S2640
Radiobiology - Tumour radiobiology
ESTRO 2026
Conclusion: Low expression of ALDH3A1 downregulates the MAPK pathway and activates autophagy. This facilitates the rapid clearance of radiotherapy-induced cytosolic damaged DNA fragments, attenuates the cGAS-STING pathway response, and reduces the secretion of inflammatory factors, thereby conferring resistance to radiotherapy-induced damage. Inhibiting autophagy can effectively ameliorate radiotherapy resistance in HNSCC. Keywords: HNSCC; Radiotherapy resistance; Autophagy Digital Poster 4519 Radiotherapy-Induced Adaptation of Circulating Tumor Cells to a New Matrix: An Acousto- Holographic Analysis (Preliminary Results) Merve SEVG İ 1 , Mehmet HALICI 2 , Huriye Ş KIZILTAN 2 , Serdar ALAY 2 , Esmahan ÇA Ğ LAR 1 , Hasan B ABD İ O Ğ LU 3 , Ya ğ mur I Ş IK 3 , Meryem AKKURT YILDIRIM 4 , Yasemin BA Ş BINAR 5 , Hüseyin ÜVET 3,6 1 Bioengineering, Yıldız Technical University, Istanbul, Turkey. 2 Radiation Oncology, Ba ş ak ş ehir Çam and Sakura City Hospital, Istanbul, Turkey. 3 Mechatronic Engineering, Yıldız Technical University, Istanbul, Turkey. 4 Molecular Biology and Genetics, Health Biotechnology Joint Research and Application Center of Excellence,, Istanbul, Turkey. 5 Translational Oncology, Dokuz Eylul University, Institute Of Oncology,, İ zmir, Turkey. 6 Artificial Intelligence Research and Application Center (YZAUM), Istinye University, Istanbul, Turkey Purpose/Objective: This study investigates how circulating lung cancer cells adapt to a three-dimensional ECM following radiotherapy, focusing on mechanical and molecular responses. The project builds upon our previous TÜB İ TAK 1003 program (Project No. 116E743), which developed imaging technologies based on cell mechanics. Extending this approach, we examine how radiotherapy-induced alterations influence the biomechanical and molecular adaptation of lung cancer cells. Radiotherapy not only induces DNA damage but also activates the TGF- β pathway, promoting fibrosis and invasion. Loss of p53 converts TGF- β signalling from growth-suppressive to pro- invasive, potentially enhancing metastatic behaviour. This study explores how TGF- β activation and p53 loss reshape mechanotransduction pathways, identifying potential targets for early detection and prevention of metastasis. Material/Methods: A549 (parental) lung cancer cells were exposed to 0 and 6 Gy radiation. Cell stiffness was measured using
regulated autophagy as a strategy to ameliorate radiotherapy resistance. Material/Methods: Based on TCGA and GEO database analysis and clinical sample validation, ALDH3A1 was associated with radiotherapy resistance and prognosis in HNSCC patients. Cal-27 and FaDu cell lines with ALDH3A1 knockdown (sh) or overexpression (OE) were constructed. Cell proliferation and migration were assessed using CCK-8, colony formation, and scratch assays, followed by irradiation at gradient doses to determine radiosensitivity. After 6 Gy irradiation, Cal- 27-shALDH3A1 and Cal-27-OEALDH3A1 cells were evaluated by crystal violet staining and flow cytometry for proliferation and apoptosis. In nude mouse xenografts from Cal-27-Con and Cal-27-shALDH3A1 cells, radiotherapy response was assessed by measuring tumor volume, size and weight, in vivo imaging, and ALDH3A1/Ki67 IHC. Transcriptome sequencing of irradiated Cal-27-shALDH3A1 cells revealed gene expression changes, validated by immunofluorescence and Western Blot. Ultrastructure was observed by TEM, and ALDH3A1 enzymatic activity was tested via point mutation. Hydroxychloroquine, an autophagy inhibitor, was applied to examine its effect on radiosensitivity and pathway activity. Results: Integrated bioinformatics and clinical analyses established that low ALDH3A1 expression correlates with radiotherapy resistance and unfavorable prognosis in HNSCC patients. Cellular functional assays demonstrated that irradiated Cal-27- shALDH3A1 cells exhibited significantly enhanced proliferative and migratory capacities in CCK-8, crystal violet staining, and scratch wound assays, while flow cytometric analysis revealed no substantial alterations in apoptosis. In the murine xenograft model, tumors derived from Cal-27-shALDH3A1 cells manifested accelerated growth kinetics and substantially larger volumes compared to control cohorts. Transcriptome sequencing implicated MAPK signaling pathway downregulation in ALDH3A1 knockdown-mediated radioresistance. Western blot validation confirmed attenuated p-MAPK expression concurrent with upregulated autophagy-related proteins ATG5 and LC3b, indicating MAPK pathway inhibition potentially triggers autophagic activation. Furthermore, combined western blot and immunofluorescence analyses revealed diminished DNA damage marker γ -H2AX intensity, accompanied by reduced phosphorylation levels of STAT, TBK1, and IRF3 signaling molecules. Critical dependence on canonical aldehyde dehydrogenase activity was substantiated through point mutation experiments. Notably, pharmacological autophagy inhibition via hydroxychloroquine partially reversed the radioresistant phenotype induced by ALDH3A1 deficiency.
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