially expressed genes analysis. Cellmarker database was adopted for cell cluster annotation. Functional enrichment analysis was carried out by Gene Ontology (GO) analysis. InferCNV package was conducted for copy number variation (CNV) of epithelial cell subpopulations in all chromosomal regions. Pseudotime trajectory analysis was implemented for exploring differentiation trajectory of epithelial cells subgroups during the cancer progression. CellChat analysis was used for probing the interactions between epithelial cells and NK/T cells. cellular experiments were performed using Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR), Wound-Healing Assay and transwell. Results11 major cell subpopulations were identified in ESCC and adjunct tissues. Further reclassification of epithelial cells uncovered 4 subpopulations. Enrichment analysis revealed that highly expressed genes in 4 epithelial cell subpopulations were related to cell proliferation, immune response and angiogenesis. CNV analysis found that UBD + epithelial cells and GAS2L3+ epithelial cells had a higher proportion of CNV. Cell differentiation trajectories disclosed that KRT6C+ and GSTA1+ epithelial cells were in an intermediate state of differentiation, while UBD+ and GAS2L3+ epithelial cells are in an end state of differentiation during ESCC progression. Finally, we found that four epithelial cell subpopulations all inhibited NK/T cells through NECTIN2-TIGIT and CLEC2B-KLRB1. Low ATF3 and DDIT3 mRNA expression inhibited ESCC cell migration and invasion. ConclusionHere, we obtained a through epithelial cell atlas of ESCC at single-cell resolution, explored the role of epithelial cell in ESCC progression, and unveiled immunosuppressive signals to NK/T cells in promoting ESCC. Our findings expand the comprehension of epithelial cells and offer a theoretical guidance for future anti-epithelial cell treatment of ESCC.

Decreased plasma gelsolin fosters a fibrotic tumor microenvironment and promotes chemoradiotherapy resistance in esophageal squamous cell carcinoma

BackgroundStromal fibrosis is highly associated with therapeutic resistance and poor survival in esophageal squamous cell carcinoma (ESCC) patients. Low expression of plasma gelsolin (pGSN), a serum abundant protein, has been found to correlate with inflammation and fibrosis. Here, we evaluated pGSN expression in patients with different stages of cancer and therapeutic responses, and delineated the molecular mechanisms involved to gain insight into therapeutic strategies for ESCC.MethodsCirculating pGSN level in ESCC patients was determined by enzyme-linked immunosorbent assay analysis, and the tissue microarray of tumors was analyzed by immunohistochemistry staining. Cell-based studies were performed to investigate cancer behaviors and molecular mechanisms, and mouse models were used to examine the pGSN-induced tumor suppressive effects in vivo.ResultsCirculating pGSN expression is distinctively decreased during ESCC progression, and low pGSN expression correlates with poor therapeutic responses and poor survival. Methylation-specific PCR analysis confirmed that decreased pGSN expression is partly attributed to the hypermethylation of the GSN promoter, the gene encoding pGSN. Importantly, cell-based immunoprecipitation and protein stability assays demonstrated that pGSN competes with oncogenic tenascin-C (TNC) for the binding and degradation of integrin αvβ3, revealing that decreased pGSN expression leads to the promotion of oncogenic signaling transduction in cancer cells and fibroblasts. Furthermore, overexpression of pGSN caused the attenuation of TNC expression and inactivation of cancer-associated fibroblast (CAF), thereby leading to tumor growth inhibition in mice.ConclusionsOur results demonstrated that GSN methylation causes decreased secretion of pGSN, leading to integrin dysregulation, oncogenic TNC activation, and CAF formation. These findings highlight the role of pGSN in therapeutic resistance and the fibrotic tumor microenvironment of ESCC.

