The combination of radiotherapy and immunotherapy exerts synergistic antitumor in a range of human cancers, and also in esophageal cancer. Radiotherapy-induced tumor immune microenvironment (TIME) reprogramming is an essential basis for the synergistic antitumor between radiotherapy and immunotherapy. Radiotherapy can induce autophagy in tumor cells and immune cells of TIME, and autophagy activation is involved in the modification of immunological characteristics of TIME. The TIME landscape of esophageal cancer, especially ESCC, can be affected by radiotherapy or autophagy regulation. In this review, we depicted that local radiotherapy-induced autophagy could promote the maturation, migration, infiltration, and function of immune cells by complicated mechanisms to make TIME from immune "cold" to "hot", resulting in the synergistic antitumor of RT and IO. We argue that unraveling the relevance of radiotherapy-initiated autophagy to driving radiotherapy reprogramming TIME will open new ideas to explore new targets or more efficiently multimodal therapeutic interventions in ESCC.

Inflammation and immune biomarkers can promote angiogenesis and proliferation and metastasis of esophageal squamous cell carcinoma (ESCC). The degree of pathological grade reflects the tumor heterogeneity of ESCC. The purpose is to develop and validate a nomogram based on enhanced CT multidimensional radiomics combined with inflammatory immune score (IIS) for predicting poorly differentiated ESCC.

A total of 266 ESCC patients from the retrospective study were included and randomly divided into a training set (N=186) and a validation set (N=80), and a complete data set (N=266), and overall survival was determined to follow up after surgery. The tumor imaging was segmented to form intratumoral and peritumoral 3 mm areas of 3D volume of interest (VOI) on CT arterial and venous phases, and 3404 radiomics features were extracted. Finally, the radiomics scores were calculated for arterial phase intratumoral (aInRads), peritumoral 3 mm (aPeriRads3), and venous phase intratumoral (vInRads), peritumoral 3 mm (vPeriRads3). Logistic regression was used to fuse the four cohorts of scores to form a Stacking. Additionally, sixteen inflammatory-immune biomarkers were analyzed, including aspartate aminotransferase to lymphocyte ratio (ALRI), aspartate aminotransferase to alanine aminotransferase ratio (AAR), neutrophil times gamma-glutamyl transpeptidase to lymphocyte ratio (NγLR), and albumin plus 5 times lymphocyte sum (PNI), etc. Finally, IIS was constructed using ALRI, AAR, NγLR and PNI. Model performance was evaluated by area under receiver operating characteristic curve (AUC), calibration curve, and decision curve analyse (DCA).

Stacking and IIS were independent risk factors for predicting poorly differentiated ESCC (P<0.05). Ultimately, three models of the IIS, Stacking, and nomogram were developed. Compared with the Stacking and IIS models, nomogram achieved better diagnostic performance for predicting poorly differentiated ESCC in the training set (0.881vs 0.835 vs 0.750), validation set (0.808 vs 0.796 vs 0.595), and complete data set (0.857 vs 0.823 vs 0.703). The nomogram achieved an AUC of 0.881(95%CI 0.826-0.924) in the training set, and was well verified in the validation set (AUC: 0.808[95%CI 0.705-0.888]) and the complete data set (AUC: 0.857[95%CI 0.809-0.897]). Moreover, calibration curve and DCA showed that nomogram achieved good calibration and owned more clinical net benefits in the three cohorts. KaplanMeier survival curves indicated that nomogram achieved excellent stratification for ESCC grade status (P<0.0001).

The nomogram that integrates preoperative inflammatory-immune biomarkers, intratumoral and peritumoral CT radiomics achieves a high and stable diagnostic performance for predicting poorly differentiated ESCC, and may be promising for individualized surgical selection and management.

The original manuscript contained in the research is included in the article. Further inquiries can be made directly to the corresponding author.

Head and neck squamous cell carcinoma (HNSC) exhibits profound intratumoral heterogeneity, driven by dynamic interactions between malignant cells and the tumour microenvironment (TME). Using consensus non-negative matrix factorisation (cNMF) on multi-site HNSC single-cell transcriptomes, we resolving conserved meta-programs define cellular ecosystems. Six major epithelial programmes emerged, including a differentiation-associated programme (Epi_Diff) correlated with SPDEF activity and favourable patient prognosis, and an invasive programme (Epi_pEMT) potentially controlled by TEAD4-mediated ECM remodelling, exhibiting partial EMT markers (VIM, TGFB1). Compartment-specific crosstalk analysis revealed Epi_pEMT cells may coordinate with mCAF1 fibroblasts and TAM(SPP1) through COL1A1-CD44 and SPP1-CD44 signalling, suggesting potential formation of a pro-invasive niche. Conversely, Epi_Diff cells may interact with NK/T cells through CEACAM5-CD8A and CCL5-ACKR2, and may contribute to inhibit immune infiltration. Multi-compartment correlation analysis revealed three ecosystem-level patterns: (1) Inverse association between Epi_Diff and Epi_pEMT (Spearman R = -0.43); (2) Negative correlation between mCAF1 abundance and cCAF frequency (R = -0.48); (3) TAM(SPP1) dominance inversely correlating with both TAM(C1Q) (R = -0.43) and NK/T infiltration (R = -0.36). These axes suggest a potential hierarchical ecology framework where lineage-specific polarisation and inter-compartment synergies may collectively govern disease progression.

