ESTRO 2024 - Abstract Book

S5260 ESTRO 2024 Kyle Kim 1 , George Skoufos 2 , Frank Chinga 1 , Joanna Griffin 3 , Steven Feigenberg 1 , Bonnie Ky 4 , Constantinos Koumenis 1 , Ioannis Verginadis 1 1 University of Pennsylvania Perelman School of Medicine, Department of Radiation Oncology, Philadelphia, USA. 2 University of Thessaly, Department of Electrical & Computer Engineering, Volos, Greece. 3 University of Pennsylvania Perelman School of Medicine, Department of Medicine, Cardiovascular Institute, Philadelphia, USA. 4 University of Pennsylvania Perelman School of Medicine, Department of Medicine, Philadelphia, USA Radiobiology - Normal tissue radiobiology With more than 50% of all cancer patients receiving radiation therapy (RT) at some point within their course of illness, radiation therapy continues to be the cornerstone of cancer treatment strategies. However, a significant number of patients who receive RT for malignant tumors of the thoracic region, including breast, lung, mediastinal lymphoma, and esophageal cancers, incidental doses of radiation to the heart and vascular structures have been linked to the development of radiation-induced heart disease (RIHD). Resultantly, patients often develop long-term cardiovascular consequences, quite notably the development of radiation- induced myocardial fibrosis (RIMF). Characterized by the accumulation of collagen within the heart’s extracellular matrix, RIMF can result in life-threatening complications such as pericarditis, cardiomyopathy, and congestive heart failure. To further understand the underlying mechanisms of RIMF, we have developed a clinically relevant mouse model of image-guided radiation-induced cardiotoxicity [1]. This model enables the delivery of focal radiation to the apex of the heart whereupon we have observed induction of fibrosis primarily in perivascular region in irradiated region at 24 weeks post-RT. Utilizing this model, we have observed an increased expression of the major integrated stress response (ISR) effector, known as activating transcription factor 4 (ATF4), in RT-treated hearts. Building on our previous research, which demonstrated that ATF4 regulates fibroblast functionality by controlling various steps in the collagen biosynthetic pathway [2], we investigated whether altered ATF4 expression contributes to myocardial fibrosis following localized RT. We generated conditional ATF4 knockout ( Atf4 fl/fl ) mice and crossed them with C57BL/6 syngeneic mice expressing a tamoxifen inducible Cre transgene under the control of the ubiquitously expressed Rosa26 promoter (Rosa26:CreERT2) [2]. Tamoxifen administration resulted in near complete excision of Atf4 mRNA in the lung, spleen, heart and liver, as determined by qPCR. To investigate the potential involvement of ATF4 in the initiation and progression of fibrotic events following focal heart irradiation, we conducted an RNA-seq analysis comparing irradiated (40 Gy) and nonirradiated hearts from Atf4 wt/wt and Atf4 Δ/Δ mice at 3 weeks after RT. To investigate the role of ATF4 in M1/M2 polarization, we performed immunofluorescent (IF) staining on heart lysates from Atf4 wt/wt and Atf4 Δ/Δ mice 3 weeks after 40 Gy RT. Tissue lysates were also analyzed using a mouse-specific multiplex cytokine microbead assay. Moreover, we performed IF staining for the profibrotic marker TGF-β1 of irradiated heart samples from Atf4 wt/wt and Atf4 Δ/Δ mice. These tissues were further co-stained for CD31 as an endothelial cell marker and αSMA and PDGFRα as common markers of fibroblast activation. Furthermore, to assess the role of ATF4 in the long-term consequences, irradiated hearts from Atf4 wt/wt and Atf4 Δ/Δ mice were collected at 16 weeks post-RT and stained for Masson’s trichrome. Finally, we assess the cardiac functionality and its correlation with the amount of fibrotic tissue, we performed 2D echocardiography at 4 and 8 weeks after focal 40 Gy RT. Purpose/Objective: Material/Methods:

Results:

Reactome analysis of the 100 most upregulated genes in Atf4 wt/wt hearts revealed