Abstract Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, yet their long-term efficacy remains limited only to a subset of patients. A major barrier is the immunosuppressive tumor microenvironment (iTME), which hinders the infiltration and function of tumor-infiltrating lymphocytes (TILs). This challenge is particularly pronounced in neuroblastoma (NB), the most common extracranial solid tumor in infants and leading cause of childhood cancer deaths. We previously demonstrated that remodeling the NB iTME with a combination of anthracyclines and ICIs enhances anti-tumor immunity. Building on this, our current aim is to identify novel immunomodulatory agents (IAs) that selectively inhibit NB growth by boosting immune-mediated tumor control. To achieve this, we performed a high-throughput phenotypic screen of a chemical library containing clinical- and preclinical-stage compounds, including FDA-approved drugs with repurposing potential. The screen employed a co-culture system that mimics the iTME, combining PBMCs with MYCN-amplified and non-MYCN-amplified NB cell lines. Tumor growth and viability were measured through multiplexed imaging and biochemical assays. Compounds that reduced tumor viability while promoting immune activation were further evaluated in dose-response studies using NB spheroids, with immune activation further confirmed by flow cytometry. This strategy yielded 12 promising IAs capable of significantly inhibiting NB growth in the presence of immune cells. Some treatments reduced NB cell confluence by up to 40% and increased IFNγ and granzyme B production by cytotoxic CD8+ T cells, indicating potent immune activation. Six compounds were effective in both MYCN-amplified models, and two demonstrated broad activity across human and murine NB models. These two also demonstrated efficacy in the murine 9464D NB model by recruiting macrophage subsets capable of supporting an anti-tumor immunity. We propose that combining these IAs with existing immunotherapies could enhance NB treatment by reshaping the iTME to promote immune infiltration and tumor clearance. This strategy may also hold promise for other cancers characterized by a strongly immunosuppressive microenvironment. Citation Format: Gloria Bedini, Francesca Rita. Pellegrino, Martina Ardito, Fabio Pastorino, Doriana Fruci, Doriana Fruci. High-throughput phenotypic screening reveals novel immunomodulators that enhance immunotherapy in high-risk neuroblastoma abstract. In: Proceedings of the AACR Immuno-Oncology Conference (AACR IO): Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2026 Feb 18-21; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2026;14(2 Suppl):Abstract nr C046.
Bedini et al. (Wed,) studied this question.