Immune regulation is a key function of the skin, a barrier tissue that exhibits spatial compartmentalization of innate and adaptive immune cells. Recent advances in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) have facilitated systems-based investigations into the molecular and cellular features of skin immunity at single-cell resolution, identifying cell types that maintain homeostasis in a coordinated manner, and those that exhibit dysfunctional cell-cell interactions in disease. Here, we review how technological innovation is uncovering the multiple scales of heterogeneity in the immune landscape of the skin. The microanatomic scale encompasses the skin's diverse cellular components and multicellular spatial organization, which govern the functional cell interactions and behaviors necessary to protect the host. On the macroanatomic scale, understanding heterogeneity in cutaneous tissue architecture across anatomical sites promises to unearth additional functional immune variation and resulting disease consequences. We focus on how single-cell and spatial dissection of the immune system in experimental models and in humans has led to a deeper understanding of how each cell type in the skin contributes to overall immune function in a context-dependent manner. Finally, we highlight translational opportunities for adopting these technologies, and insights gleaned from them, into the clinic.

The comprehension of intricate molecular mechanisms underlying how external stimuli promote malignancy is conducive to cancer early prevention. Esophageal squamous cell carcinoma (ESCC) is considered as an external stimuli (hot foods, tobacco, chemo-compounds) induced cancer, characterized by stepwise progression from hyperplasia, dysplasia, carcinoma in situ and invasive carcinoma. However, the underlying molecular mechanism governing the transition from normal epithelium to neoplastic processes in ESCC under persistent external stimuli has remained elusive. Herein, we show that a positive correlation between p38 and ERK1/2 activation during the progression of ESCC. We identify that phosphorylation of ACSL4 at T679 by p38 enhances its enzymatic activity, resulting in increased production of myristoyl-CoA (C14:0 CoA). This subsequently promotes Src myristoylation and activates downstream ERK signaling. Our results partially elucidate the role of ACSL4 in mediating stress-induced signaling pathways that activate growth cascades and contribute to tumorigenesis.

It remains undetermined regarding the impact of neoadjuvant therapy on immunogenic cell death (ICD) and subsequent tumor microenvironment (TME) remodeling in esophageal squamous cell carcinoma (ESCC). And it is of paramount significance to identify beneficiaries from neoadjuvant therapy in treatment-naïve ESCC. In this study, 88 ESCC samples undergoing neoadjuvant therapy plus surgery (NA+S) or surgery alone (SA) were subjected to bulk-RNA sequencing. A five-gene RINscore incorporating ICD-related signature genes with TME-based hub genes was established to predict clinical outcomes and pharmacological responses, in which SLAMF7 and IL1R1 were selected out as co-expressed genes. The regulatory mechanism of the repressive co-transcription factor BATF of SLAMF7 and IL1R1 was further demonstrated. Our data demonstrated that NA+S led to high abundance in kinds of T helper cells, nature killer T cells and M1-like macrophages with increased CD8+T cells infiltration compared with SA. ICD phenotypes were further characterized in treatment-naïve ESCC to determine their differences in TME and potential benefits from NA. Our findings not only offered novel insights into the distinct TME and ICD profiles of ESCC undergoing different therapeutic modes, but also provided the RINscore, which may aid oncologists in determining individualized (neo)adjuvant immunotherapy regimen.

Ferroptosis is a unique modality of regulated cell death induced by excessive lipid peroxidation, playing a crucial role in tumor suppression and providing potential therapeutic strategy for cancer treatment. Here, we find that aldehyde dehydrogenase-ALDH3A1 tightly links to ferroptosis in squamous cell carcinomas (SCCs). Functional assays demonstrate the enzymatic activity-dependent regulation of ALDH3A1 in protecting SCC cells against ferroptosis through catalyzing aldehydes and mitigating lipid peroxidation. Furthermore, a specific covalent inhibitor of ALDH3A1-EN40 significantly enhances the ferroptosis sensitivity induced by the ferroptosis inducer. The combination of EN40 and a ferroptosis inducer exhibits a synergistic effect, effectively inhibiting the proliferation of SCC cells/organoids and suppressing tumor growth both in vitro and in vivo. On mechanism, high expression of ALDH3A1 is transcriptionally governed by TP63, which binds to super-enhancer of ALDH3A1. Collectively, our findings reveal a yet-unrecognized function of ALDH3A1 exploited by SCC cells to evade ferroptosis, and targeting ALDH3A1 may enhance the effect of ferroptosis-induced therapy in SCCs.

