ABSTRACT: Subcutaneous white adipose tissue (scWAT), a key metabolic and endocrine organ, is inevitably exposed during radiotherapy (RT). Although RT is a cornerstone of cancer treatment, its clinical use is limited by damage to surrounding healthy tissues. Ultra-high-dose-rate FLASH-RT has emerged as a promising approach that preserves tumor control while reducing normal tissue toxicity. Notably, conventional (CONV) RT has been associated with long-term metabolic dysfunction and white adipose tissue (WAT) impairment, particularly following childhood exposure; however, the impact of FLASH-RT on WAT remains unknown. Here, we compared the effects of FLASH- and CONV-RT on adipocyte function and scWAT homeostasis, integrating molecular, structural, and functional analyses. Experiments were conducted using the human SGBS preadipocyte/adipocyte cell line and a mouse model of proximal hind limb irradiation, employing a dedicated linear accelerator capable of delivering both modalities. In vivo analyses were performed 70 days after irradiation. In vitro, RT impaired adipogenic differentiation in a dose-dependent manner, with a relative sparing effect of FLASH-RT at 4–8 Gy, while mature adipocytes exhibited radioresistance with partial protection at 8 Gy. In vivo, both irradiation modalities reduced fat mass without affecting body weight, with a more pronounced loss following CONV-RT. Transcriptomic profiling of inguinal scWAT by RNA sequencing revealed a marked divergence between treatments. CONV-RT induced extensive transcriptional reprogramming of scWAT. Upregulated genes were enriched in inflammatory and immune-related pathways, as well as processes associated with chemotaxis, oxidative stress, and macrophage recruitment. Conversely, downregulated genes were linked to neuronal function, angiogenesis, and differentiation-related pathways, indicating a compromised neurovascular and adipogenic environment. In contrast, FLASH-RT elicited minimal transcriptional changes, with only three genes differentially expressed and no significant enrichment of biological processes. These molecular findings were supported by histological and ultrastructural analyses, which showed increased cellular damage, vacuolization, lipid spill-over, and reduced PLIN1 expression, predominantly in CONV-treated mice. In conclusion, our data suggest that WAT homeostasis is highly sensitive to conventional RT, which induces extensive transcriptional remodeling associated with inflammation and tissue dysfunction, whereas FLASH-RT largely preserves the scWAT transcriptome, tissue structure, and function, supporting its potential to mitigate long-term metabolic complications in cancer survivors.
