Esophageal squamous cell carcinoma (ESCC) is associated with poor prognosis due to aggressive invasion and therapy resistance. Cancer-associated fibroblasts (CAFs) are key stromal components that promote tumor progression; however, their specific roles in ESCC remain unclear. Using a direct co-culture model of ESCC cell lines (TE-9, -10, and -15) and mesenchymal stem cells (MSCs) to generate CAF-like cells, we identified biglycan (BGN) as a significantly upregulated gene in CAF-like cells via cDNA microarray analysis. Public single-cell RNA sequencing data also demonstrated elevated BGN expression in CAF clusters. We confirmed that CAF-like cells exhibited elevated BGN expression and secretion at both the mRNA and protein levels. Recombinant human BGN enhanced ESCC cell proliferation and migration by activating Erk and NF-κB signaling pathways, effects abrogated by TLR4 blockade. Furthermore, BGN promoted CAF marker expression in MSCs, M2-like macrophage polarization, and enhanced proliferation and migration abilities in both cell types. Immunohistochemical analysis of 66 ESCC tissues revealed that high stromal BGN expression correlated with greater tumor invasion, lymphatic invasion, and shorter disease-free survival. These findings indicate that CAF-derived BGN promotes ESCC progression via TLR4-mediated signaling and modulates stromal cell behavior, highlighting its potential as a prognostic biomarker and therapeutic target.

Cancer-associated fibroblasts (CAFs) in the esophageal squamous cell carcinoma (ESCC) microenvironment promote cancer progression through cell-cell interactions with cancer cells. A method has been established to generate CAF-like cells by direct co-culturing human bone marrow-derived mesenchymal stem cells (MSCs), a known source of CAFs, with ESCC cells. Using this method, cDNA microarray analyses were conducted on monocultured and co-cultured ESCCs or MSCs. In this study, bone marrow stromal cell antigen 2 (BST2) was studied, which is remarkably expressed in both ESCC cells and CAF-like cells after co-culture and may contribute to cell-cell interactions. In a public database of single-cell RNA-sequencing data from ESCC tissues, BST2 was markedly expressed in cancer cells and CAFs, and the results strongly suggested an interaction between CAFs and BST2-expressing cancer cells. The co-cultured ESCC cells and CAF-like cells showed enhanced proliferation, migration, and adhesion via the extracellular signal-regulated kinase signaling pathway, and BST2 was involved in these phenotypes. Furthermore, BST2 contributed to adhesion between MSCs and ESCC cells in direct co-culture. Immunohistochemistry demonstrated that high BST2 expression, especially in the cancer stroma, was significantly associated with adverse clinicopathologic factors and poor prognosis in patients with ESCC. In conclusion, BST2 contributes to ESCC progression by facilitating cell-cell interactions between cancer cells and CAFs within the tumor microenvironment.

Small‐cell lung carcinoma (SCLC) is a highly aggressive tumor with a dismal prognosis among lung cancers. Treatment is failing because of primary or rapidly developing secondary resistance. Recent molecular studies have suggested four SCLC subtypes with unique therapeutic vulnerabilities—ASCL1, NEUROD1, POU2F3, and YAP. These subtypes still distinguish themselves by considerable intra‐tumoral heterogeneity and the influence of the microenvironmental context, which are both considered to be responsible for treatment resistance. We integrated in situ hybridization‐based spatial transcriptomics with histological and protein‐expression analyses of 18 specimens, comprising 16 SCLCs, one atypical carcinoid, and one normal lung specimen. Systematic case‐by‐case analysis revealed multiple coexisting tumor states, including neuroendocrine and non‐neuroendocrine components, regional hypoxic states, and case‐specific FABP7 ‐positive and loss‐of‐chromosome‐Y‐associated populations. Integrative transcriptome clustering, copy number estimation, and exploratory trajectory analysis revealed transcriptional relationships among spatially coexisting tumor states, including phenotypically distinct tumor components with molecular features consistent with close clonal relationships in selected heterogeneous tumors. Furthermore, we identified immune signatures and uncovered a previously unrecognized MMP12 ‐positive tumor‐associated macrophage population, enriched at the tumor interface and spatially associated with necrotic and hypoxic regions. In vitro co‐culture experiments further showed that direct contact with SCLC cells increased MMP12 expression in macrophages, with hypoxia further enhancing its expression under specific conditions. Together, our study delineates the complex spatial architecture of SCLC by defining specific cellular programs and adjacent immune phenotypes that provide an organizational framework for understanding its marked intratumoral heterogeneity.