Summary Project 3 Late toxicity of thoracic irradiation limits curative treatment of lung cancer and compromises long-term life quality. Radiation induced lung fibrosis (RILF) is among the paradigm organs at risk (OAR) models for which evidence for substantial reduction in late toxicity of electron FLASH irradiation was successfully demonstrated. Moreover, the physiological oxygen condition has been postulated to govern the FLASH protective effect in normal tissues while relatively hypoxic tumors demonstrate similar level of sensitivity. The only possibility to provide ultra-high dose rate FLASH irradiation for deep-seated thoracic malignancies will be to utilize particles. Therefore, this project aims to provide evidence if Carbon-, Proton- and Electron FLASH will spare OAR (lung, vascular, heart and esophagus) following thoracic irradiation from early/late toxicities while demonstrating non-inferiority in terms of local control of non-small cell lung cancer (NSCLC) tumors. Whole thoracic irradiation (WTI) and focal irradiation are performed with carbon ions, protons and electron (reference particle) FLASH vs. S-PRT. The impact of FLASH on lung microvascular damage and M2 polarized inflammatory response in fibrotic lung tissue as well as in-field heart- and GI-toxicity (esophagus) will be examined. Reduced oxygen dependence of high- LET carbon ion FLASH could be further instrumental in exploration of the impact of transient hypoxia for the emergence of FLASH effect. In addition to LET modulation with carbon ions, further development of an ultra- rapid optical sensor for O2 is envisioned to online monitor, prove or disprove the postulated oxygen dependence of FLASH effect in-vitro and in-vivo. Based on increasing application of salvage reirradiation of thoracic malignancies, the impact of FLASH in sparing OAR toxicity post exposure to initial fractionated WTI will be studied and surrogates of tissue radiation memory, i.e. molecular as well as senescent-cells like phenotypic switches will be deconvoluted at single cell resolution. Considering potential differences in pathophysiology of FLASH, the relevance of TGFbeta, CTGF and endostatin as key players of RILF in mitigating FLASH effects will be evaluated. In context of tumor control, the consequence of intratumoral oxygenation heterogeneity on FLASH effect will be studied. Assuming that in analogy to normal tissue, well perfused tumor regions may be spared by FLASH, demonstration of non-inferiority of F- vs. S-PRT in tumor growth inhibition will be of utmost significance for clinical translation of FLASH. In addition to OER effect, implication of intertumoral heterogeneity on F-PRT efficacy will be elucidated by studying relevant pathways involved in ROS homeostasis rendering tumor resistant to S-RT in NSCLC patients. The relevance of LET and partial oxygen pressure on FLASH effect will be further systematically studied in 3D in-vitro tumor models and microvascular organoids. Based on preliminary data that interferon signaling might be differentially affected by FLASH, the cascade of cytosolic cGas/STING/IFN activation is examined and its potential consequence for inferior outcome in combination strategies with immune- check-point blockade, as recently approved standard regimen for NSCLC, will be evaluated.

Project 3 Narrative: This project aims to provide evidence for Carbon-, Proton and Electron FLASH sparing effects of normal organs at risk for development of early/late toxicities following thoracic irradiation. In parallel, non-inferiority of FLASH vs. standard irradiation in achieving local tumor control of non-small cell lung cancer will be evaluated. 1