Abstract
Background: Osteosarcoma is the most common primary malignant tumor of bone and occurs predominantly in children and adolescents during periods of accelerated skeletal growth. Within the tumor microenvironment (TME), vascular smooth muscle cells (VSMCs) may acquire osteogenic characteristics. Nuclear factor of activated T cells c1 (NFATc1) is central to skeletal biology, yet its behavior in VSMCs in osteosarcoma remains insufficiently defined. Methods: We profiled fresh osteosarcoma tissues by single-cell RNA sequencing on the 10x Genomics Chromium platform. Mechanical and enzymatic dissociation yielded single-cell suspensions; viable cells (> 85%) were enriched; libraries were prepared with Single Cell 3' v3 reagents and sequenced on NovaSeq 6000. Reads were processed with Cell Ranger (GRCh38). Following log-normalization, 2,000 highly variable genes were retained. Harmony integration reduced batch structure. We computed 50 principal components, generated UMAP/t-SNE embeddings, and applied k-nearest neighbor/Louvain clustering (k = 20; resolution = 0.5). Cell types were assigned from differentially expressed markers cross-checked with CellMarker and PanglaoDB. NFATc1 distribution and co-expression patterns were assessed by Pearson correlation. Results: Nine major cell classes were identified, including VSMCs, fibroblasts, macrophages, T/NK subsets, B cells, neutrophils, mast cells, and inflammatory myeloid cells. NFATc1 expression was most prominent in VSMCs, with a bimodal distribution within this lineage. In VSMCs, NFATc1 co-varied with SERGEF, APOE, and CCS, pointing to coordinated cytoskeletal remodeling, lipid handling, and copper homeostasis during osteogenic reprogramming. Conclusions: At single-cell resolution, NFATc1 marks a VSMC fraction with osteogenic features in osteosarcoma. The associated network suggests testable links to metabolism and matrix remodeling; functional perturbation will be required to establish causality.