Spatial transcriptomic sequencing of fresh frozen canine osteosarcoma tumor samples

Osteosarcoma (OSA) is the most common malignant bone tumor in dogs and serves as a translational model for human pediatric OSA due to shared molecular features. This aggressive and metastatic cancer exhibits significant heterogeneity, which has limited therapeutic advances. Spatial transcriptomics offers a powerful method to investigate intratumoral complexity and the tumor microenvironment. In this study, we used the 10x Genomics Visium platform to profile spatial gene expression in 14 fresh-frozen canine OSA (cOSA) tumor samples from 11 dogs, including primary, metastatic, and recurrent tumors. Samples were stratified by survival outcomes (short-, median-, and long-term). Spatially variable genes and conserved transcriptional clusters were identified, and integration with published single-cell cOSA data enabled deconvolution of cell type proportions and spatial co-localization. Analysis of 18,834 tissue spots revealed intra-tumoral subpopulations and spatial clustering patterns. Macrophage-related gene expression was enriched in long-term survivors. Deconvolution uncovered co-localization of immune cells such as mast cells with osteoclasts and NKT cells with regulatory dendritic cells. This study maps the spatial transcriptomic landscape of cOSA, highlighting immune microenvironment features and gene expression patterns associated with survival. These findings provide insights into OSA biology and may guide future therapeutic strategies in both canine and human patients.