Abstract A031: Targeting prostate cancer bone metastasis with iNKT immunotherapy

Abstract Introduction: For men with metastatic castration resistant prostate cancer (mCRPC), there is an urgent need for improved therapeutics as the disease is highly lethal. Approximately 90% of mCRPC patients develop bone metastases. The bone tumor microenvironment (TME) is highly complex, involving dynamic interactions between tumor cells and bone-resident stromal populations that drive pathological remodeling, therapy resistance and create immunosuppression. Invariant natural killer T cells (iNKT cells) are a subset of “donor unrestricted” T cells that do not mediate alloreactivity and have strong potential as off-the-shelf immunotherapy agents. iNKT cells home to bone marrow and have been shown to modulate the bone TME in addition to directly killing tumor cells. To further investigate the mechanisms of iNKT modulation of the prostate cancer (PCa) bone TME, we used a microphysiological system (MPS), an advanced fully human 3D in vitro culture model that recreates the PCa bone microenvironment. Methods: The bone MPS includes 9 different primary cell types cultured in an optimized media formulation. The MPS comprises a main chamber filled with a collagen matrix containing osteoblasts, osteoclasts, adipocytes, mesenchymal stem cells, macrophages, fibroblasts, and PCa organoids, surrounding an engineered blood vessel mimic. iNKT cells were embedded within the bone stroma or added through the endothelial microvessel for trafficking readouts. MPS were analyzed using fluorescence microscopy to evaluate tumor cell viability (calcein-AM/ethidium homodimer), and trafficking of iNKT cells into the TME from the vasculature. Media was removed from the MPS for cytokine secretion analysis using multiplex bead-based ELISA and RNA was isolated from cells for gene expression analysis using qPCR. Results: We demonstrated that iNKT cells migrated into the bone TME from the vasculature, with migration increased in the presence of PCa tumor organoids. iNKT cells invaded into the tumor organoids and induced tumor cell death. Co-culture experiments demonstrated that this iNKT cell mediated tumor cell death was dependent on the presence of osteoclasts. qPCR revealed upregulation of FasL and chemokines CCL2, CCL4, CXCL9, and CXCL11 in the iNKT condition compared to control, suggesting activation of M1-type macrophages and induction of FasL-mediated cytotoxicity. Multiplex bead-based ELISA analysis showed upregulation of M-CSF, GM-CSF, G-CSF, IL-1α, TNFα, MIP-1α, IL-2, IL-9, MCP-3, and RANTES, consistent with enhanced pro-inflammatory reprogramming, and recruitment of effector immune cells. Conclusions: These findings suggest that iNKT cells are a promising immunotherapy for treatment of PCa bone metastases. In addition to killing tumor cells, iNKT cells can modulate the PCa bone metastatic niche which could contribute towards overcoming tumor-induced immunosuppression and promoting anti-tumor immunity. Citation Format: Cristina Sánchez-de-Diego, Nikhila S. Bharadwaj, Marcos Lares, Nikolett Lupsa, Erika Heninger, Joshua M. Lang, David J. Beebe, Jenny E. Gumperz, Sheena C. Kerr. Targeting prostate cancer bone metastasis with iNKT immunotherapy abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr A031.