Comprehensive single cell transcriptomics analysis of murine osteosarcoma uncovers function in metastasis, genomic instability and immune activation and reveals additional target pathways.

Osteosarcoma (OS) is the most common primary pediatric bone malignancy. One promising new therapeutic target is , encoding a substrate recognition factor of the SCF E3 ubiquitin ligase responsible for ubiquitination and proteasome degradation of substrate p27, thus driving cellular proliferation. We have shown previously that knockout of in an immunocompetent transgenic mouse model of OS improved survival, drove apoptosis, and induced tumor inflammation. Here, we applied single-cell RNA-sequencing (scRNA-seq) to study primary OS tumors derived from Osx-Cre driven conditional knockout of and . We showed that murine OS models recapitulate the tumor heterogeneity and microenvironment complexity observed in patient tumors. We further compared this model with OS models with functional disruption of : one with knockout and the other with the Skp2-p27 interaction disrupted (resulting in p27 overexpression). We found reduction of T cell exhaustion and upregulation of interferon activation, along with evidence of replicative and endoplasmic reticulum-related stress in the disruption models, and showed that interferon induction was correlated with improved survival in OS patients. Additionally, our scRNA-seq analysis uncovered decreased activities of metastasis-related gene signatures in the -disrupted OS, which we validated by observation of a strong reduction in lung metastasis in the knockout mice. Finally, we report several potential mechanisms of escape from targeting in OS, including upregulation of targets, DNA copy number amplification and overexpression of alternative E3 ligase genes, and potential alternative lineage activation. These mechanistic insights into OS tumor biology and function suggest novel targets for new, synergistic therapies, while the data and our comprehensive analysis may serve as a public resource for further big data-driven OS research.