A spatiotemporal comparative analysis on tumor immune microenvironment characteristics between neoadjuvant chemotherapy and preoperative immunotherapy for ESCC

The average five-year survival rate for esophageal cancer, a common malignant tumor of the digestive system, is barely 20%. The majority of esophageal squamous cell carcinoma (ESCC) patients had already progressed to a locally advanced or even advanced stage at initial diagnosis, making routine surgery ineffective. Chemotherapy and immunotherapy are important neoadjuvant treatments for ESCC, however, it remains unknown how treatment will affect the immunological microenvironment, especially at the spatial level. Here, we presented the TME characters of ESCC from the temporal and spatial dimensions using scRNA-seq and ST, investigated the changes of immune cell clusters in the TME under neoadjuvant chemotherapy and preoperative immunotherapy, and explored the potential mechanisms. It was found that compared with chemotherapy, immunotherapy combined with chemotherapy increased the level of T cell proliferation, partially restored the function of exhausted T cells, induced the expansion of specific exhausted CD8 T cells, increased the production of dendritic cells (DCs), and supported the immune hot microenvironment of the tumor. We also found that CD52 and ID3 have potential as biomarkers of ESCC. Particularly, CD52 may be served as a predictor of the efficacy to screen the advantaged population of different regimens. Through multiple pathways, CAF2 and CAF5’s antigen-presenting role affected the other fibroblast clusters, resulting in malignant transformation. We analyzed the immune microenvironment differences between the two regimens to provide a more thorough description of the ESCC microenvironment profile and serve as a foundation for customized neoadjuvant treatment of ESCC.

Single-Cell Transcriptomics Reveals STAT1 Drives LHPP Downregulation in Esophageal Squamous Cell Carcinoma Progression.

Esophageal Squamous Cell Carcinoma (ESCC) poses a significant health challenge in China due to its high incidence and mortality. Single-cell transcriptomics has transformed the approach to cancer research, allowing detailed analysis of cellular and molecular heterogeneity. The interaction between the transcription factor STAT1 and the tumor suppressor LHPP is crucial, potentially influencing cancer progression and therapeutic outcomes. This study aims to explore the molecular mechanisms and cellular dynamics underlying ESCC, with a focus on the role of transcription factors, particularly STAT1, and its regulatory relationship with LHPP, in the pathogenesis of ESCC. Utilizing single-cell transcriptomics data sourced from the public database GEO (GSE160269), we identified major cell types and their transcriptomic changes in ESCC patients. Differential gene expression profiles were examined to understand the dynamics of the tumor microenvironment (TME). A cohort of 21 ESCC patients was recruited to validate the findings. Furthermore, in ESCC cell lines, we validated the transcriptional regulatory relationship between STAT1 and LHPP. Our analysis identified six major cell types within the ESCC microenvironment, revealing significant changes in cellular composition and gene expression profiles associated with tumorigenesis. Notably, a novel association between STAT1's regulatory role over LHPP was observed, suggesting a complex regulatory loop in ESCC pathogenesis. Elevated STAT1 and reduced LHPP expression were confirmed in patient samples with STAT1 negatively regulates LHPP expression at the promoter level; when the promoter binding motif regions were mutated, the transcriptional repression ability on LHPP was weakened. The study highlights STAT1 as a core regulator in ESCC, directly influencing LHPP expression. The findings offer novel insights into the molecular mechanisms driving ESCC, shedding light on the cellular alterations and gene regulation dynamics within the ESCC microenvironment. This research provides a foundation for developing targeted therapeutic strategies, potentially utilizing the STAT1-LHPP axis as a focal point in ESCC treatment and prognosis.

Clinical Significance and Prognostic Value of the Expression of LAG-3 and FGL1 in Esophageal Squamous Cell Carcinoma.

In this retrospective study, we analyzed the expression of lymphocyte activating gene 3 (LAG-3) and fibrinogen-like protein 1 (FGP1) mRNA and the corresponding proteins in 78 patients with esophageal squamous cell carcinoma (ESCC) to evaluate the clinical significance and prognostic value. mRNA and protein expression were analyzed by reverse transcription PCR and Western blotting, respectively. The expression of LAG-3 and FGL1 mRNA and the corresponding proteins in tumor tissues were significantly increased in comparison with the normal esophageal mucosa. The overexpression of LAG-3 significantly correlated with the content of tumor-infiltrating lymphocytes (TILs), tumor differentiation, and TNM stage. The overexpression of FGL1 also significantly correlated with TILs, TNM stage, and lymph node metastasis. Kaplan-Meier survival analysis showed that tumor diameter, TNM stage, lymph node metastasis, LAG-3 and FGL1 protein expression were related to the progression-free survival (p<0.05). Multivariate Cox regression showed that the level of FGL1 and TNM stage were independent prognostic factors of progression-free survival. We speculated that the tumor microenvironment of ESCC induces immunosuppression due to up-regulated expression of LAG-3 and FGL1 in the tumor tissues, which promotes tumor growth.

