Background: Tertiary lymphoid structures (TLSs) play key roles in tumour adaptive immunity. However, the prognostic value and molecular properties of TLSs in oesophageal squamous cell carcinoma (ESCC) patients have not been studied.Methods: The prognostic values of the presence and maturation status of tumour‐associated TLSs were determined in 394 and 256 ESCC patients from Sun Yat‐sen University Cancer Center (Centre A) and the Cancer Hospital of Shantou University Medical College (Centre B), respectively. A deep‐learning (DL) TLS classifier was established with haematoxylin and eosin (H&E)‐stained slides using an inception‐resnet‐v2 neural network. Digital spatial profiling was performed to determine the cellular and molecular properties of TLSs in ESCC tissues.Results: TLSs were observed in 73.1% of ESCCs from Centre A via pathological examination of H&E‐stained primary tumour slides, among which 42.9% were TLS‐mature and 30.2% were TLS‐immature tumours. The established DL TLS classifier yielded favourable sensitivities and specificities for patient TLS identification and maturation evaluation, with which 55.1%, 39.5% and 5.5% of ESCCs from Centre B were identified as TLS‐mature, TLS‐immature and TLS‐negative tumours. Multivariate analyses proved that the presence of mature TLSs was an independent prognostic factor in both the Centre A and Centre B cohorts (p < .05). Increased proportions of proliferative B, plasma and CD4+ T helper (Th) cells and increased B memory and Th17 signatures were observed in mature TLSs compared to immature ones. Intratumoural CD8+ T infiltration was increased in TLS‐mature ESCC tissues compared to mature TLS‐absent tissues. The combination of mature TLS presence and high CD8+ T infiltration was associated with the best survival in ESCC patients.Conclusions: Mature TLSs improve the prognosis of ESCC patients who underwent complete resection. The use of the DL TLS classifier would facilitate precise and efficient evaluation of TLS maturation status and offer a novel probability of ESCC treatment individualization.

Background: Different intratumoral microbiotaexist in different tumors and play a crucial function in carcinogenesis. However, whether they impact clinical outcomes in esophageal squamous cell carcinoma (ESCC) and their mechanism remain unclear.Methods: 16S rDNA amplicon sequencing was performed on surgically resected samples from 98 ESCC patients to analyze intratumoral microbiome abundance and composition. Multiplex fluorescent immunohistochemistry staining was used to profile the phenotypes of immune infiltrates in the tumor microenvironment (TME).Results: Patients with higher intratumoral Shannon index had significantly worse surgical outcomes. When patients were divided into short-term survivors and long-term survivors based on the median survival time, both intratumoral alpha-diversity and beta-diversity were found to be significantly inconsistent, and the relative abundance of Lactobacillus and Leptotrichia emerged as the two microorganisms that probably influenced the survival of ESCC patients. Only Lactobacillus in ESCC was validated to significantly worsen patients’ prognoses and to be positively correlated with the Shannon index. Multivariate analysis revealed that the intratumoral Shannon index, the relative abundance of Lactobacillus, and the pathologic tumor–node–metastasis (pTNM) stage were independently associated with patients’ overall survival. Furthermore, the relative abundance of both Lactobacillus and Shannon index was positively correlated with the proportions of PD-L1⁺ epithelial cells (ECs) and tumor-associated macrophages (TAMs). The Shannon index was negatively correlated with the proportions of natural killer (NK) cells in the TME.Conclusions: A high abundance of intratumoral Lactobacillus and bacterial alpha-diversity was associated with the formation of the immunosuppressive TME and predicted poor long-term survival in ESCC patients.