Esophageal cancer is a major cause of cancer-related death, often preceded with chronic inflammation and injuries. The NFκB/IKKβ pathway plays a central role in inflammation, yet its role in early esophageal carcinogenesis remains unclear. This study investigated the role of epithelial IKKβ in early esophageal carcinogenesis. Mice were treated with the carcinogen 4-nitroquinoline-1-oxide (4-NQO) or a vehicle for one month to induce precancerous lesions. Esophagi were harvested and examined through histological, protein, flow cytometry, and RNA analyses. Histological analysis revealed that 4-NQO treatment led to increased inflammation, intraepithelial CD45+ immune cells, and elevated IKKβ phosphorylation levels. Mice with esophageal epithelial-specific Ikkβ deletion (4-NQO/Ikkβ EEC-KO ) showed delayed progression to a precancerous state, with reduced immune cell recruitment compared to 4-NQO/controls. Immunophenotyping showed decreased recruitment of T cells, including CD4+, CD8+ and regulatory (Tregs) T cells, and increased recruitment of macrophages in 4-NQO/Ikkβ EEC-KO mice compared to 4-NQO/controls. RNA sequencing data identified 262 differentially expressed genes in 4-NQO/Ikkβ EEC-KO mice, implicating pathways related to inflammation and wound healing. Notably, the chemokine CXCL9, a T cell chemoattractant, was significantly upregulated in 4-NQO control mice, but not in 4-NQO/Ikkβ EEC-KO mice. Further analysis identified IFNγ as an upstream regulator of Cxcl9 expression, and neutralization of IFNγ reduced Cxcl9 expression levels in 4-NQO treated mice. Additionally, in vitro studies demonstrated that IFNγ upregulates Cxcl9 in an NF-κB dependent manner in esophageal keratinocytes. These findings suggest that epithelial IKKβ regulates the immune microenvironment in early esophageal carcinogenesis through the IFNγ/CXCL9 axis and influencing T cell recruitment and inflammatory responses.

Cancer development involves the co-evolution of cancer cells and their surrounding microenvironment, yet the dynamics of this interaction within the physical architecture remains poorly understood. Here, we present a spatial transcriptomic map at single-cell resolution, encompassing 127 multi-stage fields of view from 43 patients, to chart the evolutionary trajectories of human esophageal squamous cell carcinoma (ESCC). By analyzing 6.4 million cells, we reveal that ESCC progression is driven by a proliferative epithelial cell subpopulation that acquires dedifferentiated and invasive characteristics. At the late precancerous stage, these cells disrupt the epithelial-stromal interface and recruit normal fibroblasts via JAG1-NOTCH1 signaling, transforming them into cancer-associated fibroblasts (CAFs). This interaction leads to the formation of a "CAF-Epi" (CAF and epithelial cell) niche at the tumor edge that shields the tumor from immune surveillance. The CAF-Epi niche formation is a key indicator of progression in ESCC and other squamous cell carcinomas and patient outcomes.

Cancer cells exhibit high heterogeneity and lineage plasticity, complicating studies of tumorigenesis and development of therapies. Recently, preneoplastic cells, although histologically normal, have been shown to possess high plasticity and early genetic alterations, yet their origins and lineage trajectories remain unclear. Herein, we introduce a lineage-tracing tool integrating genetic barcoding with single-cell RNA sequencing to map preneoplastic esophageal cell lineages. We identified preneoplastic precursor cells (PNPCs) as a distinct progenitor-like population with unique transcriptional profiles and high plasticity, contributing to proliferative and basal cell populations. To enhance lineage mapping, we developed the eXamined Ridge (XR) score, accurately identifying high-plasticity cells. Nfib and Qk emerged as conserved PNPC markers, peaking in early preneoplasia and declining after malignant transformation. These findings reveal PNPCs as key players in early tumorigenesis and highlight their potential as biomarkers for early cancer detection and therapeutic intervention, offering new strategies for preventing esophageal cancer progression.

Squamous cell cancers (SCCs) of the head and neck, esophagus, and lung, referred to as aero-upper digestive SCCs, are prevalent in the United States and worldwide. Their incidence and mortality are projected to increase at alarming rates, posing diagnostic, prognostic, and therapeutic challenges. These SCCs share certain epigenetic, genomic, and genetic alterations, immunologic properties, environmental exposures, as well as lifestyle and nutritional risk factors, which may underscore common complex gene-environmental interactions across them. This review focuses upon the frequent shared epigenetic, genomic, and genetic alterations, emerging preclinical model systems, and how this collective knowledge can be leveraged into perspectives on standard of care therapies and mechanisms of resistance, nominating new potential directions in translational therapeutics.

Esophageal squamous cell carcinoma (ESCC) is a highly heterogeneous and aggressive malignancy. The progression, invasiveness, and metastatic potential of ESCC are shaped by a multitude of cells within the tumor microenvironment (TME), including tumor cells, immune cells, endothelial cells, as well as fibroblasts and other cell types. Recent advancements in single-cell sequencing technologies have significantly enhanced our comprehension of the diverse landscape of ESCC. Single-cell multi-omics technology, particularly single-cell transcriptome sequencing, have shed light on the expression profiles of individual cells and the molecular characteristics of distinct tumor cell populations. This review summarizes the latest literature on single-cell research in the field of ESCC, aiming to elucidate the heterogeneity of tumor cells, immune cells, and stromal cells at the single-cell level. Furthermore, it explores the impact of cellular interactions within the TME on the progression of ESCC. By compiling a comprehensive overview of single-cell omics research on ESCC, this article aims to enhance our understanding of ESCC diagnosis and treatment by elucidating the intricate interplay within the TME. It explores the cellular composition, spatial arrangement, and functional attributes of the ESCC TME, offering potential therapeutic targets and biomarkers for personalized treatment strategies.

