Progression from reflux esophagitis (RE) through Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) represents a key inflammation-associated pathway of esophageal tumorigenesis. The specific cellular transitions that underlie this progression are incompletely defined. The study used single-cell transcriptomics to map epithelial and microenvironmental changes across the RE–BE–EAC continuum and to identify candidate molecular drivers and intercellular communication pathways that accompany malignant transformation.
The authors performed single-cell RNA sequencing of clinical RE, BE, and EAC tissues and integrated these data with public EAC datasets. The combined dataset comprised 51,283 cells spanning the disease continuum. Analytical approaches included clustering of epithelial and microenvironmental cell types, pseudotime trajectory inference to reconstruct differentiation paths, copy number alteration (CNA) analysis to identify malignant epithelial cells, and ligand–receptor interaction inference to explore putative intercellular signaling networks. Supplementary data and raw datasets were deposited in public repositories (OMIX018259; GEO: GSE292971, GSE273127, GSE197677).
Epithelial cells were classified into distinct subpopulations, including Basal epithelial cells and more differentiated epithelial states. Pseudotime analysis positioned Basal cells along two major differentiation trajectories. One trajectory led toward mature epithelial phenotypes, while the alternative branch progressed through proliferative and gastric-type metaplastic states and ultimately toward epithelial populations exhibiting copy number alterations consistent with malignancy. This bifurcation highlights a putative path from basal progenitors through metaplasia to malignant epithelial states within the RE–BE–EAC sequence.
Along the malignant pseudotime branch, expression of MMP7 progressively increased. MMP7 was enriched in epithelial clusters classified as cancer-associated, suggesting that its upregulation is a feature of cells on the route to malignancy. The study emphasizes MMP7 as an epithelial marker associated with proliferative and migratory transcriptional programs in the malignant-associated epithelial compartment.
To support the transcriptomic association, the authors performed knockdown experiments targeting MMP7 in established esophageal adenocarcinoma cell lines OE19 and OE33. Reduction of MMP7 decreased both proliferation and migration in these cell lines, providing functional evidence that MMP7 expression is linked to proliferative and migratory phenotypes in esophageal cancer cells. Details on experimental conditions, knockdown efficiency, and quantification methods were reported in the source document; further mechanistic characterization was not described beyond these functional assays and should be validated longitudinally.
In parallel with epithelial transitions, the microenvironment underwent notable remodeling across progression. Features reported include an increased profile of T-cell exhaustion, polarization of macrophages toward tumor-associated states, and enrichment of cancer-associated fibroblast (CAF)-like myofibroblasts. Collectively these changes indicate a shift toward an immune-suppressive and stromally remodelled microenvironment accompanying epithelial evolution from RE/BE to EAC.
Ligand–receptor interaction analysis highlighted an epithelial-centered communication network. In particular, interactions involving MDK (midkine) and receptors SDC2, LRP1, and NCL were predicted between malignant epithelial cells and fibroblast, T-cell, and myeloid compartments. These MDK-related axes were proposed as candidate signaling pathways mediating cross-talk that could support stromal remodeling and immune modulation in esophageal carcinogenesis. The analysis indicates putative paracrine interactions but does not provide in vivo validation of the MDK signaling effects; the authors note these as candidates for further functional study.
The single-cell atlas across RE, BE, and EAC identifies a bifurcating epithelial differentiation path with a malignant branch marked by increasing MMP7 expression and acquisition of copy number alterations. Functional knockdown in cell lines linked MMP7 to reduced proliferation and migration, supporting its association with malignant epithelial behavior. Concurrent microenvironmental shifts toward T-cell exhaustion, tumor-associated macrophage polarization, and CAF-like myofibroblast enrichment delineate an immune-suppressive, stromally remodeled niche accompanying malignant progression. Predicted MDK-centered ligand–receptor interactions outline candidate epithelial-to-stroma/immune communication pathways for further longitudinal and mechanistic validation.
Data supporting these findings are available in OMIX018259 and GEO accessions GSE292971, GSE273127, and GSE197677. The authors declare no competing interests. Funding sources reported include grants from the National Natural Science Foundation of China and multiple provincial and institutional funds.
Limitations and next steps: the study is presented as a preprint and has not been peer reviewed. While cell-line knockdown provided functional support for MMP7 involvement, longitudinal in vivo validation and mechanistic dissection of MDK-related signaling were not reported and are needed to establish causality and therapeutic relevance.