Single‐nucleus <scp>RNA</scp> sequencing identifies a novel tenogenic heterologous differentiation in endometrial carcinosarcomas: implications for diagnosis and tumor classification

Carcinosarcomas (CSs) are aggressive biphasic tumors characterized by epithelial and mesenchymal components, whose histogenesis and differentiation dynamics remain poorly understood. We present single-nucleus RNA sequencing (snRNA-seq) analysis of six CSs (five endometrial and one ovarian) and two normal endometrial samples, profiling over 96,298 cells. By integrating transcriptomic data with inferred copy number variations (CNVs), immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and in situ hybridization (ISH) validation, we resolved the complex cellular architecture of these tumors, identified lineage-specific programs, and revealed unexpected differentiation trajectories. snRNA-seq was used to further refine the histopathological classification of three cases by uncovering heterologous differentiation not previously recognized: one rhabdomyogenic, one osteogenic, and, notably, one exhibiting a novel tenogenic program, defined by the expression of SCX, MKX, and TNMD. All CSs displayed a prominent mesenchymal compartment comprising both undifferentiated fibroblast-like cells and distinct lineage committed populations, including rhabdomyoblasts (Rhab), tenoblasts (Teno), osteoblasts (Osteo), and chondroblasts (Chond). In some tumors, multiple mesenchymal identities co-existed, and in others, differentiation gradients (e.g. immature versus mature rhabdomyoblasts) were observed. These patterns underscore the cellular plasticity and multilineage potential of the sarcomatous component. Furthermore, the expression of specialized interface markers (COL22A1, NCAM1, ACAN, CHRNG, MUSK) suggests that some tumors use structured developmental programs reminiscent of the muscle-tendon junction, enthesis, or neuromuscular junction. CNV analysis revealed tumor-specific genomic alterations with clonal and subclonal patterns linked to differentiation state, which were validated by FISH. Altogether, this study demonstrates that CSs are not static biphasic tumors but rather complex ecosystems with extensive developmental plasticity. Our findings redefine their classification and support the use of single-nucleus approaches to uncover hidden differentiation trajectories in highly heterogeneous cancers, including the discovery of a previously unreported tenogenic lineage. Our results challenge the diagnosis of homologous CS when only morphological criteria are applied. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.