Abstract Introduction: Neutrophil-mediated immunosuppression limits immunotherapy in Castration-Resistant Prostate Cancer (CRPC), but its mechanisms are unclear. This study aims to identify the drivers of this immunosuppression and evaluate whether targeting Master Regulator (MR) proteins can reprogram neutrophils, remodel the tumor microenvironment (TME), and enhance PD-1 blockade efficacy. Methods: We used orthotopic CRPC models treated with MR inhibitors and anti-PD-1 therapy to assess neutrophil depletion and immune remodeling. Spatial profiling with the 5,000-plex Xenium panel along with cell types annotation via a curated scRNA-seq atlas, revealed a detailed map of tissue organization. Treatment effects on immune populations and spatial reorganization, particularly neutrophils, were quantified. VIPER-based analysis defined MR-driven neutrophil functional states, and spatial mapping was conducted using Xenium data. Finally, ligand-receptor interactions between neutrophils and tumor cells were explored through cell-cell communication network analysis. Summary of unpublished data: We conducted a pilot Xenium study across four treatment arms (vehicle, Trametinib, anti-PD1, and combination) to assess MR inhibition’s effect on immune responses through cell abundance and spatial reorganization. Anti-PD1 induced broad immune recruitment, especially neutrophils, while Trametinib had minimal effect. Combination therapy reduced neutrophil abundance below baseline, attenuating checkpoint blockade-induced neutrophil accumulation. NK/T cells, monocytes, and dendritic cells increased, while macrophages remained stable. Spatial analysis showed that anti-PD1 caused tumor cells to marginalize, while combination therapy partially restored integration. Neutrophils transitioned from marginalization to forming an immunological barrier around tumor cells, and NK/T cells interacted mainly with stromal/myeloid cells, indicating immune exclusion. Despite upregulating PD-L1 in all treatments, combination therapy induced PD-L1 expression without barrier formation, suggesting that spatial organization, not just ligand expression, drives neutrophil-mediated immune exclusion. Conclusion: Treatments reshape tumor-immune architecture without disrupting tissue organization, relying on both immune cell abundance and their spatial distribution within the TME. Anti-PD1 induces broad immune infiltration, but neutrophils form a barrier limiting tumor penetration, which is relieved by the MR-inhibitor Trametinib. Neutrophils upregulate PD-L1 across all treatments, peaking after anti-PD1, while Trametinib and combination therapy drive PD-L1 expression without barrier formation. Citation Format: Melania Franchini, Florencia Picech, Cory Abate-Shen, Andrea Califano. Targeting master regulators to reprogram neutrophils and enhance PD-1 blockade efficacy in castration-resistant prostate cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6859.
Franchini et al. (Fri,) studied this question.