Introduction Ultra-high dose rate (UHDR) radiotherapy has shown significant promise in reducing normal tissue toxicities with equivalent tumour control compared to conventional (CONV) dose rates termed the FLASH effect. In this study, we compared radiation-induced lung injury and changes in the pulmonary circulation following localised right lung irradiation at UHDR and CONV exposures using photons. Methods Acute and late pulmonary radiation responses were assessed in pneumonitis (C3H/NeJ) and fibrosis prone (C57BL/6) mice. 18 Gy was delivered to the right lung at CONV (0.05 Gy/s), FLASH (76.2 Gy/s) and FLASH split dose (FSD, average 2.6 Gy/s) exposures. Early responses (72 h and 4 weeks after irradiation) were investigated by immunohistochemistry (Ly6G, F4/80), fluorescence staining (EdU) and serum profiling (cytokines and lipid peroxidation assays). Fibrosis was assessed at 30 weeks after irradiation by cone-beam CT (CBCT) analysis and quantification of collagen, senescence (p21) and TGF-β levels. Changes in pulmonary vasculature and transthoracic echocardiography (TTE) was assessed in C57BL/6 mice at 30 weeks to characterise changes in the pulmonary circulation associated with pulmonary hypertension (PH). Results At early timepoints, UHDR exposures led to reduced immune cell infiltration (neutrophils and macrophages) and tissue damage in comparison to CONV and FSD groups. Serum analysis supported FLASH sparing with decreased levels of markers associated with chronic inflammation and increased markers of tissue repair in comparison to CONV and FSD groups. At 30 weeks after irradiation at a UHDR, the lung parenchyma was structurally similar to controls (p = 0.33) with minimal collagen deposition supported by CBCT tissue density analysis. In contrast, CONV and FSD animals showed significant collagen deposition (p < 0.01) with thickened alveolar septa and reduced alveolar air spaces. UHDR exposures significantly reduced functional and histological metrics of PH (p < 0.05) compared to CONV. Conclusions UHDR photons significantly reduce radiation-induced pneumonitis and fibrosis with secondary effects on the pulmonary circulation sparing PH.
Introduction The modern management of locally advanced rectal cancer (LARC) involves sequential radiotherapy and chemotherapy, termed Total Neoadjuvant Therapy (TNT). Although TNT significantly improves tumour regression and achieves complete response rates of approximately 30 %, treatment efficacy remains limited in part by tumour-intrinsic resistance to apoptosis. We therefore sought to investigate the effects of radiotherapy on TNFα-associated apoptotic signalling in rectal cancer and evaluate whether inhibition of Inhibitor of Apoptosis Proteins (IAPs) could enhance treatment response. Methods Transcriptomic profiling was performed on rectal tumour biopsies obtained before and after two weeks of neoadjuvant radiotherapy (n = 42). Gene Set Enrichment Analysis was used to identify pathways altered following radiotherapy. The clinically relevant IAP antagonist, tolinapant, was evaluated in rectal cancer models to assess effects on cIAP1/2 degradation, radiation- and chemotherapy-induced apoptosis, and formation of 5-fluorouracil (5FU)-induced drug-tolerant persister (DTP) cells. Mechanistic studies were performed in the presence of TNFα, and in vivo efficacy was assessed in SW837 xenograft models. Results Radiotherapy induced robust upregulation of TNFα/TNFR1 signalling and apoptosis-associated gene sets. Gene Set Enrichment Analysis confirmed enrichment of both “TNFα signalling via NFκB” and “Apoptosis” pathways. Upregulation of TNF, TNFRSF1A, RIPK1, TRADD, and CFLAR (FLIP) was observed alongside downregulation of DIABLO, suggesting that radiotherapy primes tumours for TNFα-mediated apoptosis but that elevated antiapoptotic signalling may suppress this response. Tolinapant degraded cIAP1/2 and significantly enhanced radiation-induced apoptosis in rectal cancer models. Co-treatment with TNFα further amplified apoptosis through a caspase-8-dependent mechanism. The combination of tolinapant and radiation was well-tolerated in vivo and reduced SW837 xenograft tumour volume compared to radiation alone; however, this difference did not meet statistical significance. Tolinapant also potentiated chemotherapy-induced apoptosis and prevented the emergence of 5FU-induced DTP rectal cancer cells. Conclusion These findings identify TNFα-associated apoptotic signalling as a potential radiation-induced vulnerability in rectal cancer and provide a mechanistic rationale for combining radiotherapy with IAP antagonists. Tolinapant also enhanced apoptosis induced by chemotherapy whilst suppressing drug-tolerant persisters, supporting further investigation of IAP antagonism as a strategy to improve TNT efficacy in the neoadjuvant management of rectal cancer.
