Neuroblastoma is the most prevalent solid tumor in childhood and the most common tumor in infants less than 1 year of age. In spite of aggressive multi-modal therapeutic approaches, the 5-year survival rate in patients with high-risk neuroblastoma remains poor at <50%. Recent advances have focused on harnessing the immune system to combat neuroblastoma. Antibodies that abrogate the immune checkpoint signaling pathway such as anti-CTLA-4 and anti-PD-L1 suppress neuroblastoma growth in certain mouse models of disease. Furthermore, the dinutuximab antibody that targets ganglioside GD2 expressing cells has been demonstrated to trigger neuroblastoma regression using a mechanism that involves several immune subsets. Although evidence suggest the host immune response can be exploited to fight neuroblastoma, the key immune subsets, their relative contribution, and mechanism of action to suppress neuroblastoma growth have yet to be fully characterized. To this end, we used flow cytometry to immunophenotype the intratumoral immune response in the neuro-2a model of neuroblastoma. This was accomplished by developing two 10-color panels to quantify the proportions of various myeloid and T cell subsets respectively. Using the myeloid panel, our results showed that total neuro-2a tumor immune cells were predominantly myeloid lineage-derived (CD11b+). A combination of Ly-6G and Ly-6C antibodies demonstrated that myeloid-derived suppressor cells (MDSCs) comprised the bulk of the myeloid cells and were more heavily skewed towards the granulocytic phenotype versus monocytic. An MDSC exclusion gate and a combination of F4/80, MHC class II, and CD206 antibodies enabled the analysis of tumor-associated macrophage subsets. Our results revealed that neuro-2a tumors contained relatively few activated M1 macrophages, which are typically associated with anti-tumor responses, and instead were dominated with a pro-tumor M2 macrophage response. The T cell antibody panel revealed that CD4+ T cells outnumbered CD8+ T cells by 2-fold. Regulatory T cells (Tregs) in the CD4+ T cell fraction were identified by a combination of FoxP3 and CD25 antibodies. The frequency of Tregs varied but represented <50% of total CD4+ T cells. Finally, the activation state of CD8+ T cells was examined using Ki-67, CTLA-4, and PD-1 antibodies. Analysis demonstrated that the majority of CD8+ T cells in neuro-2a tumors were actively proliferating based on the expression of Ki-67. However, most of the proliferating CD8+ T cells also co-expressed PD-1 suggesting a loss of anti-tumor functionality and an exhausted phenotype. This study provides a comprehensive immune profile of neuro-2a tumors and supports a platform with which to test new single agent and combination therapies designed to treat neuroblastoma.

Citation Format: David D. Draper, Matt Thayer, Alden Wong, Dan Saims, Scott C. Wise. Comprehensive 10 color flow cytometry analysis of the neuroblastoma intratumoral immune response using the murine syngeneic neuro-2a tumor model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1668. doi:10.1158/1538-7445.AM2017-1668