Abstract PR016: Multi-omic profiling reveals endothelial cell plasticity in metastatic osteosarcoma

Abstract

Metastatic osteosarcoma is an aggressive malignant bone tumor that primarily affects pediatric, adolescent, and young adult populations and remains an understudied disease with significant unmet medical need. Although recent work has begun to define the complex biology of the osteosarcoma microenvironment, most studies have focused on macrophages, lymphocytes, and, to a lesser extent, fibroblasts, leaving the tumor vasculature comparatively underexplored. To address this gap, we performed single-nuclei multiome profiling, integrating RNA expression and chromatin accessibility, on 24 human metastatic osteosarcoma specimens. These analyses revealed that endothelial cells within the metastatic microenvironment frequently adopt a hybrid endothelial–mesenchymal transcriptional state suggestive of endothelial-to-mesenchymal transition. Joint copy-number analysis further identified a subset of malignant cells that cluster with endothelial cells, consistent with vascular mimicry. Gene regulatory network analysis suggested that a subset of endothelial cells progress from a hybrid endothelial–mesenchymal state toward an osteoblast-like state and confirmed endothelial-associated regulatory programs in osteosarcoma vascular mimics. In addition, immunoregulatory gene expression patterns suggest that endothelial cells may contribute to immune modulation within the metastatic osteosarcoma microenvironment. To functionally assess these observations, we performed complementary in vitro and in vivo assays. Modified transwell co-culture experiments showed that osteosarcoma-derived factors induce osteoblast-associated gene expression programs in lung microvascular endothelial cells. In vivo, endothelial lineage tracing combined with GFP-positive syngeneic murine osteosarcoma cells demonstrated that endothelial-lineage cells can acquire osteoblast-like features and become embedded within osteoid-rich tumor regions. Together, these findings support a model in which endothelial and tumor cells exhibit substantial plasticity and lineage infidelity in metastatic osteosarcoma. More broadly, this work identifies the tumor vasculature as an active and dynamic component of the metastatic osteosarcoma microenvironment and provides a foundation for investigating how endothelial cell-state transitions contribute to osteosarcoma progression and metastatic niche remodeling.