S2528
Radiobiology – Immuno-radiobiology
ESTRO 2026
Digital Poster Highlight 2125 Opposing two-fraction regimens combining low and high doses of ionizing radiation elicit differential immune responses Irene Vetrugno 1 , Irma Telarovic 2 , Alba Sanchez- Fernandez 1 , Nathan Torelli 1 , Jan Unkelbach 1 , Martin Pruschy 1 1 Radiation Oncology, University of Zurich, Zurich, Switzerland. 2 Radiation Oncology, University Hospital Zurich, Zurich, Switzerland Purpose/Objective: Radiotherapy exerts direct cytotoxic effects on cancer cells, but also induces immunogenic responses in the tumor microenvironment, eliciting both immune stimulatory and immune suppressive dynamics. These effects are influenced by fraction size, fractionation regimen and timing of radiotherapy-immunotherapy co-administration. In this study, two opposing two- fraction regimens were investigated, in which the same cumulative physical dose is applied to the tumor, but low and high dose fractions were administered in opposite order. Material/Methods: Using two murine tumor models (MC38, B16F10-Luc) we investigated how dose sequencing affects the immune dynamics interplay in the tumor microenvironment and characterized the tumor microenvironment in response to low (6 Gy) and high (12 Gy) single doses of ionizing radiation and to two opposing fractionation regimens (6 + 12 Gy versus 12 + 6 Gy). Furthermore, we assessed the effect of the different radiotherapy regimens on tumor growth and survival and strategically combine the two-fraction regimens with an immune checkpoint blockade. Results: We demonstrated that the two opposing fractionation regimens generated distinct tumor microenvironments, depending on the sequence of low and high dose fractions. While the 12 + 6 Gy regimen resulted in a TME enriched with CD8+ T cells with increased effector function, tumors treated with the 6+ 12 Gy regimen exhibited an enhanced proportion of suppressive CD4+ FOXP3+ Tregs, thereby shaping ionizing radiation-induced antitumor immunity. By combining an immune checkpoint blockade with radiotherapy, we effectively counteracted the immune suppressive effect, predominantly associated with the 6+12 Gy regimen. We demonstrated superior tumor control and a strengthened immunological memory in response to this combinatorial approach and corroborated these findings in a secondary tumor model. Conclusion: The sequence of low and high radiation doses impacts the immunologic response and must be carefully
cytometry are employed for TM production, T-cell activation/infiltration and tumor cell killing. Results: We found that siRNA-mediated silencing of CD98hc- associated AATs increased residual γ H2AX foci 24 h post-irradiation in Cal33, FaDu cells; regulated DNA repair signaling in Cal33 and FaDu cells 1–24 h after X- ray exposure and led to intracellular ROS accumulation, which was further enhanced upon RT specifically in CD98hc-depleted cells. A combination of CD98hc-targeted UniCAR-T cell therapy with proton or photon radiotherapy significantly enhances tumor cell killing in the radioresistant and parental HNSCC models. Additionally, proton irradiation combined with CD98hc-TM yields more pronounced viability reduction in the Cal33 RR model. The immunohistochemical analysis of 3D spheroids confirmed that UniCAR-T cells can infiltrate 3D tumor spheroids and this infiltration depends on the presence of the CD98hc-TM. The combination of photon-RT with UniCAR-mediated targeting of CD98hc- TM enhances tumor cell cytotoxicity in relatively radioresistant HNSCC PDTOs compared to immunotherapy alone. Conclusion: CD98hc regulates tumor radioresistance and antitumor immunity. Combining photon or proton irradiation with CD98hc-targeted UniCAR-T based immunotherapy enhances therapeutic efficacy, supporting the development of next-generation radioimmunotherapy for HPV-negative HNSCC. References: 1- Marcus et al. 2021, Charged Particle and Conventional Radiotherapy: Current Implications as Partner for Immunotherapy2- Mirjolet et al. 2021, Impact of proton therapy on antitumor immune response3- Digomann et al. 2019, The CD98 Heavy Chain Is a Marker and Regulator of Head and Neck Squamous Cell Carcinoma Radiosensitivity4- Digomann et al. 2019, SLC3A2/CD98hc, autophagy and tumor radio resistance: a link confirmed5- Köseer et al. 2022, Validation of CD98hc as a Therapeutic Target for a Combination of Radiation and Immunotherapies in Head and Neck Squamous Cell Carcinoma6- Kahya et al. 2021, Amino Acid Transporters on the Guard of Cell Genome and Epigenome Keywords: CD98hc, Proton RT, UniCAR immunotherapy
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