Abstract 3940: Development of immunocomplex pediatric tumor organoids for onco-immunotherapy research

Abstract Aggressive pediatric solid tumors urgently require effective and specific therapies. In the clinic, promising new therapies, including immunotherapies, have shown limited efficacy against pediatric solid tumors. One challenge is the lack of reliable preclinical models for the evaluation of new therapies. While patient-derived organoids recapitulate primary tumor heterogeneity, and some structural properties, they fail to preserve immune cells, limiting their utility to evaluate antibody-based or cell-based immunotherapies. To address this limitation, we aimed to generate tumor immune organoids (TIOs) using an air liquid interface culture system. By optimizing the media and matrix conditions, we successfully developed osteosarcoma (OS), medulloblastoma (MB) and neuroblastoma (NBL) TIOs from mouse models that mimic the architecture and composition of human primary tumors. Flow cytometric and confocal microscopy analyses demonstrated that TIOs were viable and preserved not only tumor cells but stromal cells including immune cells for more than 130 days in NBL, 14 days in MB and 30 days in OS. Singe-cell RNA sequencing of primary tumors and TIOs after several days in culture showed preservation of the immune landscape, including T cells, myeloid cells and NK population, as well as their specific phenotypes. To evaluate the feasibility of using TIOs to test onco-immune agents, we treated NBL TIOs with anti-CD47 and anti-GD2 antibodies alone or in combination, hoping to replicate our previous work showing that blockade of GD2 and CD47 exerted a synergistic antitumor effect (Theruvath et al, Nat Med 2021). Importantly NBL TIOs responded to single agents with less tumor cell killing than the combination treatment, confirming that the immune compartment within the TIOs is biologically functional, enabling the study of tumor-immune interactions. Next, we tested chimeric antigen receptor T-cell (CAR-T) targeting GD2 in NBL, GPC2 in MB and B7H3 in OS. First, we optimized the delivery of the CAR-T cells to the culture without disrupting the architecture of the TIOs, facilitating the study of CAR-T interplay with TME populations and CAR-T antitumor evaluation. Notably, we successfully established patient-derived MB TIOs from a pediatric patient, that preserved neoplastic, stromal and immune compartments, and co-cultured them with GD2.BBz CAR-T cells. Using confocal live imaging we tracked the antitumor activity of CAR-T cells in the native 3D MB microenvironment. Our findings highlight the invaluable potential of using patient-derived TIOs as a platform to predict clinical responses to immunotherapies. In the context of cellular therapies, co-culture with autologous CAR-T cells provides a unique opportunity to study the mechanisms of resistance and efficacy in pediatric solid tumors, facilitating the development and testing of more effective and personalized treatments. Citation Format: Naiara Martinez Velez, Roel Polak, William Wang, Maria Fernanda Henao Echeverri, Diane Cruz Manjarrez, Moksha Desai, Yiyun Cheng, Peng Xu, Cara Coleen Rada, Bita Sahaf, Hiroyuki Yoda, William Weiss, Elena Sotillo, Calvin J. Kuo, Crystal L. Mackall. Development of immunocomplex pediatric tumor organoids for onco-immunotherapy research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3940.