SLC1A5, unrelated to prognosis, was associated with CD8+ T-cell exclusion in the tumor microenvironment of squamous cell carcinoma

SLC1A5, short for solute carrier family 1 member 5, is a neutral amino acid transporter whose expression has been reported to be upregulated in various cancers, including esophageal squamous cell carcinoma (ESCC). Despite this, little has been described regarding the immunological involvement of SLC1A5 expression in the tumor microenvironment of ESCC. Given this, we adopted in silico analyses together with a wet lab strategy to investigate the prognostic and clinicopathological meaning of SLC1A5 expression in ESCC. In silico analyses of SLC1A5 expression data available from The Cancer Genome Atlas (TCGA) database revealed that SLC1A5 expression was unrelated to the prognosis of ESCC, which holds true when extended to other types of squamous cell carcinoma (SCC), including head and neck squamous cell carcinoma (HNSC) and lung squamous cell carcinoma (LUSC). Further analyses revealed that SLC1A5 expression correlated markedly with the infiltration density of effector CD8+ T cells in ESCC, and the same was true for HNSC and LUSC when extrapolated. As experimental confirmation, multiplexed immunofluorescent staining was undertaken to verify the correlation between SLC1A5 expression and infiltration of CD8+ T cells in a tissue microarray prepared from ESCC and matched normal control tissues. Our data confirmed that SLC1A5 expression was not associated with prognosis but was associated with the exclusion of CD8+ T cells. Taken together, all the data we curated strongly support the notion that SLC1A5 expression is associated with CD8+ T-cell exclusion in the tumor microenvironment of SCC.

PD-1H Expression Associated With CD68 Macrophage Marker Confers an Immune-Activated Microenvironment and Favorable Overall Survival in Human Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal carcinoma (EC) in China. Although the PD-1 inhibitor pembrolizumab has been approved to treat patients with EC, its therapeutic efficacy is limited. Thus, additional immunotherapeutic targets for EC treatment are needed. Programmed Death-1 Homolog (PD-1H) is a negative checkpoint regulator that inhibits antitumor immune responses. Here, PD-1H expression in 114 patients with ESCC was evaluated by immunohistochemistry. Next, 12 randomly selected tumor tissue sections were used to assess the colocalization of PD-1H protein and multiple immune markers by multiplex immunohistochemistry. Our results demonstrated that PD-1H was expressed at high frequency in ESCC tumor tissues (85.1%). PD-1H protein was predominantly expressed in CD68+ tumor-associated macrophages and expressed at low levels in CD4+ T cells and CD8+ T cells in ESCC tumor tissues. Furthermore, based on ESCC data in The Cancer Genome Atlas (TCGA), the gene expression levels of PD-1H were positively associated with the infiltration levels of immune-activated cells especially CD8+ cytotoxic T cells. In contrast, the gene expression levels of PD-1H were negatively correlated with myeloid-derived suppressor cells (MDSCs). Importantly, PD-1H expression in tumor sites was significantly correlated with favorable overall survival in patients with ESCC. Collectively, our findings first provided direct information on the PD-1H expression pattern and distribution in ESCC, and positive correlation of PD-1H expression with overall survival suggested PD-1H expression levels could be a significant prognostic indicator for patients with ESCC. Future studies need to explore the immunoregulatory of PD-1H in the tumor microenvironment of ESCC.

Integrated single-cell transcriptome analysis reveals heterogeneity of esophageal squamous cell carcinoma microenvironment

The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1+ myofibroblasts with prognostic values and potential biological significance. CST1+ myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology.

Necroptosis in Esophageal Squamous Cell Carcinoma: An Independent Prognostic Factor and Its Correlation with Tumor-Infiltrating Lymphocytes.

