An integrated analysis of EwS tumor samples reveals a dynamic tumor–CAF continuum that is uniquely preserved in primary tumors and establishes a framework for identifying clinically relevant tumor-associated surface TAs, providing a foundation for the rational development of next-generation immunotherapies and precision-targeted therapies for patients with EwS.
Abstract
Ewing sarcoma (EwS) is an aggressive pediatric malignancy with poor outcomes for patients with metastatic or relapsed disease. Effective immunotherapeutic approaches, including CAR T-cell therapy, are limited by intratumoral heterogeneity, an incompletely characterized tumor microenvironment (TME), and a lack of well-defined, tumor-restricted target antigens. To address these limitations, we performed an integrated analysis of EwS tumor samples using both single-nucleus and single-cell RNA sequencing datasets derived exclusively from patient samples, including matched primary tumors and orthotopic patient-derived xenograft (PDX) models. Our analyses reveal that primary EwS tumors are largely composed of highly heterogeneous malignant cell populations occupying multiple, multidirectional transcriptional states, including neuronal-like, proliferative, angiogenic, and fibroblast-like. We demonstrate that the EwS TME contains both classical cancer-associated fibroblasts (CAFs) and abundant EwS CAF-like tumor cells that transcriptionally resemble stromal cells while retaining tumor identity. Trajectory analyses define a progressive and coordinated tumor–CAF continuum, marked by gradual loss of neuronal programs and activation of mesenchymal and extracellular matrix remodeling programs, suggesting dynamic tumor cell reprogramming that may promote invasion, immune evasion, and therapeutic resistance. Notably, this structured transcriptional continuum was prominent in primary tumors but largely absent in matched PDX models, underscoring the importance of native tumor context for capturing clinically relevant tumor–TME interactions. We also developed a systematic surface-antigen discovery pipeline and identified ten novel putative tumor-associated surface target antigens (TAs), LRRC15, ATP2B3, CACNA1I, DCHS2, DSEL, LPAR4, PRRT4, TMEM229A, UNC5A, and UNC79, none of which have been previously described in EwS tumor biology. Characterization of these surface TAs revealed distinct expression patterns across EwS tumor cells, EwS CAF-like tumor cells, and classical CAFs. Moreover, some TAs expression differed between primary and metastatic tumors and between primary patient samples and matched PDX models, highlighting the critical importance of first validating therapeutic targets in primary tissues (PT). Together, these findings redefine the cellular architecture of EwS by revealing a dynamic tumor–CAF continuum that is uniquely preserved in primary tumors and establishes a framework for identifying clinically relevant tumor-associated surface TAs. These results provide a foundation for the rational development of next-generation immunotherapies and precision-targeted therapies for patients with EwS.
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.
Shu-hang Xu, Yaorong Su, Senmin Zhang et al.· Oncoimmunology· 0 citations
Head and neck squamous cell carcinoma (HNSCC) remains clinically challenging, with limited molecularly targeted options and a strong dependence on the tumor microenvironment. Cancer-associated fibroblasts (CAFs) are major stromal regulators that shape tumor progression, extracellular matrix remodeling, invasion, and therapeutic response. However, how CAF heterogeneity and plasticity translate into distinct tumor-promoting functions and targetable vulnerabilities remains insufficiently defined. Here, we integrated patient-matched primary CAFs and normal fibroblasts with 3D functional assays, tumor–stroma co-culture models, quantitative extracellular matrix analysis, whole-proteome profiling, and pharmacological perturbation. Primary fibroblast populations displayed marked interpatient heterogeneity and context-dependent plasticity in invasion, contractility, and responsiveness to tumor-derived signals, whereas enhanced fibronectin-rich matrix deposition and disorganization emerged as a conserved CAF-associated feature. Both normal fibroblasts and CAFs promoted HNSCC cell invasion in a population-dependent manner, whereas CAFs consistently induced less compact and more dispersed tumor nest architectures. Integrative functional analyses identified distinct CAF phenotypes characterized by either invasive and matrix-remodeling activity or high responsiveness to tumor-derived cues. Proteomic profiling revealed recurrent enrichment of adhesion, cytoskeletal, and extracellular matrix programs and guided the selection of pharmacological inhibitors aimed at modulating specific CAF-mediated pro-tumoral functions. Pharmacological targeting selectively altered these functions: CHI3L1 inhibition disrupted fibronectin matrix deposition, broad phosphodiesterase inhibition increased matrix alignment, and FZD7 inhibition consistently blocked tumor-induced CAF invasion across all tested populations. These findings define functionally distinct and pharmacologically targetable CAF programs in HNSCC and support stromal-directed interventions as a rational component of future combination treatment strategies.