Neoadjuvant chemoradiotherapy (neoCRT) followed by surgery is the most common approach for locally advanced resectable esophageal squamous cell carcinoma (ESCC) patients. How neoCRT impacts ESCC tumor immune microenvironment (TIME) has not been fully understood. Single-cell RNA sequencing (scRNA-seq) was conducted to examine the neoCRT-driven cellular and molecular dynamics in 8 pre- and 7 post-neoCRT ESCC samples from 8 male patients. scRNA-seq data of about 112,000 cells were obtained. Expression programs of cell cycle, epithelium development, immune response, and extracellular structure in pre-treatment tumor cells were related to neoCRT response. Spearman correlation between CD8⁺ T cells' cytotoxicity and expression of checkpoint molecules was prominent in pre-neoCRT intermediate activated/exhausted CD8⁺ T cells. NeoCRT increased CD8⁺ T cells’ infiltration but promoted their exhaustion in both major and minor responders. NeoCRT promoted differentiation of Th but demoted that of Treg cells in major responders. Maturation of cDC1s and expression of M2 macrophage markers increased while the number of cDC2s decreased after neoCRT. Higher activities of immune-related pathways in pre-neoCRT CD8⁺ T cells and macrophages, as well as a pronounced decrease of them after neoCRT, correlated with better neoCRT response. Interactions between intermediate activated/exhausted CD8⁺ T and macrophages, cDC1s, and LAMP3⁺ cDCs decreased after neoCRT. Our comprehensive picture of the neoCRT-related immune changes provides deeper insights into immunological mechanisms associated with ESCC response to neoCRT, which may aid in future development of immune-strategies for improving ESCC treatment. This work was supported by the National Natural Science Foundation of China (82072607).

The tumor microenvironment (TME) of esophageal squamous cell carcinoma (ESCC) impacts tumor progression but is poorly understood. We obtained tumor tissues from 279 patients after esophagectomy and characterized the TME in intraepithelial and stromal regions using multiplex fluorescent immunohistochemistry (mfIHC). A heterogeneous immune population infiltrating tumor and the uninvolved esophageal tissue were observed. The infiltration of intraepithelial programmed death ligand 1 (PD-L1)-positive tumor-associated macrophages (TAMs) and stromal granzyme B⁺ activated cytotoxic T cells (aCTLs) correlated with both prolonged overall survival (OS) and disease-free survival (DFS). The intraepithelial memory T cell infiltration predicted longer OS, while intraepithelial and stromal regulatory T cell (Treg) infiltration was associated with shortened OS and DFS, respectively. Multivariate models combining immune infiltrates and clinicopathological factors outperformed tumor-node-metastasis (TNM) stage in predicting OS and DFS at 3 and 5 years. The infiltration of Treg inversely correlated with that of the antitumor effectors including CTLs, aCTLs, and natural killer (NK) cells. Intraepithelial memory T cell infiltration also negatively correlated with PD-L1 expression. In spatial analysis, intraepithelial dendritic cell (DC)-memory T cell engagement increased in high PD-L1⁺ TAM infiltration group. The characterization of the TME revealed a complex interplay between immune populations and may be employed to stratify patient for prognosis prediction and immunotherapy.

TRPV6 is over-expressed and promotes the proliferation and invasion in many cancers. The association between the expression of TRPV6 and clinical outcome in esophageal squamous cell carcinoma (ESCC) has not been studied yet. We aim to elucidate the role of TRPV6 in predicting prognosis of patients with ESCC. In the retrospective study, mRNA level of TRPV6 was examined in patients (N = 174) from Sun Yat-sen University Cancer Center (mRNA cohort) and protein level of TRPV6 was examined in patients (N = 218) from Linzhou Cancer Hospital (protein cohort). Statistical analysis was performed to test the clinical and prognostic significance of TRPV6. TRPV6 was down-regulated in ESCC tissues and cell lines. Patients with downregulation of TRPV6 trended to have a higher rate of advanced pT stage in both mRNA cohort (P = 0.089) and protein cohort (P = 0.073), though not statistically significant. No significant association was observed between TRPV6 expression and disease-specific survival (DSS) in both two cohorts. However, stratified survival analysis based on the gender showed that in mRNA cohort, downregulation of TRPV6 was associated with an unfavorable 3-year DSS in patients with male (47.3 % vs 63.6 %, P = 0.027) and with favorable 3-year DSS in patients with female (66.7 % vs 43.0 %, P = 0.031). The result was confirmed in protein cohort. Male patients with downregulation of TRPV6 had a poor 3-year DSS (20.0 % vs 57.1 %,P < 0.001) while female counterparts showed an enhanced 3-year DSS (56.1 % vs 28.6 %, P = 0.005). TRPV6 is down-regulated in ESCC. As a predictive biomarker, TRPV6 plays a Janus-like role in predicting survival of male and female ESCC patients.