Simple SummaryNecroptosis is a regulated form of necrotic cell death that plays pivotal roles in cancer biology, including tumorigenesis, metastasis, and cancer immunity. However, the significance of necroptosis in esophageal squamous cell carcinoma has remained largely unknown. In addition, its correlation with the tissue microenvironment has not yet been explored. In this study, we first investigated the diagnostic and prognostic significance of mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL), both of which are currently considered the most reliable markers for detecting necroptosis. We also investigated the correlations between the status of MLKL/pMLKL and tumor-infiltrating lymphocytes) in esophageal squamous cell carcinoma patients.Necroptosis is a pivotal process in cancer biology; however, the clinical significance of necroptosis in esophageal squamous cell carcinoma (ESCC) has remained unknown. Therefore, in this study, we aimed to verify the potential involvement of necroptosis in the clinical outcome, chemotherapeutic resistance, and tumor microenvironment of ESCC. Mixed lineage kinase domain-like protein (MLKL) and phosphorylated MLKL (pMLKL) were immunohistochemically examined in 88 surgically resected specimens following neoadjuvant chemotherapy (NAC) and 53 pre-therapeutic biopsy specimens, respectively. Tumor-infiltrating lymphocytes (TILs) were also evaluated by immunolocalizing CD3, CD8, and forkhead box protein 3 (FOXP3) in the residual tumors after NAC. High pMLKL status in the post-NAC resected specimens was significantly correlated with worse prognosis in ESCC patients. Multivariate analysis demonstrated that a high pMLKL status was an independent prognostic factor. In pre-NAC biopsy specimens, a high pMLKL status was significantly associated with a lower therapeutic efficacy. CD8+ TILs were significantly lower in the high-pMLKL group. FOXP3+ TILs were significantly higher in both high-MLKL and high-pMLKL groups. We first demonstrated pMLKL status as an independent prognostic factor in ESCC patients. Our study revealed the possible involvement of necroptosis in the immunosuppressive microenvironment, resulting in the attenuated therapeutic efficacy of NAC and eventual adverse clinical outcomes in ESCC.

Interleukin 32 Promotes Foxp3+ Treg Cell Development and CD8+ T Cell Function in Human Esophageal Squamous Cell Carcinoma Microenvironment.

Proinflammatory cytokine interleukin 32 (IL-32) is involved in infectious diseases and cancer, but what subtypes of immune cells express IL-32 and its roles in tumor microenvironment (TME) have not been well discussed. In this study, we applied bioinformatics to analyze single-cell RNA sequencing data about tumor-infiltrating immune cells from esophageal squamous cell carcinoma (ESCC) TME and analyzed IL-32 expression in different immune cell types. We found CD4+ regulatory T cells (Treg cells) express the highest level of IL-32, while proliferating T and natural killer cells expressed relatively lower levels. Knocking down of IL-32 reduced Foxp3 and interferon gamma (IFNγ) expressions in CD4+ and CD8+ T cells, respectively. IL-32 was positively correlated with Foxp3, IFNG, and GZMB expression but was negatively correlated with proliferation score. IL-32 may have a contradictory role in the TME such as it promotes IFNγ expression in CD8+ T cells, which enhances the antitumor activity, but at the same time induces Foxp3 expression in CD4+ T cells, which suppresses the tumor immune response. Our results demonstrate different roles of IL-32 in Treg cells and CD8+ T cells and suggest that it can potentially be a target for ESCC cancer immunosuppressive therapy.

Identification of prognostic genes and tumor-infiltrating immune cells in the tumor microenvironment of esophageal squamous cell carcinoma and esophageal adenocarcinoma.

BackgroundEsophageal cancer (EC) is a highly aggressive malignancy that is classified as esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Infiltrating stromal/immune cells, a major component of the tumor immune microenvironment (TIME), have prognostic significance in various cancers.MethodsIn this study we investigated genes and immune factors in the tumor microenvironment (TME) of ESCC and EAC that can serve as prognostic biomarkers. Stromal and immune scores were calculated using the Estimation of Stromal and Immune Cells in Malignant Tumor Tissues Using Expression Data (ESTIMATE) algorithm based on gene expression profiles of patient-derived tumor tissues in The Cancer Genome Atlas database. The correlation between ESTIMATE scores and survival rates in EC were analyzed. A comparison of high and low stromal/immune score groups revealed multiple differentially expressed genes (DEGs) as candidate prognostic genes; their role in immune-related biological processes was evaluated by functional and protein-protein interaction (PPI) network analyses, and the genes were validated using Gene Expression Omnibus datasets. Additionally, 22 tumor-infiltrating immune cell (TIIC) subsets were analyzed using the CIBERSORT algorithm.ResultsMedian stromal score was higher whereas immune score was lower in ESCC than in EAC (both P<0.01). Stromal score was lower in female as compared to male ESCC patients (P<0.05), and was significantly correlated with T stage (P<0.05). In EAC, median immune score was higher in female as compared to male patients (P<0.05) and was correlated with tumor-node-metastasis stage (P<0.05). The identified DEGs were mainly involved in lymphocyte (especially T-lymphocyte) activation and carbohydrate binding. Moreover, the levels of infiltrating resting-stage dendritic cells, CD8+ T cells, naïve B cells, activated mast cells, and resting memory CD4+ T cells were significantly correlated with EC prognosis (P<0.05).ConclusionsThe immune microenvironment of ESCC and EAC are quite different. We have found genes with prognostic value in multiple tumor databases.

