Canine hemangiosarcoma (HSA) represents a significant clinical challenge due to its high prevalence in certain breeds and poor prognosis. This malignancy is characterized by pronounced intra-tumor heterogeneity, which complicates diagnosis and treatment. Our research is motivated by the hypothesis that HSA stems from a circulating cancer stem cell population originating in the bone marrow, contributing to the tumor's diversity and resilience. The primary goal of our proposal is to dissect the molecular underpinnings of this malignant cell heterogeneity in canine HSA. Employing a novel approach with state-of-the-art single-cell molecular assays, we aim to simultaneously analyze mRNA abundance and chromatin accessibility within individual HSA cells. This method, incorporating 10x Multiome scRNA-seq/scATAC-seq, will provide a dual perspective on gene expression and regulatory function in both stem and differentiated tumor cells across a cohort of HSA samples (n = 4). By integrating this newly collected single-cell data with existing ChRO-seq datasets, we will map the transcriptional and epigenomic architecture of over 20 HSA tumors. Furthermore, our work will elucidate the spatial organization of HSA cell subpopulations within the tumor microenvironment using advanced spatial transcriptomics, shedding light on how malignant-stromal interactions drive colonization of the spleen. This comprehensive approach is poised to uncover novel biomarkers and therapeutic targets, paving the way for developing new tools for HSA management. Ultimately, we seek to transform the understanding of HSA biology, aiming to influence clinical practices and foster the development of improved diagnostic and therapeutic strategies in veterinary oncology.