(P 57) STUDYING HYPOXIA-INDUCED METASTASIS IN GENETICALLY ENGINEERED MOUSE MODELS OF UPS

Objective: Undifferentiated pleomorphic sarcoma (UPS) is a common soft-tissue sarcoma subtype with a predilection for lung metastasis. Current therapies offer limited therapeutic benefits for metastatic UPS, demonstrating a critical need for identifying alternative therapeutic vulnerabilities. Our group previously identified hypoxia-related gene upregulation in lung metastases compared to matched primary tumors in a genetically engineered mouse model (GEMM) of UPS. Hypoxia also promotes the expansion of mesenchymal stem cells and myotubes, both candidate UPS cells of origin, suggesting an association between hypoxia and stemness. We proposed that hypoxia promotes a stem-like state that enhances metastatic potential in UPS.

Methods: We employed a novel spontaneous GEMM of UPS via intramuscular electroporation of plasmids encoding oncogenic KrasG12D, Cas9 and a single-guide RNA (sgRNA) targeting p53 (KP model). Animals exclusively develop tumors at the site of plasmid electroporation within 3-4 weeks of tumor induction, demonstrating high spatial and temporal control of tumor initiation in this model.

Results: Hypoxic tumor regions were confirmed upon administration of pimonidazole (PIMO), a permanent hypoxia label, with some inter-tumoral variability across animals, akin to heterogeneity observed in UPS patient samples following oral PIMO administration. To study the early stages of metastasis, tumor-bearing mice were treated with PIMO 2 hours prior to terminal blood collection for isolation of pre-metastatic circulating tumor cells (CTCs). We could detect upwards of 800 CTCs in this model, and a small fraction ~8-10% were pimonidazole positive on flow cytometry analysis.

Conclusion: The KP model reliably generates spontaneous CTCs, providing a platform to interrogate the early stages of UPS metastasis. Despite robust CTC yields per animal, a minority were hypoxic. To test whether hypoxic CTCs possess enhanced metastatic capacity, we will perform competitive tail vein injections of hypoxic versus normoxic CTCs into syngeneic hosts and assess lung colonization. These studies will define hypoxia’s functional role in metastasis and evaluate its potential as a therapeutic vulnerability in UPS.