Integrative single-cell RNA sequencing analysis of publicly available datasets from non-small cell lung cancer and breast cancer is performed to systematically map transcriptional heterogeneity and regulatory networks within the TME, providing a systems-level framework of TME organization.
Abstract
Tumor progression is driven by dynamic interactions between malignant cells and the tumor microenvironment (TME), yet the regulatory mechanisms governing cellular heterogeneity and intercellular communication remain incompletely characterized. Here, we performed integrative single-cell RNA sequencing (scRNA-seq) analysis of publicly available datasets from non-small cell lung cancer and breast cancer to systematically map transcriptional heterogeneity and regulatory networks within the TME. Using a unified computational pipeline with Seurat v5, SCENIC, and ligand–receptor modeling, we resolved major cellular populations, including malignant epithelial cells, immune subsets, cancer- associated fibroblasts, and endothelial cells, and their transcriptional states. Malignant cells displayed pronounced intratumoral heterogeneity, occupying a continuum of proliferative, metabolic, and invasive phenotypes linked by pseudotime trajectories. Gene regulatory network inference identified STAT3, NF-κB, MYC, and HIF-1α as central hubs coordinating tumor-associated programs. Notably, we uncovered a cytokine-mediated immunoregulatory axis between malignant cells and tumor-associated macrophages, driven by IL6– IL6R and CCL2–CCR2 signaling. Cell–cell communication analysis further revealed coordinated networks supporting immune suppression, inflammation, and angiogenesis. These findings provide a systems-level framework of TME organization and highlight key transcriptional circuits and signaling pathways as promising targets for disrupting tumor– microenvironment crosstalk in precision oncology.
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.
Hongling Jia, Yongxuan An, Bing Chen et al.· Frontiers in Immunology· 0 citations
Cancer arises from genetic and epigenetic alterations that reshape chromatin, transcriptional regulation, and malignant cell states. To chart cancer-intrinsic regulatory programs, we build a pan-cancer single-cell atlas of 60 cancer cell lines spanning 16 tissue origins and 20 cancer types, comprising 240,957 snRNA-seq and 223,347 snATAC-seq profiles. Integrative analyses reveal cell-state heterogeneity, core gene-regulatory networks, and a conserved EMT axis transcending tissue of origin; copy-number analysis identifies transcription factor amplification and hyperactivation as drivers of state reprogramming. Comparing cutaneous melanoma with acral melanoma, a rare subtype underrepresented in previous studies, uncovers a universal inflammation-suppressive program in acral and an inflamed landscape in cutaneous melanoma, with JAK-STAT activity as the central discriminator. Integrating data across models and patient cohorts links tumor-intrinsic regulation to microenvironmental composition and therapeutic response. By profiling rare alongside common subtypes, this atlas offers a resource for mapping pan-cancer and subtype-specific regulatory programs shaping cell-state plasticity. ‘The characterization of cancer intrinsic regulatory landscape remains elusive. Here, the authors generate a pan-cancer single cell transcriptomic and epigenomic atlas and identify subtype-specific gene-regulatory programs that shape cancer cell-state plasticity.
Advanced renal cell carcinoma frequently acquires resistance to immune checkpoint blockade (ICB), underscoring the pivotal influence of the tumor immune microenvironment (TME) on therapeutic efficacy. While recent studies have implicated multicellular crosstalk within the TME as a central driver of ICB resistance, the precise multicellular programs (MCPs) that orchestrate this process remain poorly defined. Here, through integrative single-cell and spatial transcriptomic profiling of clear cell RCC (ccRCC) cohorts, we delineated a previously unrecognized MCP associated with ICB resistance, distinguished by heightened lysosomal activity, adipogenic signaling, and rewired fatty acid metabolism. Within this program, we uncover a coordinated interplay among TAM_APOE, ccRCC_CXCL14, and endothelial cells, whereby ccRCC_CXCL14 recruits TAM_APOE, which subsequently promotes tumor lipid metabolic reprogramming and angiogenesis, forming a pro-tumorigenic feedforward loop. Spatial mapping revealed a malignant gene topic colocalizing with this MCP in tumor cores, which robustly predicted both unfavorable survival and resistance in ICB-treated patients. Functional assays confirmed that the CXCL14-TAM axis promotes metabolic reprogramming, while dual CXCR4 and PD-1 blockade synergistically reverses the resistant phenotype by restoring CD8⁺ T-cell cytotoxicity. Multiplex immunofluorescence further validated the enrichment of this MCP specifically in non-responders. Collectively, our study defines a spatially organized, functionally coordinated multicellular niche that drives ICB resistance in ccRCC, establishing both a predictive biomarker for patient stratification and a mechanistic framework for therapeutic intervention.