Patients suffer from esophageal squamous cell carcinoma (ESCC), which is the ninth highly aggressive malignancy. Tumor-infiltrating immune cells (TIIC) exert as major component of the tumor microenvironment (TME), showing possible prognostic value in ESCC.

Transcriptome data and scRNA-seq data of ESCC samples were extracted from the GEO and TCGA databases. Tissue Specific Index (TSI) was defined to identify potential TIIC-RNAs from the TME. Twenty machine learning algorithms were further applied to evaluate the prognostic efficacy of TIIC signature score. Gene colocalization analysis was performed. Differences in CNV on chromosomes and SNP sites of prognostic model genes were calculated.

The most reliable model of TIIC signature score was developed based on three prognostic TIIC-RNAs. It showed a higher C-index than any other reported prognostic models. ESCC patients with high TIIC signature score showed poorer survival outcomes than low TIIC signature score. The activity of most immune cells decreased with the increase of TIIC score. TIIC signature score showed difference in the expression levels and methylation levels of DEGs. There was also significant different correlation with the degree of CNV amplification and CNV deletion of the immune checkpoint genes. Gene colocalization analysis showed two prognostic model genes (ATP6V0E1 and BIRC2). MR analysis found that rs148710154 and rs75146099 SNP sites of TIIC-RNA gene had a significant correlation between them gastro-oesophageal reflux and ESCC.

TIIC signature score was the first time developed which provided a novel strategy and guidance for the prognosis and immunotherapy of ESCC. It also gave the evidence in the important role of immune cells from the TME in the treatment of cancers.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy, characterized by a multistep pathogenic process regulated spatiotemporally within the esophageal epithelial microenvironment, including vessel normalization and immune infiltration. However, empirical evidence elucidating esophageal vascular remodeling and immune infiltration during ESCC tumorigenesis in situ is lacking. In this study, utilizing a mouse model recapitulating progressive human ESCC stages, we established a tissue clearing workflow for three-dimensional visualization and analysis of esophageal vessels and T cell distribution. Through this workflow, we delineated the spatial dynamics of vascular remodeling, CD3+ T cells, and characteristic T cell aggregates employing high-resolution light-sheet fluorescence microscopy across five ESCC pathogenic stages. Vessel remodeling might be coupled with T cell infiltration, and their interactions predominantly occurred at the inflammatory stage. These findings provided insights into research methodologies of esophageal cancer and spatiotemporal landscapes of vascular and T cell during ESCC initiation and progression.

Understanding the stepwise progression of esophageal squamous cell carcinoma (ESCC) is crucial for developing customized strategies for early detection and optimal clinical management. Herein, we aimed to unravel the transcriptional and immunologic alterations occurring during malignant transformation and identify clinically significant biomarkers of ESCC.

Digital spatial profiling (DSP) was performed on 11 patients with early-stage ESCC (pT1) to explore the transcriptional alterations in epithelial, immune cell, and non-immune cell stromal compartments across regions of distinct histology, including normal tissues, low- and high-grade dysplasia, and cancerous tissues. Furthermore, single-cell spatial transcriptomics was performed using the CosMx Spatial Molecular Imaging (SMI) system on 4 additional patients with pT1 ESCC. Immunohistochemical (IHC) analysis was performed on consecutive histological sections of 20 pT1 ESCCs. Additionally, public bulk and single-cell RNA-sequencing (scRNA-seq) datasets were analyzed, and in vitro and in vivo functional studies were conducted.

Spatial transcriptional reprogramming and dynamic cell signaling pathways that determined ESCC progression were delineated. Increased infiltration of macrophages from normal tissues through dysplasia to cancerous tissues occurred. Macrophage subtypes were characterized using the scRNA-seq dataset. Cell-cell communication analysis of scRNA-seq and SMI data indicated that the migration inhibitory factor (MIF)-CD74 axis may exhibit pro-tumor interactions between macrophages and epithelial cells. DSP, SMI, and IHC data demonstrated that DTX3L expression in epithelial cells and BST2 expression in stromal cells increased gradually with ESCC progression. Functional studies demonstrated that DTX3L or BST2 knockdown inhibited ESCC proliferation and migration and decreased M2 polarization of tumor-associated macrophages.

Spatial profiling comprehensively characterized the molecular and immunological hallmarks from normal tissue to ESCC, guiding the way to a deeper understanding of the tumorigenesis and progression of this disease and contributing to the prevention of ESCC. Within this exploration, we uncovered biomarkers that exhibit a robust correlation with ESCC progression, offering potential new avenues for insightful therapeutic approaches.

Digestive system cancers are prevalent diseases with a high mortality rate, posing a significant threat to public health and economic burden. The diagnosis and treatment of digestive system cancer confront conventional cancer problems, such as tumor heterogeneity and drug resistance. Single-cell sequencing (SCS) emerged at times required and has developed from single-cell RNA-seq (scRNA-seq) to the single-cell multi-omics era represented by single-cell spatial transcriptomics (ST). This article comprehensively reviews the advances of single-cell omics technology in the study of digestive system tumors. While analyzing and summarizing the research cases, vital details on the sequencing platform, sample information, sampling method, and key findings are provided. Meanwhile, we summarize the commonly used SCS platforms and their features, as well as the advantages of multi-omics technologies in combination. Finally, the development trends and prospects of the application of single-cell multi-omics technology in digestive system cancer research are prospected.

