(P 248) AN EX VIVO ASSAY TO PREDICT IMMUNOTHERAPY RESPONSE IN SARCOMA

Objective: Sarcomas are a heterogeneous group of mesenchymal tumors with limited response to current immunotherapies. Predictive tools to assess immune responsiveness are lacking. We developed a functional co-culture assay using our mRNA lipid particle aggregate (LPA) vaccine platform to evaluate immune-mediated tumor killing ex vivo, with potential application as a predictive platform for immunotherapy response.

Methods: Two sarcoma cell lines were studied: M3-9-M and K7M2. K7M2 was engineered to express a representative fusion oncoprotein. The cell lines were cultured into 3D spheroids. C57bl/6 and BALB/c mice were vaccinated with either total tumor or fusion-targeted mRNA-LPA which were manufactured using our standard protocol. Post-vaccination splenocytes were harvested and co-cultured with tumor spheroids in a hydrogel matrix. Confocal time-lapse microscopy tracked immune-tumor interactions over 72 hours. Interferon-gamma (IFN-γ) levels were measured in supernatants via ELISA.

Results: mRNA vaccination induced robust immune activation, with splenocytes demonstrating directed migration and tumor spheroid infiltration. Time-lapse imaging revealed progressive tumor destruction in co-culture. IFN-γ levels were significantly elevated in the groups where splenocytes were collected from vaccinated mice indicating a strong Th1 immune response. These findings were consistent across biological replicates.

Conclusion: This co-culture assay models immune-tumor interactions in a controlled, quantifiable system and may serve as a functional tool to predict immunotherapy responsiveness in sarcoma. The use of mRNA vaccination provides proof-of-concept; the use of patient tumor samples and immune cells while correlating with clinical findings will further validate the tool.