Single-cell sequencing profiling of intratumoral heterogeneity and immunosuppressive microenvironment in primary thyroid cancer and lymph node metastases
This study comprehensively maps the coevolution of malignant thyrocyte plasticity and the immunosuppressive metastatic niche in thyroid cancer and provides a robust molecular rationale for developing next-generation immunotherapeutic strategies tailored to thyroid cancer.
Abstract
ABSTRACT Metastasis is a major determinant of treatment failure and mortality in thyroid cancer, yet the interplay between malignant evolution and the immune microenvironment remains poorly characterized. Immunotherapy offers promise, but its efficacy requires a deeper understanding of tumor-associated immune infiltration and checkpoint regulation. In this study, we constructed a high-resolution transcriptomic atlas of the thyroid cancer ecosystem by analyzing 55,005 single cells from paired primary tumors and lymph node metastases. By integrating chromosomal copy number variation (CNV) inference with consensus nonnegative matrix factorization (cNMF), we deciphered the intrinsic heterogeneity of malignant epithelial cells, revealing distinct transcriptional programs and developmental trajectories driving the metastatic cascade. The metastatic niche exhibited significant reprogramming of the immunosuppressive landscape, characterized by the enrichment of FOXP3⁺ regulatory T (Treg) cells, LAMP3⁺ dendritic cells (DCs), and CCL18⁺ M2-like macrophages. Notably, while canonical checkpoints PD-1 and PD-L1/2 showed minimal expression, ligand-receptor interaction analysis identified the LAG3-LGALS3 axes as dominant immune evasion pathways mediating the crosstalk between CD8⁺ T cells and the tumor stroma. In conclusion, this study comprehensively maps the coevolution of malignant thyrocyte plasticity and the immunosuppressive metastatic niche. By uncovering the specific role of LAMP3⁺ DCs and identifying LAG3/TIGIT as critical alternative checkpoints, our findings challenge the utility of conventional PD-1 blockade in this context and provide a robust molecular rationale for developing next-generation immunotherapeutic strategies tailored to thyroid cancer. Although limited by a modest sample size, these findings provide a foundation for further investigation of the metastatic immune landscape in thyroid cancer.
The tumor microenvironment (TME) of thyroid cancer is a complex immunological ecosystem whose composition and interactions critically influence disease progression and therapy resistance. Single-cell RNA sequencing (scRNA-seq) has emerged as a transformative tool to dissect this landscape at unprecedented resolution, moving beyond bulk-tissue analyses to delineate cell-type- specific immune dysfunction, stromal crosstalk, and immunosuppressive niche formation. This review synthesizes how scRNA-seq has elucidated key immunopathological mechanisms in thyroid cancer, including the establishment of dominant immunosuppressive networks involving exhausted CD8⁺ T cells, regulatory T cells, tolerogenic dendritic cells (e.g., LAMP3⁺ DCs), and tumor-associated macrophages. We highlight the discovery of recurrent immunomodulatory signaling axes-such as MIF-(CD74+CXCR4), CCL20-CCR6, and NECTIN2-TIGIT-that orchestrate immune evasion and represent potential pharmacologic targets. Furthermore, we discuss how single-cell-derived insights are fueling the development of immune-centric biomarkers and prognostic models with implications for predicting response to immune checkpoint inhibition and other immunotherapies. However, translational advancement is hampered by the correlative nature of current findings, technical artifacts, and profound inter-tumoral heterogeneity. We argue that future efforts must prioritize the functional validation of inferred immune interactions through spatial multi-omics and experimental models, and leverage scRNA-seq data to define therapeutically actionable immune subtypes of the TME. Bridging this gap will be essential to translate deep immunological profiling into tailored immunomodulatory strategies that improve outcomes for patients with thyroid cancer.
Hong-Xi Li, Yu Chen, Xiangxiu Qi et al.· Critical reviews in oncology...· 0 citations
Distant metastasis has a major impact on melanoma mortality, yet it remains poorly defined how disseminated cells adapt to different host organs and their microenvironment. Here we applied single-nucleus RNA sequencing (snPATHO-seq) to archival FFPE melanoma metastases spanning brain, liver and lung from different patients. We built an atlas resolving malignant, immune and stromal compartments in each organ. Within the malignant compartment, we observed a differentiation axis of melanocytic, transitory, neural crest-like and neural crest-like/mesenchymal states; and meta-programs: interferon-responsive, hypoxic, cycling and stress. The immune compartment showed organ specificity: resident macrophage identity recapitulated the host tissue (microglia, Kupffer cells and alveolar macrophages). Brain-infiltrating myeloid and lymphoid cells were transcriptionally the most immunosuppressive and most cytotoxic, respectively, of the three sites. Non-malignant stromal populations, including fibroblasts, endothelial cells and pericytes, also carried distinct organ-specific transcriptional programs, suggesting that organ-of-residence effects extend beyond the malignant cells to the whole metastatic ecosystem. We provide a first multi-organ atlas of melanoma distant metastasis allowing deeper understanding of how a melanoma is remodelled by, and/or remodels, three distinct human organ environments.