Early detection and intervention of cancer or precancerous lesions hold great promise to improve patient survival. However, the processes of cancer initiation and the normal-precancer-cancer progression within a non-cancerous tissue context remain poorly understood. This is, in part, due to the scarcity of early-stage clinical samples or suitable models to study early cancer. In this Review, we introduce clinical samples and model systems, such as autochthonous mice and organoid-derived or stem cell-derived models that allow longitudinal analysis of early cancer development. We also present the emerging techniques and computational tools that enhance our understanding of cancer initiation and early progression, including direct imaging, lineage tracing, single-cell and spatial multi-omics, and artificial intelligence models. Together, these models and techniques facilitate a more comprehensive understanding of the poorly characterized early malignant transformation cascade, holding great potential to unveil key drivers and early biomarkers for cancer development. Finally, we discuss how these new insights can potentially be translated into mechanism-based strategies for early cancer detection and prevention.

Acute radiation esophagitis (ARE) is a common complication in patients with esophageal cancer undergoing radiotherapy. Therefore, it is important to construct an effective ARE risk-prediction model for clinical treatment. The present study performed a retrospective analysis of 225 patients with esophageal cancer who received radiotherapy at the First Affiliated Hospital of Anhui Medical University (Hefei, China) from January 2018 to December 2022. Univariate and logistic regression analyses were performed to screen patients with esophageal cancer after radiotherapy. The results revealed that 147 patients developed radiation esophagitis. Logistic regression analysis results demonstrated that the prognostic nutritional index [odds ratio (OR), 0.864; 95% confidence interval (CI), 0.809-0.924], neutrophil to lymphocyte ratio (OR, 1.795; 95% CI, 1.209-2.667) and platelet to lymphocyte ratio (OR, 1.011; 95% CI, 1.000-1.022) were independent predictors of ARE in patients receiving intensity-modulated conformal radiotherapy for esophagus cancer (P<0.05). A nomogram model for predicting the occurrence of ARE was established based on the three risk factors. The decision curve suggested a high net benefit value when the threshold probability was within 0.25-1.0. External verification confirmed the reproducibility and generalizability of the nomogram model. In general, the calibration curve of this model was close to the ideal curve and had excellent prediction accuracy. Therefore, it may be used as a new tool for early prediction of the ARE risk.

Gastroesophageal disorders and cancers impose a significant global burden. Particularly, the prevalence of esophageal adenocarcinoma (EAC) has increased dramatically in recent years. Barrett's esophagus, a precursor of EAC, features a unique tissue adaptation at the gastroesophageal squamo-columnar junction (GE-SCJ), where the esophagus meets the stomach. Investigating the evolution of GE-SCJ and understanding dysregulation in its homeostasis are crucial for elucidating cancer pathogenesis. Here, we present the technical quality of the comprehensive single-cell RNA sequencing (scRNA-seq) dataset from mice that captures the transcriptional dynamics during the development of the esophagus, stomach and the GE-SCJ at embryonic, neonatal and adult stages. Through integration with external scRNA-seq datasets and validations using organoid and animal models, we demonstrate the dataset's consistency in identified cell types and transcriptional profiles. This dataset will be a valuable resource for studying developmental patterns and associated signaling networks in the tissue microenvironment. By offering insights into cellular programs during homeostasis, it facilitates the identification of changes leading to conditions like metaplasia and cancer, crucial for developing effective intervention strategies.

We 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.

Epithelial 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.

Exploring the role of epithelial cell subpopulations in ESCC progression.

Seurat 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.

11 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.

Here, 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.

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal cancers worldwide. Most ESCC patients are diagnosed at an advanced stage; however, current research on in vivo animal models accurately reflecting their clinical presentation is lacking. Alcohol consumption is a major risk factor for ESCC and has been used in several disease models for disease induction. In this study, we used 4-nitroquinoline-1-oxide in combination with ethanol to induce an in vivo ESCC mouse model. Esophageal tissues were stained with hematoxylin and eosin for histopathological examination and lesion scoring. In cellular experiments, cell adhesion and migration invasion ability were observed using phalloidin staining, cell scratch and transwell assays, respectively, and the expression of epithelial-mesenchymal transition-related markers was detected using quantitative reverse transcription polymerase chain reaction and western blotting. The results showed that ethanol-exposed mice lost more weight and had an increased number of esophageal nodules. Histological examination revealed that the lesion scores of the ethanol-exposed esophageal samples were significantly higher than those of the unexposed esophageal samples. Furthermore, ethanol-exposed esophageal cancer samples had more severe lesions with infiltration of tumor cells into the muscularis propria. In vitro cellular experiments showed that ethanol exposure induced cytoskeletal microfilament formation, promoted cell migration invasion elevated the expression of N-cadherin and Snail, and decreased the expression of E-cadherin. In conclusion, ethanol exposure exacerbates ESCC, promotes tumor cell infiltration into the muscularis propria, and could be an effective agent for establishing innovative models of invasive carcinoma.

