Abstract A019: Rewiring the tumor microenvironment: Uncovering lineage drivers of immunosuppressive tumor-associated macrophages in prostate cancer bone metastasis

Bone metastases are a common and devastating complication of advanced prostate cancer (PCa), with up to 80–90% of men with castration-resistant PCa (CRPC) developing metastases to the bone. The development of bone metastasis serves as a critical indicator of worse long-term clinical outcomes. Understanding the factors driving PCa metastasis to the bone, including through myeloid cell reprogramming within the tumor microenvironment (TME), is critical. Our study examined published single-cell RNA sequencing data from 7 PCa patients with bone metastasis and 7 benign donors, focusing on myeloid cells across tumor, involved marrow, distal marrow, and benign bone factions. We used manual annotation methods and scANVI for whole integrated data annotation. Myeloid cells were sub-classed and manually annotated for classical monocytes (Mono1), intermediate monocytes (Mono2), non-classical monocytes (Mono3), myeloid dendritic cells (mDCs), tumor-infiltrating monocytes (TIMs), tumor-associated macrophages (TAMs), and osteoclasts. We applied pseudotime and used CellRank's Random-Walk Simulation for trajectory inference to determine final terminal states for the myeloid cells, i.e., tumor-associated macrophages (TAMs), myeloid dendritic cells (mDCs), and osteoclasts. Ranked driver gene lists for every sample type (benign, distal, involved, tumor) and cell type (Mono1/2/3, TIM) that were associated with the final terminal states were generated. QIAGEN Ingenuity Pathway Analysis (IPA) and Gene Set Enrichment Analysis (GSEA) were performed on driver gene lists across sample types and cell types, thus identifying upstream regulators influencing terminal state formation. IPA and GSEA identified ATF3, a stress-response regulator, and NF-κB, a central inflammatory mediator, to be significantly associated with TAM differentiation driver genes and were shared across extremes of sample types (benign & tumor) and cell types (Mono1 & TIM). CellOracle's in-silico perturbations were performed to assess the significance of ATF3 in context of the PCa bone TME while visualizing impacts on cell development. Through siRNA suppression of ATF3 in-vitro, followed by flow cytometry and qPCR for knockdown validation, our results show reduced M2/TAM differentiation with decreased associated markers, suggesting a role for ATF3 in myeloid reprogramming that may contribute to TAM-associated immunosuppression and the metastatic disease state Citation Format: Azimullah Rifai, Safiullah Rifai, Zumar Meher, Tausif Khan, Wei Guang, Linbo Wang, Mohamad Khan, Arif Hussain. Rewiring the tumor microenvironment: Uncovering lineage drivers of immunosuppressive tumor-associated macrophages in prostate cancer bone metastasis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Cancer Evolution: The Dynamics of Progression and Persistence; 2025 Dec 4-6; Albuquerque, NM. Philadelphia (PA): AACR; Cancer Res 2025;85(23_Suppl):Abstract nr A019.