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.
Abstract
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.
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.
Hong-Ling Jia, Yongxuan An, Bing Chen et al.· Frontiers in Immunology· 0 citations
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.
M. O. Odubote, Chiemeka Elochi Emeribe· bioRxiv· 0 citations
Abstract Background Clear cell renal cell carcinoma (ccRCC) is characterized by a highly heterogeneous tumor immune microenvironment. However, how tumor-intrinsic programs shape immune cell states remains incompletely understood. We aimed to identify multicellular programs (MCPs) linking tumor and immune compartments and to define tumor-derived signals that modulate immune phenotypes. Methods We applied DIALOGUE to identify MCPs across tumor and non-tumor compartments, including CD8+ T cells, CD4+ T cells, regulatory T cells, myeloid cells, endothelial cells, and fibroblasts. A total of 189,999 cells from 26 patients with sufficient tumor cell representation (>100 cells) were analyzed from our previously reported cohort (Kashima, ASCO 2024). MCP scores were derived from DIALOGUE and summarized at the sample level by averaging single-cell MCP scores. Cell–cell interactions were evaluated using CellChat and NicheNet. Spatial relationships were assessed using G4X spatial transcriptomic data (Yochum, AACR Kidney 2026). Spatial niches were defined using Seurat v5 based on k-nearest neighbor composition, and annotated as stromal, sarcomatoid, or clear cell according to cell-type composition and gene expression features. Results We identified two major MCPs, including tumor–myeloid (MCP1) and tumor–CD4+ T cell (MCP2) programs. The tumor component of MCP1 was enriched for mesenchymal and extracellular matrix (ECM)-related genes, including structural components and remodeling factors (COL5A2, MMP2, MRC2), as well as integrin-associated adhesion and cytoskeletal genes (ITGA3, ITGB1, VCL). Expression of HMGA2 further supported a dedifferentiated, EMT-like state. Clinically, a high MCP1 score was associated with sarcomatoid features (p = 0.04). In the myeloid compartment, MCP1 was characterized by immunoregulatory and tissue-remodeling markers (MRC1, MARCO, VSIG4, MS4A4A), chemokines (CCL13, CCL18, CCL23), and complement-related genes (C2, CFH, CR1), consistent with an M2-like macrophage phenotype. Cell–cell interaction analysis revealed increased communication between MCP1-high tumor cells and MARCO+ and CXCL9+ macrophage populations. Mechanistically, ECM-related signaling pathways, particularly collagen and laminin interactions, were predicted to activate CD44-mediated signaling in macrophages. These findings suggest an ECM–CD44 axis linking tumor mesenchymal programs to M2-like polarization. Spatial transcriptomic analysis further demonstrated preferential accumulation of macrophages in stromal-rich regions. Quantitative comparison across stromal, sarcomatoid, and clear cell regions showed increased macrophage infiltration in stromal and sarcomatoid areas compared to clear cell regions (3900 and 1920 vs 830 cells/mm², respectively). Differential expression analysis of macrophages across these regions showed that macrophages in sarcomatoid areas upregulated immunoregulatory and M2-associated markers, including CD163, MRC1, CSF1R, and TGFB1. CD44 expression was also significantly increased, supporting activation of ECM–CD44-associated signaling. Response to immune checkpoint inhibitors (ICIs) was evaluable in 13 patients. Although MCPs were not significantly associated with response, patients with higher myeloid MCP1 score (> mean), but not tumoral MCP1 score, showed a numerically higher response rate compared to those with low MCP1 score (4/4 vs 5/9 patients). Of note, all 4 responders in the myeloid MCP1-high group received combination therapy with CTLA-4 and PD-1 blockade. This suggests myeloid MCP1 may be associated with response to combination ICI therapy. Conclusions Our study identifies a tumor–immune MCP in which mesenchymal and ECM remodeling programs in tumor cells are associated with M2-like macrophage polarization. These findings highlight an ECM–CD44-associated signaling axis as a potential mechanism by which tumor cells shape an immunosuppressive microenvironment in ccRCC.
Katsuhiro Ito, S. Kashima, Rishabh Rout et al.· The Oncologist· 0 citations
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.
E. Putro, Alessia Carnevale, Caterina Marangio et al.· Cell Death & Disease· 0 citations
Introduction Cervical cancer is a common gynecological malignancy, having high incidence and mortality rates, especially in developing regions. The tumor microenvironment (TME) plays a crucial role in disease progression, and cancer‐associated fibroblasts (CAFs) are key components of the TME. However, the roles of CAFs in tumor‐adjacent normal tissue remain unclear. Methods Herein, we used single‐cell RNA sequencing to generate high‐resolution cellular atlases of cervical cancer and tumor‐adjacent normal tissue, systematically profiling fibroblast–cancer cell interactions and comparing fibroblasts from distinct origins. Pseudotime analysis and CytoTRACE scoring were employed to investigate the developmental trajectory of fibroblasts. Immunohistochemistry validated the inflammatory CAF (iCAF) subpopulation of C7 fibroblasts and assessed its correlations with clinicopathological features. Results We found that C7 fibroblasts secrete CXCL12 to activate the ACKR3 (CXCR7) receptor in cancer cells, thereby promoting tumor cell proliferation, invasion, and metastasis. C7 fibroblasts also highly expressed multiple oncogenic genes, including IL6, CXCL3, CXCL2, CCL2, GPC3, PLAU, TNFAIP6, PTX3, and EIF4A3, and genes associated with poor prognosis, including CXCL3, TNFAIP6, PTX3, IGSF10, and LDLR. High C7 fibroblast abundance was associated with advanced International Federation of Gynecology and Obstetrics stage, lymph node metastasis, and postmenopausal status. Discussion Our findings suggest that C7 fibroblasts being predominantly distributed in tumor‐adjacent normal tissues is significantly associated with cervical cancer progression, and that C7 fibroblasts in these tissues may serve as potential biomarkers and therapeutic targets for tumor microenvironment modulation.
Yi Liu, D. Yin, Lu-guang Wu et al.· Frontiers in Cell and Develo...· 0 citations
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.
Xiasang Chen, W. Liao, Meiqian Xu et al.· Journal of Translational Med...· 0 citations
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