PAI-1 derived from cancer-associated fibroblasts in esophageal squamous cell carcinoma promotes the invasion of cancer cells and the migration of macrophages

AbstractCancer-associated fibroblasts (CAFs) contribute to the progression of various cancers. Previously, we reported the significance of CAFs in esophageal squamous cell carcinoma (ESCC); however, the functions of CAFs in the ESCC microenvironment remain unknown. To investigate CAFs' function, we established an indirect coculture assay between human bone marrow-derived mesenchymal stem cells (MSCs) and ESCC cells. Cocultured MSCs expressed more fibroblast activation protein, one of the markers of CAFs, compared with monocultured MSCs. Therefore, we defined cocultured MSCs as CAF-like cells. To identify molecules associated with the ESCC progression in CAFs, we conducted a cDNA microarray analysis on monocultured MSCs and CAF-like cells to compare their gene expression profiles. We found that SERPINE1, which encodes plasminogen activator inhibitor-1 (PAI-1), was more abundant in CAF-like cells than in monocultured MSCs, and the PAI-1 derived from CAF-like cells induced the abilities of migration and invasion in both ESCC cells and macrophages by the Akt and Erk1/2 signaling pathways via the low-density lipoprotein receptor-related protein 1 (LRP1), which is a PAI-1 receptor. Based on immunohistochemistry assays of ESCC tissues, higher expression levels of PAI-1 and LRP1 were correlated with poor prognosis in ESCC patients. These results suggest that the PAI-1/LRP1 axis contributes to the progression of ESCC, making it a potential target for ESCC therapy.The authors show that plasminogen activator inhibitor-1 (PAI-1) derived from cancer-associated fibroblasts promotes the invasion of esophageal squamous cell carcinoma (ESCC) cells and the migration of macrophages via lipoprotein receptor-related protein 1 (LRP1). High expression of PAI-1 and/or LRP1 is associated with poor prognosis in patients with ESCC, and the PAI-1/LRP1 axis could be a target of anticancer therapy.

Induction of Immunogenic Cell Death of Esophageal Squamous Cell Carcinoma by 5-Fluorouracil and Cisplatin.

Neoadjuvant chemotherapy (NAC) using 5-FU (5-fluorouracil)/CDDP (cisplatin) is a standard therapy for stage II/III thoracic esophageal squamous cell carcinoma (ESCC) in Japan. The aim of this study was to investigate whether 5-FU/CDDP could induce immunogenic cell death in ESCC cell lines. Tumor samples for immunohistochemistry were obtained from 50 patients (mean age=63.1 years) with pathological stage 0-IVa ESCC who underwent NAC followed by surgery. Cell lines T.T and KYSE30 were used for the in vitro experiments. The concentrations of HMGB1 were elevated in the cell line supernatants treated with 5-FU/CDDP. 5-FU/CDDP treated dendritic cells (DCs) showed a mature phenotype, and enhanced T cell proliferation capacity. In addition, mature DCs were observed in surgical specimens with a histological response after treatment with 5-FU/CDDP chemotherapy. 5-FU/CDDP could induce immunogenic cell death in the tumor microenvironment of ESCC.

Identification of a Prognostic Immune Signature for Esophageal Squamous Cell Carcinoma to Predict Survival and Inflammatory Landscapes.

