S2530
Radiobiology – Immuno-radiobiology
ESTRO 2026
Germany. 2 German Cancer Consortium (DKTK), Partner-site Munich and German Cancer Research Center (DKFZ), Heidelberg, Germany Purpose/Objective: Janus Kinase inhibitors (JAKi) modulate cytokine pathways that regulate pro- and anti-inflammatory cytokine responses, such as interferon signaling. The therapeutic outcomes vary depending on the disease context and treatment. Beyond their established immunosuppressive role in autoimmune diseases, emerging evidence suggests that JAKi can also exert immunomodulatory effects that synergize with immune checkpoint inhibition (ICI). However, the impact of JAKi in the context of radioimmunotherapy (RIT) remains poorly understood. Material/Methods: We used subcutaneous MC38 colorectal carcinoma and B16.OVA melanoma models to assess how JAKi (Ruxolitinib, JAK1/2 inhibitor) or type I Interferon receptor inhibitor (anti-IFNaR1) affects acute and long- term anti-tumor immune responses after combined RIT. In bilateral tumor models, we evaluated the abscopal effect following RIT (1 × 8Gy, index tumor) with or without concurrent JAKi application, combined with ICI (anti-CTLA-4 or anti-PD-1). A bilateral B16.OVA model further examined abscopal responses and priming of antigen-specific CD8+ T cells via flow cytometry. To evaluate long-term anti-tumor immunity, unilateral models were treated with 3 × 8Gy RT ± ICI to generate long-term survivors. After three months, these mice were rechallenged with the same tumor cells to evaluate memory responses. Two experimental designs were tested: (1) JAKi administered during the initial RIT, and (2) JAKi administered during tumor rechallenge. Peripheral blood lymphocytes were profiled via flow cytometry. Results: In bilateral tumor models, the JAKi or anti-IFNaR1 significantly attenuated systemic anti-tumor immune responses following RIT, resulting in increased abscopal tumor growth in both tumor lines. As expected, IFNaR1 inhibition resulted in almost completely impaired tumor control. In both setups, blood analysis revealed a reduction in tumor-specific CD8+T cells (OVA+), characterized by decreased proliferation (Ki-67high) and diminished IFN- γ production, indicating impaired anti-tumor immunity. For long-time survivors, the timing of JAKi relative to RIT proved critical: JAKi administration during tumor rechallenge impaired tumor regression, whereas concurrent JAKi treatment during the initial RIT did not compromise established immunity. Conclusion: JAKi and anti-IFNaR1 negatively affect tumor antigen- specific immunity during initial RIT. Interestingly, JAKi limited existing anti-tumor immunity when
regard is how cold tumors can be sensitized to ICI. For this purpose, we established orthotopic mouse models of hot and cold tumors to test different ICI regimens in combination with radiation therapy (RT). Material/Methods: In vivo studies were conducted using an organoid- based orthotopic model of rectal cancer. Murine tumor organoids were generated from two sources: (I) primary tumors derived from genetically engineered mice (Vil-Cre; KrasLSL-G12D/+), and (II) ex vivo- generated tumor organoids obtained from normal tissue of genetically engineered mice (R26-CreERT2, Apcfl/wt, KrasLSL-G12D/+, Trp53fl/fl). The degree of immune infiltration into tumor tissue was determined by immunohistochemistry (IHC). Mice received fractionated RT consisting of 5 x 5 Gy RT administered on five consecutive days. Additionally, experimental groups received ICI treatment (anti-CTLA-4, anti-PD-1, or their combination) for two weeks. Disease progression was monitored using a clinical scoring system. Results: Histopathological assessment of rectal tumor lesions after orthotopic transplantation of organoids revealed lines that mimic hot tumors and lines that mimic cold tumors. RT in combination with anti-PD-1 successfully improved survival in mice bearing hot tumors. On the contrary, this therapy regimen had no effect on cold tumors. The cold tumors also showed resistance to RT combined with anti-CTLA-4. Strikingly, the combination of 5 x 5 Gy RT, anti-CTLA-4, and anti-PD-1 resulted in markedly improved tumor control and prolonged survival in mice with low immune infiltration. Conclusion: With this data, we demonstrated that depending on the transplanted tumor organoid line, our orthotopic rectal cancer model accurately replicates key features of the patients' disease, specifically the ability to recapitulate both hot tumors and cold tumors. Using this model, we showed that combining RT with dual ICI may be a promising clinical strategy to improve the outcomes of cold rectal tumors. Keywords: rectal cancer; cold tumors; immunotherapy Timing of Janus kinase inhibition determines anti- tumor immune outcomes following radiotherapy combined with immune checkpoint inhibition Sarah Diederich 1 , Julia Gissibl 1 , Vincent R. Timnik 1 , Nicole A. Schmid 1 , Maximilian Giller 1 , Sophie M. Nefzger 1 , Andreas Zoeschg 1 , Hannah Felchle 1 , Caroline N. Walther 1 , Stephanie E. Combs 1,2 , Julius C. Fischer 1 1 Department of Radiation Oncology, Klinikum Rechts der Isar, TUM-School of Medicine and Health, Munich, Digital Poster Highlight 2192
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