FLASH radiotherapy induces unique immunotranscriptomic profiles compared to conventional dose-rate radiotherapy, despite identical immune infiltration and antitumor efficacy in a murine model of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) still poses an important clinical challenge due to its aggressiveness, notwithstanding the increasing amount of treatment options. While radiation therapy (RT) is important for TNBC management, dose-limiting side effects limit its efficacy. FLASH-RT, delivered with ultra-high dose rates, has shown anti-tumour effects comparable to conventional dose rate RT (CONV-RT) but with reduced normal tissue side effects, offering the potential of dose escalation to treat aggressive tumour subtypes such as TNBC. METHODS: In this study, we investigated TNBC responses to FLASH-RT across gene transcription, immune infiltration, and anti-tumour efficacy. Using subcutaneous and orthotopic 4 T1 and MDA-MB-231 murine TNBC models, female BALB/c or Swiss nude mice received single (14 or 20 Gy) or fractionated (5 × 5.2 or 10 × 3 Gy) doses of either CONV-RT (0.2 Gy/s) or FLASH-RT (5-5.79 × 10⁶ Gy/s). Tumour growth was monitored, and 4 T1 tumours were collected at 24 h, 5 days, and 2 weeks post-irradiation for histology, flow cytometry, and bulk RNA sequencing. RESULTS: All regimens demonstrated iso-efficacy between FLASH and CONV-RT in delaying tumour growth. RNA sequencing revealed that, 24 h post-irradiation, FLASH-RT significantly preserved expression of chemokines (NES = 2.516), including CXCL9, 10 and 11 which are critical for T cell function. Additionally, FLASH-RT retained interferon alpha and gamma signalling (NES = 2.998; 3.235) via the ISGF3 complex, resulting in interferon-stimulated genes expression. While CONV-RT downregulated these genes, histology and flow cytometry showed no differences in immune cell infiltration between modalities. We hypothesize that FLASH-RT's conserved expression of negative regulators of interferon type I and II (NES = 2.108 and 1.784 respectively), but also negative regulators of inflammation (NES = 1.88), including PD-L1 and IL18BP, may underlie this discrepancy. CONCLUSIONS: In summary, while FLASH-RT and CONV-RT are equally effective in delaying tumour growth and modulating the immune response in murine TNBC, transcriptomic analysis uncovered distinct immunomodulatory profiles, suggesting nuanced mechanisms that warrant further investigation.