S2522
Radiobiology – Immuno-radiobiology
ESTRO 2026
persistent checkpoint-mediated immunosuppression by avoiding Chk1–STAT3 activation. These findings support FLASH RT as an immune-sparing modality that may enhance the therapeutic ratio and improve compatibility with immunotherapies. Future work will assess tumor-bearing models and combination strategies to translate these benefits clinically. Keywords: FLASH, lymphopenia, immune Digital Poster Highlight 673 SBRT-Induced Immune Modulation: A Comparison of Single and Multifraction Treatments in a Preclinical NSCLC Mouse Model Léa Bouchard 1 , Ryma Haroun 1 , Cleopatra Rutihinda 1 , Myriam Bouchard 2 , Ayman Jafar Oweida 1 1 Department of Medical Imaging and Radiation Sciences, Université de Sherbrooke, Sherbrooke, Canada. 2 Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Sherbrooke, Canada Purpose/Objective: SBRT is the standard of care for early-stage, node negative, inoperable NSCLC1. Yet, how different SBRT fractionation regimens shape the tumor microenvironment and influence subsequent response to immunotherapy remains poorly defined in preclinical models2. This study aims to compare the immune effects of single versus multifraction SBRT in a subcutaneous NSCLC mouse model to identify potential synergy with immune checkpoint inhibitors and guide current clinical studies1. Material/Methods: Mice bearing subcutaneous Lewis lung carcinoma (LLC) tumors were irradiated using a small-animal image-guided radiation platform. Experimental groups received either 30Gy/1 fraction (SF-SBRT) or 48Gy/4 fractions (MF-SBRT), two fractionation schedules used for patients in our institution. Tumor volume and survival of treatment groups were compared with controls using post-hoc Tukey’s multiple comparisons and log-rank tests, respectively. Flow cytometry was performed seven days after irradiation to characterize local and systemic T cell responses in tumor and spleen tissues. In the MF-SBRT group, immune effects were also quantified at different timepoints, and a tumor rechallenge was performed in mice with complete tumor regression. Differences among T cell subsets were assessed using one-way ANOVA or unpaired t-tests, with significance set at p < 0.05. Results: Both SF and MF-SBRT regimens significantly reduced tumor size compared to controls, with a stronger effect observed for MF-SBRT. Upon rechallenge, secondary tumor growth rates did not significantly
Poster Discussion 83 Thoracic FLASH Radiotherapy Mitigates Radiation- Induced Lymphopenia and Sustained Immunosuppression via Chk1–STAT3 Pathway Kevin Liu 1 , Ronghua Tao 1 , Edgardo Aguilar 2 , Min Wang 1 , Sadhna Arrgawal 1 , Denae Neill 2 , Brett Velasquez 2 , Sam Beddar 2 , Albert Koong 1 , Radhe Mohan 2 , Emil Schueler 2 , Steven Lin 1 1 Radiation Oncology, MD Anderson Cancer Center, Houston, USA. 2 Radiation Physics, MD Anderson Cancer Center, Houston, USA Purpose/Objective: Radiation-induced lymphopenia (RIL) is a common side-effect of conventional (CONV) dose-rate radiotherapy (RT) due to the extreme radiosensitivity of circulating lymphocytes. Ultra-high dose-rate (FLASH) RT can spare normal tissues while maintaining tumor control. We evaluated whether thoracic FLASH RT mitigates RIL, apoptosis, and downstream immunosuppression versus CONV RT for single- and multi-fraction irradiations. Material/Methods: C57BL/6 mice received thoracic RT as either single- fraction 17 Gy or multi-fraction 2 Gy × 5 using FLASH or CONV delivery on a Mobetron 9-MeV electron linac. Longitudinal peripheral blood was collected for flow cytometry of CD3 ⁺ , CD4 ⁺ , CD8 ⁺ , NK, and CD19 ⁺ cells; apoptotic cells were quantified by Annexin V. Immune checkpoint modulation was assessed by PD-1/PD-L1 expression and regulatory T cells at day 3, day 24, and months 2 and 5. Lung and heart fibrosis were scored at 6 months (Masson’s Trichrome). Mechanistic studies in spleen included immunofluorescence, Western blotting of Chk1 and STAT3 signaling. Statistics used two-tailed t-tests (p<0.05). Results: Across both fractionation schemes (17 Gy x 1 and 2 Gy x 5), FLASH RT significantly reduced acute lymphocyte apoptosis (1.3–6.1 × lower vs CONV) and accelerated recovery of CD4 ⁺ , CD8 ⁺ , CD3 ⁺ , NK, and B cells to near- baseline (by day 24–31), yielding higher counts than CONV at multiple time points (p<0.05). FLASH RT also markedly decreased late normal-tissue injury, with 4– 5 × less lung and heart fibrosis than CONV (p<0.0001). Whereas CONV RT induced sustained immunosuppression—elevated PD-1/PD-L1 on T and B cells and increased regulatory T cells at 2 and 5 months—FLASH maintained levels comparable to controls (p<0.05 vs CONV). Mechanistically, CONV—but not FLASH—activated the Chk1–STAT3 axis, coinciding with PD-1/PD-L1 upregulation; FLASH samples showed minimal p-Chk1/p-STAT3 signaling. Conclusion: Thoracic FLASH RT mitigates RIL, limits apoptosis, accelerates immune reconstitution, and prevents
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