Immunotherapy has achieved success in the treatment of esophageal squamous cell carcinoma (ESCC). However, studies concerning immune phenotypes within the ESCC microenvironment and their relationship with prognostic outcomes are limited. We constructed and validated an individual immune-related risk signature for patients with ESCC. We collected 196 ESCC cases, including 119 samples from our previous public data (GSE53624) to use as a training set and an independent cohort with 77 quantitative real-time polymerase chain reaction (qRT-PCR) data, which we used for validation. Head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC) cohorts were also collected for validation. A least absolute shrinkage and selection operator (LASSO) model and a stepwise Cox proportional hazards regression model were used to construct the immune-specific signature. The potential mechanism and inflammatory landscapes of the signature were explored using bioinformatics and immunofluorescence assay methods. This signature predicted different prognoses in clinical subgroups and the independent cohort, as well as in patients with HNSCC and LUSC. Further exploration revealed that the signature was associated with specific inflammatory activities (activation of macrophages and T-cell signaling transduction). Additionally, high-risk patients exhibited distinctive immune checkpoints panel and higher regulatory T cell and fibroblast infiltration. This signature served as an independent prognostic factor in ESCC. This was the first applicable immune-related risk signature for ESCC. Our results furnished new hints of immune profiling of ESCC, which may provide some clues to further optimize associated cancer immunotherapies.

Immune suppressive landscape in the human esophageal squamous cell carcinoma microenvironment

Cancer immunotherapy has revolutionized cancer treatment, and it relies heavily on the comprehensive understanding of the immune landscape of the tumor microenvironment (TME). Here, we obtain a detailed immune cell atlas of esophageal squamous cell carcinoma (ESCC) at single-cell resolution. Exhausted T and NK cells, regulatory T cells (Tregs), alternatively activated macrophages and tolerogenic dendritic cells are dominant in the TME. Transcriptional profiling coupled with T cell receptor (TCR) sequencing reveal lineage connections in T cell populations. CD8 T cells show continuous progression from pre-exhausted to exhausted T cells. While exhausted CD4, CD8 T and NK cells are major proliferative cell components in the TME, the crosstalk between macrophages and Tregs contributes to potential immunosuppression in the TME. Our results indicate several immunosuppressive mechanisms that may be simultaneously responsible for the failure of immuno-surveillance. Specific targeting of these immunosuppressive pathways may reactivate anti-tumor immune responses in ESCC.

High infiltration of B cells in tertiary lymphoid structures, TCR oligoclonality, and neoantigens are part of esophageal squamous cell carcinoma microenvironment.

Esophageal squamous cell carcinoma (ESCA) exhibits high intratumoral molecular heterogeneity posing a challenge to cancer therapy. Immune checkpoint blockade therapy has been approved for this disease, but with modest results. RNA-Seq data from paired tumor and surrounding nonmalignant tissue from 14 patients diagnosed with ESCA without previous treatment and from The Cancer Genome Atlas-ESCA cohort were analyzed. Herein, we investigated ESCA immune landscape including mutation-derived neoantigens and immune cell subpopulations. Tumor-associated antigen expression was determined by in silico analyses and confirmed by immunohistochemistry showing that PRAME, CEACAM4, and MAGEA11 proteins are expressed on tumors. Immune checkpoint molecules gene expression was higher in the tumor compared with surrounding nonmalignant tissue, but its expression varies greatly among patients. TCR repertoire and BCR transcripts analysis evidenced low clonal diversity with one TCR clone predicted to be specific for a MAGEA11-derived peptide. A high number of B-cell clones infiltrating the tumors and the abundance of these cells in tertiary lymphoid structures observed in ESCA tumors support B cells as a potential immune modulator in this tumor.

Role of FAP positive cancer-associated fibroblasts in the esophageal squamous cell carcinoma microenvironment

Role of FAP positive cancer-associated fibroblasts in the esophageal squamous cell carcinoma microenvironment

Abstract 433: Role of IL-8/CXCR2 network in the tumor microenvironment of esophageal squamous cell carcinoma

