Abstract P002: FLASH radiotherapy enhances immune activation and spares lymphocytes tumor draining lymph nodes
Authors: Saenz Francisco , Velasquez Brett , Delahoussaye Abagail , Connell Luke , Mims Nefetiti , Neill Denae , Schüler Emil , Spiotto Michael 
Published: January 2025

Abstract
Introduction: Conventional radiotherapy (CRT) causes antagonizing anti-tumor immune responses by depleting circulating immune cells. Given the normal tissue sparing associated with FLASH radiotherapy (FLASH-RT), we hypothesized that FLASH-RT rescues naïve lymphocytes while increasing the immunogenicity of cancer cells. Methods: Murine cancer cell lines TC-1 and MEER as well as the human cancer cell lines Caski, SiHA and SCC153 were irradiated using a Mobetron (IntraOp, Sunnyvale, CA) under FLASH-RT at 270.0 Gy/sec (3.00 Gy per pulse ) or CRT 0.209 Gy/sec dose rates. MEER tumors in immunocompetent C56BL/6 mice were irradiated with 27 Gy in 3 fractions. We assessed expression of immunogenic proteins calreticulin (CALR) and HMGB1 using immunofluorescence and cGAS, STING, IFNalpha and IFNbeta using qRT-PCR. Lymph node immune cells were assessed for CD8, CD4, CD62L and CD44 to assess lymphocyte subpopulations and activation. Tumors were assessed for CD3, CD8, PD1, PD-L1, F4/80 to assess immune cell infiltration 5 days after radiotherapy. Two-way ANOVA and Student’s t-test were used for paired-wise comparisons. Results: Both human and murine positive cell lines showed similar upregulation of CALR and HMGB1 and cGAS-STING family members after FLASH-RT or CRT in vitro. One cell line, TC-1, demonstrated increased expression Hmgb1 (4.2 vs 1.0-fold; p&lt;0.001 ), cGAS (1.9 vs 1.0-fold; p&lt;0.001 ) and STING (2.2 vs 0.9-fold; p&lt;0.001 ) 24h after irradiation to 9 Gy or 12 Gy using FLASH-RT. In mice bearing MEER tumors, draining lymph nodes contained greater CD8+ T cells (P&lt;0.001) and CD4+ T cells (p&gt;0.01) in tumors irradiated with FLASH-RT compared to tumors irradiated with CRT. FLASH-RT was associated with increased CD44+CD62Llo CD8+ (P&lt;0.01) and CD4+ (P&lt;0.05) lymphocytes indicating increased activated T cells in FLASH-RT treated tumors compare to CRT treated tumors. In irradiated tumors, FLASH-RT was associated with increased CD8+ tumor infiltrating lymphocytes, increased PD-1 expression on CD8+ tumor infiltrating lymphocytes and increased PD-L1 on macrophages (P&lt;0.001). Conclusion: Compared to CRT, FLASH-RT spared activated T cells in tumor draining lymph nodes and in tumors but increased checkpoint inhibitor expression in tumors. These results suggest that FLASH-RT may enhance anti-tumor immune responses by maintaining the immunogenic effects of radiotherapy that can be augmented with immune checkpoint blockade.
Citation Format: Francisco Saenz, Brett Velasquez, Abagail Delahoussaye, Luke Connell, Nefetiti Mims, Denae Neill, Emil Schüler, Michael Spiotto. FLASH radiotherapy enhances immune activation and spares lymphocytes tumor draining lymph nodes. [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P002