In esophageal cancer (EC), clinical specimen testing has uncovered a significant increase in BTB and CNC homolog 1 (BACH1) expression and a shift towards an immunosuppressive environment, alongside a notable decrease in p53 protein expression. Therefore, therapeutic strategies focusing on BACH1 inhibition and p53 upregulation appear promising. Traditional oral treatments for EC lack precision and efficacy. Here, we propose a novel approach employing tumor-targeted nanoparticles (NPs) for drug delivery. However, the formation of a drug reservoir at the esophageal site, crucial for the sustained release of therapeutics, presents significant challenges in nano-delivery systems for EC treatment. To address this, we developed a thermosensitive hydrogel composed of F127 and tannic acid, serving as a vehicle for NP loading. These NPs, synthesized through the emulsion/volatization methods of mPEG-PLGA-PLL-cRGD, facilitate in situ drug delivery. Upon contacting esophageal tissue, the hydrogel transitions to a gel, adhering to the lining and enabling sustained release of encapsulated therapeutics. The formulation encompasses NPs laden with small interfering RNA targeting BACH1 (siBACH1) and the p53 activator PRIMA-1, creating a cohesive gel-nano system. Preliminary biological assessments demonstrate that this injectable, thermosensitive gel-nano system adheres effectively to esophageal tissue and targets EC cells. For better modeling clinical outcomes, a patient-derived organoid xenograft (PDOX) model was innovated, involving transplantation of EC-derived organoids into humanized mice, reconstructed with peripheral blood mononuclear cells (PBMCs). Post-treatment analysis showed substantial EC growth inhibition (89.51% tumor inhibition rate), significant BACH1 level reduction, restored anti-tumor immune responses, and pronounced tumor apoptosis. In summary, our study introduces a thermosensitive gel-nano system for EC treatment via restoring p53 activity and boosting T-cell immunity, with potential for clinical application.

Despite recent biotechnological breakthroughs, cancer risk prediction remains a formidable computational and experimental challenge. Addressing it is critical in order to improve prevention, early detection and survival rates. Here, I briefly summarize some key emerging theoretical and computational challenges as well as recent computational advances that promise to help realize the goals of cancer-risk prediction. The focus is on computational strategies based on single-cell data, in particular on bottom-up network modeling approaches that aim to estimate cancer stemness and dedifferentiation at single-cell resolution from a systems-biological perspective. I will describe two promising methods, a tissue and cell-lineage independent one based on the concept of diffusion network entropy, and a tissue and cell-lineage specific one that uses transcription factor regulons. Application of these tools to single-cell and single-nucleus RNA-seq data from stages prior to invasive cancer reveal that they can successfully delineate the heterogeneous inter-cellular cancer-risk landscape, identifying those cells that are more likely to turn cancerous. Bottom-up systems biological modeling of single-cell omic data is a novel computational analysis paradigm that promises to facilitate the development of preventive, early detection and cancer-risk prediction strategies.

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.

The detection of copy number variations (CNVs) and somatic mutations in cancer is important for the selection of specific drugs for patients with cancer. In cancers with sporadic tumor cells, low tumor content prevents the accurate detection of somatic alterations using targeted sequencing. To efficiently identify CNVs, we performed tumor cell enrichment using tissue suspensions of formalin-fixed paraffin-embedded (FFPE) tissue sections with low tumor cell content. Tumor-enriched and residual fractions were separated from FFPE tissue suspensions of intestinal and diffuse-type gastric cancers containing sporadic tumor cells, and targeted sequencing was performed on 225 cancer-related genes. Sequencing of a targeted panel of cancer-related genes using tumor-enriched fractions increased the number of detectable CNVs and the copy number of amplified genes. Furthermore, CNV analysis using the normal cell-enriched residual fraction as a reference for CNV scoring allowed targeted sequencing to detect CNV characteristics of diffuse-type gastric cancer with low tumor content. Our approach improves the CNV detection rate in targeted sequencing with tumor enrichment and the accuracy of CNV detection in archival samples without paired blood.

Despite the promising outcomes of immune checkpoint inhibitors (ICIs), resistance to ICI presents a new challenge. Therefore, selecting patients for specific ICI applications is crucial for maximizing therapeutic efficacy. Herein, we curated 69 human esophageal squamous cell cancer (ESCC) patients' tumor microenvironment (TME) single-cell transcriptomic datasets to subtype ESCC. Integrative analyses of the cellular network and transcriptional signatures of T cells and myeloid cells define distinct ESCC subtypes characterized by T cell exhaustion, and interleukin (IL) and interferon (IFN) signaling. Furthermore, this approach classifies ESCC patients into ICI responders and non-responders, as validated by whole tumor transcriptomes and liquid biopsy-based single-cell transcriptomes of anti-PD-1 ICI responders and non-responders. Our study stratifies ESCC patients based on TME transcriptional network, providing novel insights into tumor niche remodeling and potentially predicting ICI responses in ESCC patients.

