Enhancing tumor microenvironment composition to favor PD-1 inhibition via pretreatment with low-dose per fraction radiotherapy in a mouse breast cancer model.

Abstract

e14556 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 propose a novel approach combining low-dose per fraction radiotherapy (LDRT) with PD-1 inhibition to enhance systemic antitumor effects. This study aims to investigate the mechanisms underlying tumor microenvironment (TME) modulation induced by this strategy. Methods: A syngeneic breast cancer model was established in 6-week-old C57BL/6 mice by subcutaneous injection of 4T1-luciferase tumor cells. The mice were divided into six groups (control, 0.5 Gy, 1 Gy, 1.5 Gy, 2 Gy, and 5 Gy). LDRT was administered on day 7, followed by intraperitoneal injection of anti-mPD-1 on days 8 and 11. Tumor and spleen samples were collected on day 14 for mRNA sequencing and flow cytometry. RNA-seq data were aligned using Kallisto (ver. 0.46.1) with the Mus musculus GRCm39 reference genome, and principal component analysis (PCA) confirmed the absence of batch effects. Results: LDRT delayed tumor growth and significantly reduced tumor weight, particularly at doses of 1.5 Gy and 2 Gy. Flow cytometry revealed a non-linear relationship between radiation dose and TME modulation. Intermediate doses (1.0–1.5 Gy) increased CD45⁺ immune cell infiltration and elevated the M1 macrophage ratio, whereas M2 macrophage proportions decreased at lower doses (0.5 Gy) but increased at 5 Gy. Hierarchical clustering of tumor gene expression identified three distinct patterns: genes that increased with radiation dose, genes that peaked at 1.5 Gy, and genes that decreased with dose. Gene ontology (GO) analysis of 1.5 Gy-specific genes revealed enrichment in pathways related to “cytoplasmic translation,” “response to decreased oxygen levels,” and “myeloid cell differentiation.” Spleen tissue analysis identified a distinct 1.0 Gy-specific gene group associated with “chromosome segregation” and “regulation of chromosome condensation.” PROGENy pathway analysis demonstrated significant alterations in WNT, VEGF, and androgen signaling pathways, specifically at 1.5 Gy. Conclusions: LDRT, particularly at intermediate dose ranges, delays tumor growth and reprograms the TME to favor PD-1 inhibition in a cold tumor mouse model. These effects are underpinned by transcriptional changes in key biological processes. Pathway analysis highlights notable changes in WNT, VEGF, and androgen signaling, suggesting that pretreatment with LDRT represents a promising strategy to enhance the efficacy of immune checkpoint inhibitors.