Carlos Ariel Pulido-Vicuña, Marek Piprek, Philip Georg Demaerel et al.· bioRxiv· 0 citations
Abstract Background Brain metastasis (BM) in renal cell carcinoma (RCC) remains a major clinical challenge and is frequently resistant to immune checkpoint inhibitor (ICI) therapy. The metabolic and immunological adaptations enabling tumor survival within the brain microenvironment remain poorly defined. A comprehensive, brain-specific characterization of the tumor–microenvironment is needed to understand immune dysfunction and therapeutic resistance in RCC BM. Methods We generated a large single-nucleus RNA sequencing dataset comprising 184,037 nuclei from 14 RCC BM patients, including matched primary kidney tumors (n = 8) and extracranial metastases (n = 5). Cell populations were identified across tumor, immune, and stromal compartments. Comparative analyses identified BM-specific transcriptional, metabolic, and immune programs. Spatial transcriptomic profiling was conducted on 12 BM samples (13,128 cells) to validate cellular localization and interactions. Ligand–receptor inference was applied to reconstruct intercellular communication. Results RCC BM is associated with extensive immune remodeling of the brain microenvironment and stromal involvement. Tumor cells show neural-like features with evidence of neuroglial cells infiltration, while stromal populations display immunomodulatory phenotypes beyond structural roles. This landscape includes expansion of immunosuppressive myeloid populations, depletion of dendritic cells, absence of tertiary lymphoid structures, and CD8+ T cells exhibiting terminal exhaustion. Across compartments, we observed coordinated metabolic shifts, including enhanced OXPHOS and MYC-associated programs. Spatial and ligand–receptor analyses confirmed interactions providing mechanistic insight and informing therapeutic targeting. Conclusion RCC BM represents a biologically distinct tumor entity shaped by neural adaptation, metabolic reprogramming, and immune dysfunction. Immunosuppressive myeloid signaling, T cell exhaustion, and impaired antigen presentation establish a brain-specific microenvironment limiting immune checkpoint efficacy. These findings highlight context-dependent resistance mechanisms and identify actionable pathways to guide brain-tailored immunotherapy. Importantly, this work supported clinical trials approval testing lenvatinib plus pembrolizumab and zanzalitinib in RCC BM patients.
M. I. Ali, Z. Akpinar, Jose A. Ovando-Ricardez et al.· Neuro-Oncology Advances· 0 citations
BACKGROUND
Prostate cancer (PCa) bone metastases cause significant morbidity and mortality in advanced disease. The tumor microenvironment (TME) of bone metastases drives disease progression and therapeutic resistance, yet comprehensive characterization of its cellular heterogeneity remains limited. This study aims to characterize cellular populations and molecular signatures of PCa bone metastases using single-cell RNA sequencing (scRNA-seq) data from the Gene Expression Omnibus (GEO) database.
METHODS
scRNA-seq data from PCa bone metastasis samples were obtained from GEO. Quality control, normalization, dimensionality reduction, and cell type identification were performed using Seurat. Differential expression, pseudotime trajectory, pathway enrichment, gene regulatory network, and cell-cell communication analyses were conducted to investigate molecular mechanisms of bone metastasis progression.
RESULTS
Single-cell analysis identified distinct cellular populations within the bone metastatic TME, including malignant epithelial cells, fibroblasts, endothelial cells, osteoblasts, osteoclasts, and immune cells. Clustering revealed heterogeneous transcriptional signatures, while pseudotime analysis uncovered developmental transitions between cell states. Key transcription factors, enriched pathways related to bone remodeling, angiogenesis, and immune regulation, and critical signaling interactions between cancer and stromal cells were identified.
CONCLUSION
This study provides comprehensive insights into the cellular composition and molecular architecture of the PCa bone metastatic TME, revealing distinct cell populations, type-specific gene signatures, and cell-cell communication networks driving bone metastasis progression.
Jun Zhao, Desheng Wu· SLAS technology· 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
Multiple myeloma progresses from precursor states to active disease, and studying tumor microenvironment (TME) evolution across these stages is key to understanding immune dysregulation and therapeutic resistance. Here, we integrated paired single-cell RNA, T-, and B-cell receptor sequencing data from bone marrow samples of 235 patients spanning the disease spectrum. This dataset revealed dynamic changes in the abundance and functional states of diverse immune populations, including T-, natural killer, B-, and myeloid cells. Using non-negative factorization of cell-subset composition, we identified five reproducible TME subtypes, or "ecotypes," defined by coordinated cellular architectures. These ecotypes captured structured variation beyond disease stage and were associated with distinct cell-cell communication networks, cytokine signaling landscapes, transcription factor programs, and shared gene modules, reflecting coordinated immune adaptation to microenvironmental constraints. By linking tumor features with immune changes and ecotype distributions, we identified context-dependent associations influenced by both disease biology and treatment effects on the TME. We found that an ecotype enriched for bone marrow-resident populations and limited immune infiltration was associated with tumor expansion and inferior clinical outcomes, whereas ecotypes reflecting T-cell functional states showed distinct associations with immunotherapy response and survival. Our study provides a comprehensive single-cell immune atlas of multiple myeloma and its precursors, offering insights into TME organization and progression, and may guide development of stage-specific or ecotype-targeted immunotherapies.
M. Dang, Luz Yurany Moreno Rueda, Maria Jose Acevedo Calado et al.· Blood· 0 citations