Subtherapeutic-Dose of Ionizing Radiation Reprograms the Pre-Metastatic Lung Niche, Accelerating Its Formation and Promoting Metastasis

Pre-metastatic niche (PMN) formation is a critical step in metastatic progression, yet the biological effect of subtherapeutic-dose of ionizing radiation (SDIR) following radiotherapy on this process remains unclear. Using a 4T1 breast cancer mouse model, we investigated the effects of SDIR (3×0.3 Gy) on lung PMN development and metastasis formation. Lung PMNs were exposed to SDIR on days 8–10 post-tumor induction, followed by mastectomy on day 18, and analyzed on day 24. SDIR significantly increased total metastatic volume (TMV) in lungs, suggesting an accelerated PMN formation. Mechanistically, SDIR upregulated Bv8 expression, enhanced neutrophil recruitment, and increased MMP9, S100A8, and IL-6 production in the PMN by day 11, though differences between irradiated and non-irradiated PMN were no longer evident by day 14. Notably, Bv8 inhibition prevented SDIR-induced neutrophil recruitment but did not reduce TMV, indicating additional mechanisms in SDIR-driven metastasis. Proteomic analysis revealed SDIR-induced metabolic reprogramming, extracellular matrix (ECM) remodeling, and upregulation of adhesion-related pathways, driving a microenvironment that accelerates tumor invasion and metastatic outgrowth. By demonstrating that SDIR reprograms the pre-metastatic lung microenvironment, this study highlights the need to integrate organ-specific radiation exposure into predictive models of metastasis and identifies metabolic and immune-stromal pathways as potential targets for therapeutic intervention.