Llara Prieto-Fernández, A. Martínez-Carrillo, Lucas de Villalaín et al.· bioRxiv· 0 citations
Background Sarcomas show heterogeneous responses to immune-checkpoint blockade (ICB), and cancer-associated fibroblasts (CAFs) are considered to shape the tumor immune microenvironment, yet CAF programs that predict ICB outcomes in sarcoma remain unclear. Methods We investigated the interaction between different cell types in the sarcoma tumor microenvironment and their effects on immune-checkpoint blockade response, with a focus on identifying signature genes and molecular mechanisms that distinguish tumor-promoting from tumor-suppressive CAFs at the single-cell level. We analyzed single-cell data from two different sarcoma cohorts, transcriptome profiles of 206 sarcoma patients recruited from The Cancer Genome Atlas, and predicted immune-checkpoint blockade (ICB) response data inferred using the TIDE algorithm from 64 TCGA sarcoma patients. Results We found 134 stem-like CAF-related signature genes in the recurrent group and eight signature genes in the metastasis group, defining three CAF subtypes (myofibroblastic CAF, antigen-presenting CAF, and inflammatory CAF). SIG134 and SIG8 were associated with TIDE-inferred ICB response in subtype-specific analyses: SIG134 in STLMS and ULMS, and SIG8 in MFS, STLMS, and ULMS. In addition, the MDK-NCL ligand-receptor signal transduction pathway was linked to the infCAF subtype and myoCAFs in metastatic sarcoma. Furthermore, SIG4 (MDK, SDC2, LRP1, and NCL) was highly expressed in inflammatory CAFs. Conclusions Single-cell-derived CAF signatures may reflect sarcoma subtype-specific stromal programs associated with predicted ICB response and clinical outcome. SIG4 is proposed as a candidate prognostic signature that warrants further validation.
Ji-Yong Sung, Jin-Hong Kim, Yi-Jun Kim· PLoS ONE· 0 citations
Neuroendocrine tumors (NETs), including aggressive subtypes such as neuroendocrine prostate cancer (NEPC) and small cell carcinomas, are characterized by marked heterogeneity, therapeutic resistance, and limited availability of clinically relevant models. The development of patient-derived systems that faithfully recapitulate tumor biology remains a major barrier to advancing precision oncology in this disease. Here, we applied conditional reprogramming (CR) technology to establish patient-derived neuroendocrine tumor cell cultures (CR-NETs) from primary and metastatic specimens. Using co-culture with irradiated feeder cells and ROCK inhibition, CR-NETs were rapidly expanded from small clinical samples, including biopsies and effusions, with high efficiency and short turnaround time. Comprehensive characterization demonstrated that CR-NETs retain key neuroendocrine features, including expression of canonical markers (e.g., CHGA, SYP), lineage plasticity signatures, and patient-specific genomic alterations. Functional assays revealed preserved tumor heterogeneity and differential responses to standard-of-care agents and targeted therapies. Notably, CR-NETs exhibited distinct vulnerabilities in epigenetic regulation and metabolic pathways, consistent with neuroendocrine differentiation states. Importantly, CR-NETs enabled real-time drug sensitivity testing, identifying patient-specific therapeutic susceptibilities. Collectively, our findings demonstrate that conditional reprogramming provides a robust and scalable approach to generate clinically relevant NET models, enabling functional precision oncology, biomarker discovery, and therapeutic optimization. This platform may facilitate rapid clinical decision-making and accelerate the development of targeted strategies for patients with neuroendocrine malignancies.
Jenny Li, Akshay Sood, Debasish Sundi, Timothy Gauntner, Shang-Jui Wang, Lingbin Meng, Qingqing Wu, Peng Wang, Eric Singer, Anil V. Parwani, Cheryl Lee, Xuefeng Liu. Conditional Reprogramming Enables Rapid Establishment of Patient-Derived Neuroendocrine Tumor Models for Functional Precision Oncology [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr PR005.
Jenny Li, Akshay Sood, Debasish Sundi et al.· Cancer Research· 0 citations
It is identified that lymph node metastases in high-risk neuroblastomas display distinctive cellular heterogeneity and plasticity, marked by mesenchymal-like and stem-like states and heightened epithelial-to-mesenchymal transition activity compared to primary adrenal tumors.
Laiman N. Wu, Janet L. Oblinger, Da-Zhuan Xin et al.· Science Advances· 0 citations
While recent research has increasingly focused on the role of fibroblasts and macrophages in osteosarcoma, the tumor vasculature remains poorly understood, particularly in metastatic disease. To address this gap, we performed single-nuclei multi-ome (RNA+ATAC) sequencing on 24 human metastatic osteosarcoma specimens. We found that endothelial cells adopt a hybrid endothelial-mesenchymal state resembling endothelial-to-mesenchymal transition (EndMT) and that a subset of diploid endothelial cells expresses osteoblastic transcriptional profiles and gene regulatory networks (GRN). Joint copy-number analysis further identified osteosarcoma cells with endothelial transcriptional programs and GRNs, consistent with vascular mimicry. In vitro assays and syngeneic lineage-tracing experiments validated that tumor educated endothelial cells acquire osteoblast-like features. Together, these findings reveal substantial plasticity among endothelial and osteosarcoma cells in human and murine metastatic osteosarcoma, provide new insight into the how the metastatic microenvironment shapes the tumor vasculature, and challenge current models of osteosarcoma biology.
Julian Burks, Ying Wu, K. Bhuvaneshwar et al.· bioRxiv· 0 citations