Abstract Background: IL-8, a pro-inflammatory cytokine, and its receptor, CXCR2, are expressed in various cancer cells. The IL-8/CXCR2 network is believed to be involved in angiogenesis, tumor cell proliferation and invasion. However, its role in esophageal squamous cell carcinoma (ESCC) remains unclear. In this study, we investigated the role of the IL-8/CXCR2 network in the tumor microenvironment of ESCC. Methods: CXCR2 expression was investigated immunohistochemically in 82 primary ESCCs with R0 resection but without any preoperative treatment. We evaluated the intensity of the staining in four grades; grades 3 (strong) and 2 (medium) were deemed “positive”, and 1 (weak) and 0 (negative) as “negative”. The concentration of IL-8 and CXCR2 were investigated using the human ESCC cell lines TE1, 4, 5, 6, 8, 9, 10, 11, 14 and 15. IL-8/CXCR2 signaling was stimulated by IL-8 addition, and suppressed by SB225002 (Cayman Chemical) treatment, a selective antagonist of CXCR2. Stable IL-8 over-expressing TE4 cells were established, and cell proliferation assays were performed. To evaluate tumor growth in vivo, we subcutaneously inoculated 1.0×106 TE4 cells or transfectant into nude mice, and half of the transfectant-inoculated mice received 1mg/kg of SB225002 by intraperitoneal injection thrice weekly. The size and incidence of subcutaneous tumors were recorded. Result: Thirty-two (38.6%) patients were positive for CXCR2 immunostaining and 50 (61.4%) patients were negative. CXCR2 expression was related to poor survival in ESCC patients (P = 0.063). IL-8 concentration was high in TE4, 5, 10 and 14 supernatant, and low in that of TE1, 6, 8, 9, 11 and 15. CXCR2 expression was high in TE1, 4, 5, and 14 cells, and low in TE6, 8, 9, 10, 11 and 15. We chose TE1 and TE4 cells for further investigation. Stimulation or inhibition of the IL-8/CXCR2 network resulted in significant enhancement or suppression of cell proliferation in both cell lines (P&amp;lt;0.05) in a dose-dependent manner. IL-8 over-expressing TE4 cells showed significantly higher proliferative activity than the parental line (P&amp;lt;0.05), which was markedly suppressed by inhibition of the IL-8/CXCR2 network using SB225002 (P&amp;lt;0.05). In vivo, tumor sizes were smaller in the SB225002-treated group than in the untreated group. Conclusion: Our results demonstrated that stimulation of the IL-8/CXCR2 network clearly enhanced ESCC cell proliferation, while its inhibition obviously suppressed ESCC cell proliferation in vitro. These results indicate that control of IL-8/CXCR2 network signaling may be a new therapeutic strategy for ESCC. Citation Format: Masazumi Inoue, Hiroya Takeuchi, Sachiko Matsuda, Tomohiko Nishi, Kazumasa Fukuda, Rieko Nakamura, Tsunehiro Takahashi, Norihito Wada, Hirofumi Kawakubo, Yoshiro Saikawa, Yuko Kitagawa. Role of IL-8/CXCR2 network in the tumor microenvironment of esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2015-433

Cyr61 promotes CD204 expression and the migration of macrophages via MEK/ERK pathway in esophageal squamous cell carcinoma.

Tumor-associated macrophages (TAMs) are known to be involved in the progression of various human malignancies. We previously demonstrated that CD204 was a useful marker for TAMs contributing to the angiogenesis, progression, and prognosis of human esophageal squamous cell carcinoma (ESCC). We also showed that conditioned media of ESCC cell lines induced CD204 expression in THP-1 human monocytic leukemia cells. Here, we performed a cDNA microarray analysis between THP-1 cells stimulated with TPA (macrophage [MΦ]-like THP-1 cells) treated with and without conditioned medium of ESCC cell line to clarify the molecular characteristics of TAMs in ESCC. From the microarray data, we discovered that Cyr61 was induced in CD204-positive-differentiated THP-1 cells (TAM-like THP-1 cells). In the ESCC microenvironment, not only cancer cells but also TAMs expressed Cyr61. Interestingly, the expression levels of Cyr61 showed a significant positive correlation with the number of CD204-positive macrophages in ESCCs by immunohistochemistry. Recombinant human Cyr61 (rhCyr61) promoted cell migration and induced the expression of CD204 along with the activation of the MEK/ERK pathway in MΦ-like THP-1 cells. Pretreatment with a MEK1/2 inhibitor significantly inhibited not only the Cyr61-mediated migration but also the CD204 expression in the MΦ-like THP-1 cells. These results suggest that Cyr61 may contribute to the expression of CD204 and the promotion of cell migration via the MEK/ERK pathway in TAMs in the ESCC microenvironment.