ESTRO 2025 - Abstract Book

S4002

Radiobiology - Tumour radiobiology

ESTRO 2025

2637

Digital Poster Hypoxic tumor cells acquire a cellular quiescence phenotype that protects them against radiation-induced cell death Apostolos Menegakis 1,2 , Johanna Erbani 3 , Menno Boon 1 , Otilia Ciobanu 1,4 , Wietske Jeninga 5 , Anoek Friskes 5 , Rolf Harkes 6 , Bram van den Broek 6 , Monique de Jong 2 , Zeno Gouw 2 , René H. Medema 7 , Leila Akkari 1 , Jan-Jakob Sonke 2 1 Division of Tumor Biology and Tumor Immunology, Netherlands Cancer Institute (NKI), Amsterdam, Netherlands. 2 Department of Radiation Oncology, Netherlands Cancer Institute (NKI), Amsterdam, Netherlands. 3 Division of Tumor Biology and Tumor Immunology, Netherlands Cancer Institute (NKI), Amsteram, Netherlands. 4 epartment of Radiation Oncology, Netherlands Cancer Institute (NKI), Amsterdam, Netherlands. 5 Division of Cell Biology, Netherlands Cancer Institute (NKI), Amsterdam, Netherlands. 6 Bioimaging facility, Netherlands Cancer Institute (NKI), Amsterdam, Netherlands. 7 Director of research, Princess Maxima Center for Pediatric Oncology, Utrecht, Netherlands Purpose/Objective: Previously, using a lineage-tracing hypoxia reporter system we showed that pre-treatment hypoxic cells enrich in the regrowing spheroids and xenograft tumors following irradiation (IR) (1). Here, we aim to understand how hypoxic tumor cells evade radiation-induced cell death. Material/Methods: The lung Adenocarcinoma cell line (H1299), originally carrying the hypoxia reporter, was transduced with the Fluorescent Ubiquitination-base cell cycle reporters (FUCCI) to visualize the different cell cycle phases. Standard colony forming assays were performed at normoxic, chronic hypoxic or hypoxic and reoxygenated conditions prior to IR. To estimate the DNA repair capacity of the cells at the different conditions, immunofluorescent staining for DNA damage response (DDR) proteins was performed and subsequently the 53BP1 protein was endogenously tagged with a Halo tag to visualize the radiation-induced DNA double-strand breaks (DSBs) by means of live cell imaging. Results: Tumor cells cultured for 10 days in 1% O2 atmosphere are progressively arrested in the G1/G0 phase of the cell cycle, entering a cellular quiescent phase, in a duration-dependent manner and display higher radiosensitivity compared normoxic cells. Incubating the hypoxic cells for 2h in almost anoxic conditions (0.001%O2) during irradiation increased their radioresistance, consistent with the oxygen fixation theory (Figure 1). However, cells previously cultured in hypoxia and fully reoxygenated prior to IR (3h) are still more radioresistant compared to normoxic. When FUCCI cels were sorted based on the cell Cycle Profile we observed that G1 phase cells (the dominating fraction of previously hypoxic cells) exhibited higher radioresistance compared to S and G2 phase cells upon reoxygenation. In live cell imaging, hypoxic cells that were reoxygenated prior to IR displayed lower fraction of catastrophic events during mitosis and less chromosomal abnormalities during mitosis compared to normoxic cells leading to a higher fraction of daughter cells committed to cell cycle progression. To correlate the DNA damage induction, clearance and the cell cycle phase with the cellular outcome, we have tagged endogenously 53BP1 DNA repair protein and we are currently performing live-cell imaging. We aim to trace individual cells and generate cellular pedigrees that link cellular fitness with the cell cycle phase that the cells are irradiated in normoxic and hypoxic/reoxygenated conditions.