Single cell transcriptomics analyses reveal functional heterogeneity and anti-tumor role of mast cells in esophageal squamous cell carcinoma

Single-cell RNA sequencing reveals pro-tumorigenic intestinal mast cell programs in colorectal cancer

These findings identify a tumor-adapted MC state that orchestrates immune evasion and tissue remodeling during CRC progression, supporting the notion that MC are reprogrammed toward an immune-suppressive and pro-tumorigenic phenotype.

Single-cell RNA sequencing uncovers an immunosuppressive program in PDAC mast cells: tumour-driven activation, proliferation, and association with Treg recruitment

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly immunosuppressive tumour microenvironment (TME), which contributes to its resistance to immunotherapy. Although mast cells (MCs) have been implicated in PDAC progression, their functional heterogeneity and candidate signaling models of immune modulation remain poorly understood. Methods Through an integrated analysis of multiple single-cell RNA sequencing (scRNA-seq) datasets, we identified a significant enrichment of MCs in PDAC tissues and established a characteristic gene signature (TPSAB1, TPSB2, CPA3, HPGDS, KIT, LTC4S) for their precise identification. Results Single-cell RNA-seq analysis categorized MCs in PDAC into resting, activated, and proliferating subpopulations. To functionally validate these transcriptomic predictions, in vitro co-culture experiments showed that pancreatic cancer cells promote the activation and proliferation of MCs. Cell–cell communication analysis suggested that activated MCs preferentially interact with regulatory T cells (Tregs) via the MIF–(CD74/CXCR4) signaling axis. This interaction was associated with an immunosuppressive T-cell landscape, characterized by an expanded population of Tregs exhibiting a highly activated immunosuppressive phenotype. Spatial transcriptomics and immunofluorescence validated the confirmed the spatial proximity of MCs and Tregs in PDAC tissues. Clinically, high expression of MC-Treg signature genes correlated with poor patient survival. Conclusions Our study suggests that MCs are key orchestrators of immunosuppression in PDAC, predicted to interact with Tregs through the MIF–CD74/CXCR4 axis, offering a novel rationale for targeting the MC–Treg axis in future immunotherapeutic strategies.

Single-cell transcriptomics and functional analyses identified SPIN.DOC as a regulator for Wnt signaling and cancer stemness in colorectal cancer

Single-cell transcriptomics deciphers cancer-associated fibroblast heterogeneity and immune regulatory mechanisms in the bladder cancer tumor microenvironment

Background Bladder cancer (BLCA) is a common malignancy, with muscle-invasive bladder cancer (MIBC) associated with a 5-year survival rate below 50%. Cancer-associated fibroblasts (CAFs) are heterogeneous stromal components of the tumor microenvironment (TME) that contribute to tumor progression, therapy resistance, and immune evasion. However, the molecular basis of CAF diversity and immune regulation in BLCA remains incompletely understood. Methods We analyzed single-cell transcriptomic data from GEO dataset GSE135337 using dimensionality reduction, unsupervised clustering, differential expression analysis, GO/KEGG/GSEA, diffusion pseudotime inference, and LIANA ligand–receptor analysis. TCGA-BLCA bulk transcriptomic data (n = 408 total; n = 401 after stage filtering) were used for score validation. RT–qPCR was performed to assess selected myCAF- and iCAF-associated genes in RT4 and T24 bladder cancer cells. Results Analysis of 9,253 post-QC cells across two non-paired specimens (6,035 adjacent-tissue; 3,218 tumor) yielded 9 DE-marker-annotated cell populations. Among 1,923 retained adjacent-tissue fibroblasts, two transcriptionally distinct states were identified: Homeostatic fibroblasts (n = 1,197; enriched for COL1A1, DCN) and CCL2-high activated-like fibroblasts (n = 726; CCL2 log2FC = 0.806, FDR = 8.08 × 10−11). No fibroblast-like cells were retained in the tumor specimen. Diffusion pseudotime positioned CCL2-high cells at a later within-sample state (median DPT 0.813 vs. 0.672; FBLN1 rho = −0.808). Exploratory LIANA analysis identified 1,347 fibroblast-outgoing ligand–receptor interactions. A pre-specified six-gene myCAF score (ACTA2, COL1A1, MMP11, MYL9, TAGLN, TPM2) tracked pathological stage in TCGA-BLCA (P = 1.60 × 10−12) and showed an unadjusted overall survival association (HR = 1.21, 95% CI: 1.02–1.42, P = 0.025) that attenuated to non-significance after age and stage adjustment (HR = 1.03, P = 0.748); the association was not replicated in GSE31684 (n = 93; log-rank P = 0.443). RT–qPCR confirmed higher expression of ACTA2, POSTN, MMP11, FAP, IL6, and CXCL12 in T24 versus RT4 cells (4.33–16.56-fold; all BH-adjusted q < 0.01). Conclusion Single-cell transcriptomics of publicly available BLCA data identifies two transcriptionally distinct fibroblast states in adjacent tissue and supports an exploratory ligand–receptor interaction framework. A six-gene myCAF-associated score tracks pathological stage but does not independently predict overall survival after covariate adjustment and was not replicated in an independent cohort. These findings constitute an exploratory computational framework warranting prospective validation with primary CAF populations and adequately powered multi-specimen cohorts.