Development of new diagnostic methods is essential for disease diagnosis and treatment. In this work, we present a stimuli-responsive incremental DNA machine auto-catalyzed CRISPR-Cas12a (SRI-DNA machine/CRISPR-Cas12a) feedback amplification for ultrasensitive molecular detection of miRNA-21, which is an important biomarker related closely to the initiation and development of cancers, such as esophageal cancer. Strategically, the powerful SRI-DNA machine and efficient trans-cleavage activity of the CRISPR-Cas12a system are ingeniously integrated via a rationally designed probe termed as stem-elongated functional hairpin probe (SEF-HP). The SRI-DNA machine begins with the target miRNA, the trigger of the reaction, binding complementarily to the SEF-HP, followed by autonomously performed mechanical strand replication, cleavage, and displacement circuit at multiple sites. This conversion process led to the amplified generation of numerous DNA activators that are complementary with CRISPR RNA (CrRNA). Once formed the DNA activator/CrRNA heteroduplex, the trans-cleavage activity of the CRISPR-Cas12a was activated to nonspecific cleavage of single-stranded areas of a reporter probe for fluorescence emission. Under optimal conditions, the target miRNA can be detected with a wide linear range and an excellent specificity. As a proof-of-concept, this SRI-DNA machine/CRISPR-Cas12a feedback amplification system is adaptable and scalable to higher-order artificial amplification circuits for biomarkers detection, highlighting its promising potential in early diagnosis and disease treatment.

Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide, and its development comprises a multistep process from intraepithelial neoplasia (IN) to carcinoma (CA). However, the critical regulators and underlying molecular mechanisms remain largely unknown.

To explore the genes and infiltrating immune cells in the microenvironment that are associated with the multistage progression of ESCC to facilitate diagnosis and early intervention.

A mouse model mimicking the multistage development of ESCC was established by providing warter containing 4-nitroquinoline 1-oxide (4NQO) to C57BL/6 mice. Moreover, we established a control group without 4NQO treatment of mice. Then, transcriptome sequencing was performed for esophageal tissues from patients with different pathological statuses, including low-grade IN (LGIN), high-grade IN (HGIN), and CA, and controlled normal tissue (NOR) samples. Differentially expressed genes (DEGs) were identified in the LGIN, HGIN, and CA groups, and the biological functions of the DEGs were analyzed via Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The CIBERSORT algorithm was used to detect the pattern of immune cell infiltration. Immunohistochemistry (IHC) was also conducted to validate our results. Finally, the Luminex multiplex cytokine analysis was utilized to measure the serum cytokine levels in the mice.

Compared with those in the NOR group, a total of 681541, and 840 DEGs were obtained in the LGIN, HGIN, and CA groups, respectively. Using the intersection of the three sets of DEGs, we identified 86 genes as key genes involved in the development of ESCC. Enrichment analysis revealed that these genes were enriched mainly in the keratinization, epidermal cell differentiation, and interleukin (IL)-17 signaling pathways. CIBERSORT analysis revealed that, compared with those in the NOR group, M0 and M1 macrophages in the 4NQO group showed stronger infiltration, which was validated by IHC. Serum cytokine analysis revealed that, compared with those in the NOR group, IL-1β and IL-6 were upregulated, while IL-10 was downregulated in the LGIN, HGIN, and CA groups. Moreover, the expression of the representative key genes, such as S100a8 and Krt6b, was verified in external human samples, and the results of immunohistochemical staining were consistent with the findings in mice.

We identified a set of key genes represented by S100a8 and Krt6b and investigated their potential biological functions. In addition, we found that macrophage infiltration and abnormal alterations in the levels of inflammation-associated cytokines, such as IL-1β, IL-6, and IL-10, in the peripheral blood may be closely associated with the development of ESCC.

Oesophageal squamous cell carcinoma (ESCC) is a leading cause of cancer-related deaths worldwide because existing treatments are often insufficient. Therefore, new, reliable biomarkers must be identified. CTSL overexpression is closely associated with tumour progression and poor prognosis. However, the role and mechanism of CTSL as an oncogene in ESCC remain unclear.

Genome-wide association study (GWAS) data were used for Mendelian randomization analysis to investigate the possible relationships between CTSL and ESCC. The correlation between CTSL expression and prognosis was analysed using GEO, TCGA, and GEPIA data. We compared CTSL expression among the cell types using single-cell sequencing. Correlations between CTSL and the tumour microenvironment, immune cell infiltration, tumour mutational load, immunological checkpoints, and treatment sensitivity in patients with ESCC were investigated. Finally, using mouse models and cellular investigations, we assessed the effects of CTSL on the growth, apoptosis, and metastasis of ESCC tumour cells.

CTSL was overexpressed in ESCC and correlated with prognosis. We also discovered its close association with cell immunity, especially with tumour-associated macrophages and immune checkpoints in the tumour microenvironment. CTSL may play a key role in ESCC development by affecting M2 macrophage polarisation. CTSL and the M2 macrophage marker genes showed significant positive correlations. Mendelian randomization analysis confirmed a relationship between CTSL and ESCC. Finally, our in vitro and in vivo experiments demonstrated that CTSL promoted the proliferation and migration of ESCC cells, validating our bioinformatic analysis.

CTSL emerged as a crucial gene in ESCC that influences patient prognosis and immunity, particularly in association with M2 macrophages. Therefore, targeting or modulating CTSL levels may provide new therapeutic strategies for patients with ESCC.

