IMMU-06. INVESTIGATING IMMUNOSUPPRESSIVE MECHANISMS IN THE TUMOR MICROENVIRONMENT OF HIGH-RISK NEUROBLASTOMA; AN IMMUNOCOMPETENT, MYCN-DRIVEN, NON-GERMLINE GEM MODEL

Combining anti-GD2 ganglioside monoclonal antibody with immune-activating cytokines improves survival in high-risk neuroblastoma, a breakthrough for immunotherapy in this disease. However, approaches such as immune checkpoint blockade, anti-GD2 CAR-T cells, and tumor vaccines have thus far shown limited success. While a better understanding of the tumor microenvironment (TME) promises to improve the success of immunotherapy strategies, few immunocompetent models exist for high-risk neuroblastoma. We used a 30-parameter mouse-specific CyTOF mass cytometetry panel to characterize the TME of neuroblastoma arising in TH-MYCN transgenic mice. The TME was dominated by tumor-associated macrophages (TAMs) and devoid of T, B or NK cells; recapitulating the TME of human high-risk neuroblastoma. The TH-MYCN model is highly penetrant only in the 129/SvJ strain however, a challenge to leveraging immunological tools available mainly in the C57/BL6 background. We therefore created a non-germline GEM model in which primary trunk neural crest cells isolated from C57BL6/J embryos were modified genetically by transducing MYCN (MYCN-nGEMM). Orthotopic transplantation in renal capsules resulted in neuroblastoma with 50% penetrance. Co-expression of ALKF1174Lwith MYCN resulted in 80% penetrance and decreased latency. Cell lines could be derived robustly from these tumors and serially transplanted with 100% penetrance. CyTOF analysis of MYCN-nGEMM tumors also revealed a highly immunosuppressive TME similar to TH-MYCN tumors, with 80% of immune cells being TAMs, and only 5% T-cells. MYCN-nGEMM tumors were then treated with various immunotherapies, including anti-GD2 antibody, checkpoint inhibitors, and a combination of both, and changes in the TME were analyzed by CyTOF. In summary, we have characterized an immune-intact model for MYCN-driven neuroblastoma that can leverage modern methods to rapidly modify the murine genome, enabling customization of this mouse model for immunological research. We propose this model to understand the immunosuppressive mechanisms of MYCN-driven neuroblastoma tumors, and evaluate the potential of combining MYCN-directed therapies with immunotherapy.