(592e) Biomaterial Scaffolds for Combined Focal Ablation and Immunotherapy to Target Disseminated Cancer

Metastasis continues to be the primary cause of mortality for patients suffering from cancer. Focal therapies such as hyperthermia, cryosurgery, and irreversible electroporation (IRE) have been successful at eliminating solid tumors, but often fail to mitigate local recurrence and have a very limited effect on disseminated tumor cells. With recent advances in immunotherapy, both solid tumor vaccine and immune checkpoint blockade have emerged as potential cancer therapies. Furthermore, ongoing efforts to combine focal therapy with immunotherapy to address both local and systemic cancer are beginning to show some promise. However, the mechanisms by which focal tumor ablation therapy and immunotherapy synergize are not known. Polymeric scaffolds have previously been shown to capture metastasizing cancer cells in vivo and are likely to provide a biomechanically consistent platform suitable for the clinical application of a variety of focal therapies in vivo. We sought to identify the optimal focal therapy for generating a robust CD8⁺ cytotoxic T cell-mediated immune response when applied to tumor cells within the scaffold. We chose to evaluate this strategy using the B16 mouse melanoma model, taking advantage of transgenic mouse T cells which are specific for the melanoma antigen tyrosinase-related-protein-2 (TRP-2). Focal therapies were first studied in vitro to quantify differences in solubilized protein released from B16 cells upon treatment. BCA total protein assays performed on the released supernatant following heating, cryo-freezing, and IRE showed that cryo and IRE achieved similar levels of protein release as the full lysis control, while heat-treated samples yielded 29 ± 16% of the protein amount in the control. To assay immune presentation of the TRP-2 antigen, the soluble proteins released from heat, cryo and IRE lysates were incubated with bone marrow-derived dendritic cells, which were then combined with purified naive (CD44^low) TRP-2-specific T cells labeled with CellTrace Violet (CTV). Cryo and IRE derived B16 extracts promoted similar levels of T cell activation, as measured by upregulation of CD44 and dilution of CTV (proliferation) at high cell equivalents. Interestingly, IRE achieved significantly higher T cell activation at lower cell equivalents, suggesting that the mode of cell lysis can promote differential tumor antigen release or immune presentation. Applying the focal therapies to tissue-laden polycaprolactone scaffolds in vivo showed at least a 79% reduction in viability by WST-1 assay and visible cellular damage by histology following all three methods of ablation. Future in vivo studies will study the effects of focal treatment on scaffolds containing B16 cells in combination with local and systemic immunomodulation to generate a cytotoxic T cell response capable of targeting both local and systemic disease and protecting against secondary challenge.