Single-cell transcriptomics and functional validation reveal a TUBB4B-associated malignant epithelial state in nasopharyngeal carcinoma

Intratumoral heterogeneity of malignant epithelial cells limits prognostic stratification and therapeutic development in NPC, yet transcriptionally distinct malignant epithelial states and their associated molecular programs remain poorly characterized. We aimed to identify clinically relevant malignant epithelial states and define their associated molecular programs in NPC. Fresh normal nasopharyngeal tissue, primary NPC, and nodal metastases were profiled by scRNA-seq, yielding 27,330 cells. Malignant epithelial states were characterized by transcriptomic reclustering, cell-cycle analysis, CNV inference, pseudotime trajectory analysis, and pathway enrichment analyses. An independent public scRNA-seq cohort (GSE150825) was reanalyzed to validate the identified malignant epithelial state and its associated molecular programs. TUBB4B function was examined using knockdown and overexpression assays, cell-cycle analysis, clonogenic assays, and xenograft experiments. Clinical relevance was evaluated by immunohistochemistry and immunofluorescence in NPC tissue sections and by survival analysis in an independent NPC tissue microarray cohort with long-term follow-up. We identified a malignant epithelial state occupying an early branch of the inferred pseudotime trajectory, characterized by increased inferred CNV burden and increased cell-cycle engagement, and further distinguished by coordinated microtubule- and cilium-associated transcriptional programs and selective enrichment of TUBB4B expression. Reanalysis of GSE150825 identified a phenotypically similar malignant epithelial state subpopulation with concordant microtubule- and cilium-associated pathway enrichment. TUBB4B localized to mitotic structures; its knockdown reduced the proportion of cells in G2/M phase, whereas overexpression increased PCNA expression, clonogenic growth, and xenograft tumor growth. In clinical tissue sections, TUBB4B expression was higher in NPC than in normal nasopharyngeal epithelium and was accompanied by an increased Ki-67-positive area. In the tissue microarray cohort, high cytoplasmic TUBB4B expression was associated with significantly worse overall survival and progression-free survival using a data-derived cut-off, and remained associated with overall survival after adjustment for staging in multivariable Firth penalized Cox models, although this association was attenuated using an alternative cut-off. We identify a TUBB4B-linked malignant epithelial transcriptional program in NPC associated with proliferation, inferred CNV burden, and adverse clinical outcome in this cohort. These findings provide a transcriptionally resolved framework for understanding malignant epithelial heterogeneity in NPC progression and nominate TUBB4B as a candidate prognostic biomarker and potential therapeutic vulnerability warranting further validation.

Integrative single-nucleus transcriptomic analysis identifies UBE2C+ proliferative tumor cells and immune-stromal regulatory crosstalk in small cell lung cancer

Background Small cell lung cancer (SCLC) is an aggressive neuroendocrine malignancy characterized by rapid proliferation, early dissemination, and limited durable benefit from current chemoimmunotherapy. Although immune checkpoint blockade has modestly improved clinical outcomes, the regulatory logic linking malignant cell states to the tumor immune microenvironment remains incompletely understood. Here, we applied an integrative single-nucleus transcriptomic framework to dissect tumor cell heterogeneity, regulatory programs, and immune-stromal communication networks in SCLC. Methods Publicly available Single-nucleus RNA sequencing data from primary and metastatic SCLC samples were analyzed using Seurat-based clustering, inferCNV-based malignant cell identification, differential expression analysis, pathway enrichment, metabolic and stemness scoring, pseudotime trajectory reconstruction, CellChat-mediated cell-cell communication inference, and transcription factor regulatory module analysis. A UBE2C-enriched proliferative tumor cell subpopulation was prioritized for functional validation. siRNA-mediated UBE2C knockdown was performed in DMS114 and NCI-H446 SCLC cell lines, followed by qRT-PCR, CCK-8, colony formation, transwell migration, and Annexin V/PI apoptosis assays. Results Using snRNA-seq, we identified multiple cell types and resolved a UBE2C+ subpopulation with marked proliferative features. UBE2C+ subpopulation displayed strong G2/M-phase enrichment, elevated mitotic and cell cycle programs. And pseudotime analysis positioned C3 UBE2C+ tumor cells at a proliferative state during tumor cell state evolution. Cell-cell communication analysis suggested that this subpopulation might interact with macrophages and fibroblasts through GRN-SORT1 and THBS1-CD47/CD36 signaling axes, indicating a potential link between proliferative tumor states and candidate communication axes. Transcription factor module analysis further revealed enrichment of cell cycle-associated regulators, including MYBL2, NFYB, E2F2, TGIF1, and RXRG, in the C3 subpopulation. Functionally, UBE2C knockdown significantly suppressed proliferation, clonogenic growth, and migration while increasing apoptosis in SCLC cells. Conclusions This study identified UBE2C+ proliferative tumor cells as a functionally relevant malignant subpopulation in SCLC and links this state to immune-stromal communication networks within the tumor microenvironment. By integrating single-nucleus transcriptomics, regulatory network inference, intercellular communication analysis, and in vitro validation, our findings nominate UBE2C as a potential candidate functional regulator and provide a systems-level framework for investigating the cancer-immunity regulome in SCLC.