Work

674 Single-cell transcriptomic analysis of the prostate cancer tumor microenvironment reveals radiotherapy-induced innate immune vulnerabilities

Year: 2026

Type: conference-abstract

Abstract: Background Myeloid inflammation is correlated with poor oncologic outcomes and therapeutic resistance across solid tumors. Inflammatory myeloid cells are highly abundant within the prostate cancer tumor microenvironment (TME), which likely drives clinical immunotherapy resistance. Although stereotactic body radiotherapy (SBRT) can reprogram an immunologically ‘cold’ TME and instigate T cell recruitment, whether these mechanisms occur in prostate cancer remains unclear. To this end, we leverage single-cell RNA sequencing (scRNA-seq) to identify novel innate immune vulnerabilities that can alleviate immunosuppression driven by tumor-associated macrophages (TAMs) and neutrophils.Methods We used the syngeneic RM9 prostate cancer model implanted subcutaneously into male C57BL/6J mice. Tumor-bearing mice were treated with SBRT using the X-RAD SmART platform, alone or combined with neutralizing antibodies against PD-1 (clone RMP1-14), CD47 (clone MIAP301), and Ly6G (clone 1A8). CD45 + tumor-infiltrating leukocytes (TILs) were isolated from tumors at multiple post-SBRT timepoints by magnetic-activated cell sorting and processed for scRNA-seq per 10x Genomics protocols. Data were analyzed by Seurat (v5.2.1) and BBrowserX. Tumor volumes were compared by two-sided unpaired t tests or two-way ANOVA.Results We sequenced 24,173 TILs from RM9 tumors at days 0, 3, 7, and 10 post-SBRT. Unsupervised clustering revealed a rapid influx of TAMs, classical neutrophils, and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), with a concomitant decrease in dendritic cells, B, and T cells. Subclustering monocyte-derived populations demonstrated a significant increase in chronically inflamed TAMs (Inflam-TAMs) and classical tumor-infiltrating monocytes (TIMs), but a decrease in type-2 conventional dendritic cells (cDC2s). We observed the upregulation of the matricellular protein thrombospondin-1 ( Thbs1) in Inflam-TAMs and TIMs, peaking at day 3 post-SBRT. PMN-MDSCs were enriched for a ferroptosis gene signature (Ptgs2, Hmox1, Hilpda) known to impair T cell function. In clinical datasets, elevated Thbs1 and Hilpda expression inversely correlated with prognosis. Although neutrophil depletion failed to enhance the antitumor response to SBRT, dual innate immune checkpoint targeting via anti-PD-1 and anti-CD47 significantly accelerated tumor regression compared to SBRT alone (P < 0.001) or SBRT combined with anti-PD-1 (P < 0.01).Conclusions Using scRNA-seq, we generated a comprehensive atlas of the prostate cancer immune TME following ablative radiotherapy, revealing multiple distinct pathways of myeloid inflammation that actively suppress T cell immunity. We demonstrate that targeting these innate inflammatory pathways enhances the antitumor efficacy of SBRT and generates potential synergy with IO. Paralleling our previous results in breast cancer, these data support a conserved and targetable mechanism of TME reprogramming to overcome clinical immunotherapy resistance.

Source: Journal for ImmunoTherapy of Cancer

DOI: 10.1136/jitc-2026-sitc2026.0674