The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-β, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-β signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

The efficacy of neoadjuvant chemo-immunotherapy (NAT) in esophageal squamous cell carcinoma (ESCC) is challenged by the intricate interplay within the tumor microenvironment (TME). Unveiling the immune landscape of ESCC in the context of NAT could shed light on heterogeneity and optimize therapeutic strategies for patients.

We analyzed single cells from 22 baseline and 24 post-NAT treatment samples of stage II/III ESCC patients to explore the association between the immune landscape and pathological response to neoadjuvant anti-PD-1 combination therapy, including pathological complete response (pCR), major pathological response (MPR), and incomplete pathological response (IPR).

Single-cell profiling identified 14 major cell subsets of cancer, immune, and stromal cells. Trajectory analysis unveiled an interesting link between cancer cell differentiation and pathological response to NAT. ESCC tumors enriched with less differentiated cancer cells exhibited a potentially favorable pathological response to NAT, while tumors enriched with clusters of more differentiated cancer cells may resist treatment. Deconvolution of transcriptomes in pre-treatment tumors identified gene signatures in response to NAT contributed by specific immune cell populations. Upregulated genes associated with better pathological responses in CD8 + effector T cells primarily involved interferon-gamma (IFNγ) signaling, neutrophil degranulation, and negative regulation of the T cell apoptotic process, whereas downregulated genes were dominated by those in the immune response-activating cell surface receptor signaling pathway. Natural killer cells in pre-treatment tumors from pCR patients showed a similar upregulation of gene expression in response to IFNγ but a downregulation of genes in the neutrophil-mediated immunity pathways. A decreased cellular contexture of regulatory T cells in ESCC TME indicated a potentially favorable pathological response to NAT. Cell-cell communication analysis revealed extensive interactions between CCL5 and its receptor CCR5 in various immune cells of baseline pCR tumors. Immune checkpoint interaction pairs, including CTLA4-CD86, TIGIT-PVR, LGALS9-HAVCR2, and TNFSF4-TNFRSF4, might serve as additional therapeutic targets for ICI therapy in ESCC.

This pioneering study unveiled an intriguing association between cancer cell differentiation and pathological response in esophageal cancer patients, revealing distinct subgroups of tumors for which neoadjuvant chemo-immunotherapy might be effective. We also delineated the immune landscape of ESCC tumors in the context of clinical response to NAT, which provides clinical insights for better understanding how patients respond to the treatment and further identifying novel therapeutic targets for ESCC patients in the future.

Differential expression (DE) analysis is a necessary step in the analysis of single-cell RNA sequencing (scRNA-seq) and spatially resolved transcriptomics (SRT) data. Unlike traditional bulk RNA-seq, DE analysis for scRNA-seq or SRT data has unique characteristics that may contribute to the difficulty of detecting DE genes. However, the plethora of DE tools that work with various assumptions makes it difficult to choose an appropriate one. Furthermore, a comprehensive review on detecting DE genes for scRNA-seq data or SRT data from multi-condition, multi-sample experimental designs is lacking. To bridge such a gap, here, we first focus on the challenges of DE detection, then highlight potential opportunities that facilitate further progress in scRNA-seq or SRT analysis, and finally provide insights and guidance in selecting appropriate DE tools or developing new computational DE methods.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8+ T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8+ T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8+ T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer.

Autophagy plays roles in esophageal pathologies both benign and malignant. Here, we aim to define the role of autophagy in esophageal epithelial homeostasis.

We generated tamoxifen-inducible, squamous epithelial-specific Atg7 (autophagy related 7) conditional knockout mice to evaluate effects on esophageal homeostasis and response to the carcinogen 4-nitroquinoline 1-oxide (4NQO) using histologic and biochemical analyses. We fluorescence-activated cell sorted esophageal basal cells based on fluorescence of the autophagic vesicle (AV)-identifying dye Cyto-ID and then subjected these cells to transmission electron microscopy, image flow cytometry, three-dimensional organoid assays, RNA sequencing, and cell cycle analysis. Three-dimensional organoids were subjected to passaging, single-cell RNA sequencing, cell cycle analysis, and immunostaining.

Genetic autophagy inhibition in squamous epithelium resulted in increased proliferation of esophageal basal cells under homeostatic conditions and also was associated with significant weight loss in mice treated with 4NQO that further displayed perturbed epithelial tissue architecture. Esophageal basal cells with high AV level (Cyto-IDHigh) displayed limited organoid formation capability on initial plating but passaged more efficiently than their counterparts with low AV level (Cyto-IDLow). RNA sequencing suggested increased autophagy in Cyto-IDHigh esophageal basal cells along with decreased cell cycle progression, the latter of which was confirmed by cell cycle analysis. Single-cell RNA sequencing of three-dimensional organoids generated by Cyto-IDLow and Cyto-IDHigh cells identified expansion of 3 cell populations and enrichment of G2/M-associated genes in the Cyto-IDHigh group. Ki67 expression was also increased in organoids generated by Cyto-IDHigh cells, including in basal cells localized beyond the outermost cell layer.

Autophagy contributes to maintenance of the esophageal proliferation-differentiation gradient. Esophageal basal cells with high AV level exhibit limited proliferation and generate three-dimensional organoids with enhanced self-renewal capacity.