Multiple myeloma (MM) persists within a specialized bone marrow niche in which malignant plasma cells, immune dysfunction, inflammatory signaling, and metabolic stress reinforce one another. To resolve this ecosystem at compartment-level resolution, we integrated public single-cell RNA sequencing data with pathway scoring, cell-cell communication inference, independent clinical validation, multiplex immunofluorescence, and metabolic perturbation experiments. Analysis of 95,940 bone marrow cells identified 32 annotated populations and revealed broad microenvironmental remodeling in MM, including plasma-cell expansion, altered cytotoxic and dendritic-cell compartments, TAM-associated inflammatory programs, and lineage-specific hematopoietic perturbations. Hallmark pathway analysis identified recurrent immunometabolic programs, including IL6-JAK-STAT3, TNFα-NFκB, mTORC1 signaling, oxidative phosphorylation, unfolded protein response, hypoxia, and checkpoint/exhaustion-associated pathways. CellChat analysis showed disease-associated rewiring of ligand-receptor networks involving malignant plasma cells, TAMs, dendritic cells, and T/NK subsets, with checkpoint-enriched communication and clinically relevant plasma-cell and CD274 survival associations. Serum IL-6 was elevated in an independent clinical validation cohort. Multiplex immunofluorescence confirmed PD-L1-positive plasma cells, C1QA/C1QB/C1QC-positive TAMs expressing LAG3, and CD8-positive/LAG3-positive cytotoxic T cells. Mechanistically, malignant plasma cells showed transcriptional activation of LDH-associated glycolytic/lactate programs and ASCT2/SLC1A5-GLS-linked glutamine-metabolic programs, nominating these pathways as functional vulnerabilities. Accordingly, the LDH inhibitor galloflavin and the ASCT2/SLC1A5 glutamine-transport inhibitor V-9302 suppressed RPMI-8226 viability in dose- and time-dependent manners; their combination produced synergistic anti-myeloma activity supported by Bliss synergy, combination-index analysis, observed-versus-expected inhibition, and apoptosis-related validation. Together, these findings support an IL6-centered immunometabolic communication circuit linking malignant plasma cells, TAMs, and dysfunctional T cells, and identify cooperative glycolytic/lactate and glutamine-dependent metabolic vulnerabilities with therapeutic relevance in MM.
Delong Lang, Jing Wu, Jiayou Zhang et al.· Frontiers in Immunology· 0 citations
Introduction Cutaneous squamous cell carcinoma (cSCC) is a common keratinocyte-derived malignancy whose progression is strongly influenced by the tumor microenvironment. Although recent studies have begun to describe cellular diversity in squamous cell carcinomas, the regulatory architecture coordinating epithelial, immune, and stromal interactions in cSCC remains incompletely understood. Methods We performed single-cell RNA sequencing on matched tumor tissues, adjacent normal skin, and peripheral blood from patients with cSCC to construct an integrated cellular landscape of the tumor ecosystem. Lineage trajectory analysis, transcription factor activity inference, and ligand–receptor modeling were used to characterize cellular differentiation, regulatory programs, and intercellular communication within the cSCC microenvironment. Results Analysis of 80,736 single cells revealed extensive heterogeneity across epithelial, immune, and stromal compartments. Trajectory analysis of the myeloid lineage suggested a tumor-associated differentiation continuum extending from circulating monocytes toward a specialized angiogenic macrophage state characterized by transcriptional programs associated with extracellular matrix remodeling and immunoregulatory signaling. Concurrently, malignant keratinocyte populations displayed inferred copy-number alterations and activation of invasive transcriptional programs, accompanied by the emergence of distinct cancer-associated fibroblast subsets associated with stromal remodeling. Ligand–receptor modeling further identified extensive predicted communication networks linking macrophages, fibroblasts, and malignant epithelial cells within the tumor niche. Furthermore, transcription factor activity inference highlighted the nuclear receptor NR1H3 as a candidate regulator associated with the angiogenic macrophage program, and the NR1H3-associated transcriptional signature correlated with poor survival across multiple squamous cell carcinoma cohorts. Discussion These findings define the cellular organization and molecular programs that shape the cSCC microenvironment and provide insights into the coordinated immune, stromal, and epithelial interactions involved in cSCC progression.
Yong He, Ting Tian, Liming Li et al.· Frontiers in Immunology· 0 citations
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.
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.
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).
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.
Yandong He, Wen-Long Lu, Guanqun Ju et al.· Frontiers in Cell and Develo...· 0 citations