Immune-modulatory effects of low-dose radiotherapy through macrophage polarization and transcriptional rewiring in triple-negative breast cancer.

BACKGROUND

Despite recent advancements with immune checkpoint inhibitors, a significant proportion of patients, particularly those with triple-negative breast cancer, exhibit resistance to these therapies. To address this challenge, we investigated whether adding low-dose-per-fraction radiotherapy (LDRT) could remodel the tumor microenvironment (TME) and enhance the efficacy of PD-1 blockade.

METHODS

Luciferase-expressing 4T1 breast-cancer cells were inoculated subcutaneously at multiple sites in 6-week-old BALB/c mice. The mice were divided into seven groups: (i) untreated control; (ii) anti-PD-1 antibody alone (0 Gy); and (iii) anti-PD-1 combined with single-fraction LDRT of 0.5, 1.0, 1.5, 2.0 or 5.0 Gy. Radiation was delivered to one tumor focus, followed by intraperitoneal injection of anti-mPD-1. Directly irradiated tumors (RT tumor) and non-irradiated tumors (NT tumor), and spleen samples were collected for mRNA sequencing and flow cytometry analysis.

RESULTS

LDRT could suppress tumor progression under PD-1 inhibition dependent manner, with 1.0-1.5 Gy providing considerable tumor growth delay in RT tumors. Flow cytometry revealed a non-linear relationship between radiation dose and TME modulation, where intermediate doses shifted macrophages towards an M1-like phenotype, whereas higher doses restored an M2-like state. Transcriptomic clustering revealed three gene-expression trajectories: monotonically increasing with dose, peaking at 1.5 Gy, or decreasing with dose. Genes maximally induced at 1.5 Gy were enriched for cytoplasmic translation, hypoxia response and myeloid differentiation. In spleen tissue, a 1.0 Gy-specific signature implicated chromosome-segregation and ubiquitin-ligase pathways. Pathway analysis further demonstrated significant alterations in WNT, VEGF, and androgen signaling, particularly at 1.5 Gy

CONCLUSION

LDRT, particularly at intermediate dose ranges, delays tumor growth and reprogram the TME to favor PD-1 inhibition in a cold tumor model. These effects are underpinned by transcriptional changes in key biological processes, suggesting that pretreatment with LDRT represents a promising strategy to enhance the efficacy of immune checkpoint inhibitors.