Single-Nuclei Multiome (ATAC + Gene Expression) Sequencing of a Primary Canine Osteosarcoma Elucidates Intra-Tumoral Heterogeneity and Characterizes the Tumor Microenvironment
Osteosarcoma (OSA) is a highly aggressive bone tumor primarily affecting pediatric or adolescent humans and large breed dogs. Canine OSA shares striking similarities with its human counterpart, making it an invaluable translational model for uncovering the disease's complexities and de-veloping novel therapeutic strategies. Tumor heterogeneity, a hallmark of OSA, poses significant challenges to effective treatment due to the evolution of diverse cell populations that influence tumor growth, metastasis, and resistance to therapies. In this study, we apply single-nuclei mul-tiome sequencing, encompassing ATAC (Assay for Transposase-Accessible Chromatin) and GEX (Gene Expression, or RNA) sequencing, to a treatment-naïve primary canine osteosarcoma. This comprehensive approach reveals the complexity of the tumor microenvironment by simultane-ously capturing the transcriptomic and epigenomic profiles within the same nucleus. Furthermore, these results are analyzed in conjunction with bulk RNA sequencing and differential analysis of the same tumor and patient-matched normal bone. By delving into the intricacies of OSA at this un-precedented level of detail, we aim to unravel the underlying mechanisms driving intra-tumoral heterogeneity, opening new avenues for therapeutic interventions in both human and canine patients. This study pioneers an approach that is broadly applicable, while demonstrating sig-nificant heterogeneity in the context of a single individual's tumor. Overall design: Single nuclei were extracted from a primary, treatment-naive canine osteosarcoma tumor and subjected to 10x Genomics single nuclei multiome (ATAC + GEX) sequencing to evaluate intra-tumoral heterogeneity and characterize the tumor microenvironment.