2835 - Exploration of the Effectiveness and Mechanism of Reversal of Radiotherapy Resistance in Head and Neck Squamous Cell Carcinoma by High-Energy X-Ray Based FLASH Radiotherapy

H. S. Li¹, R. Tang², Z. J. Qin¹, X. Y. Zhang², Z. Wei¹, Z. R. Liu¹, L. Zhu¹, Z. Zhang², and X. Peng¹; ¹Department of Biotherapy, Cancer Center, West China Hospital of Sichuan University, Chengdu, China, ²The Institute of Fluid Physics, China Academy of Engineering Physics, Mianyang, China

Purpose/Objective(s): Radiotherapy resistance in head and neck squamous cell carcinoma (HNSCC) severely affects the prognosis and quality of life of patients. The purpose of this study was to evaluate the efficacy and underlying mechanism of high-energy X-ray-based FLASH radiotherapy (FLASH-RT) in reversing radiotherapy resistance of HNSCC. Materials/

Methods: Radioresistant cell and animal models established by our team were irradiated with FLASH-RT (88 Gy/s) or conventional RT (Conv-RT) (3.095 Gy/min) using a high-power petal accelerator independently developed by the Chinese Academy of Engineering Physics. Radiosensitive groups were also included as controls. Cellular experiments included cell counting kit assays, wound healing assays, and transwell assays to evaluate malignant phenotypes, and live/dead fluorescence staining, apoptosis assays, ?-H2AX detection, and JC-1 mitochondrial membrane potential detection to evaluate cell viability and damage. In animal experiments, Ki-67 staining, Tunel staining, and ?-H2AX detection were used to assess tumor proliferation and DNA damage repair, while immunofluorescence (IF) was used to evaluate tumor immune microenvironment (TIME). Transcriptome sequencing was performed to elucidate the underlying mechanism.
Results: Dose calibration and irradiation pendulum were performed, and the irradiation dose was stable (target dose ± 0.3 Gy) and uniform (within 2.0 × 2.0 cm²). Cellular experiments confirmed that FLASH-RT significantly inhibited the proliferation, migration and infiltration of cells in the radioresistant group, while increasing the degree of apoptosis and DNA damage (p <0.05), compared with Conv-RT. Interestingly, these differences were not observed in the radiosensitive group. Animal experiments also confirmed that FLASH-RT significantly inhibited tumor growth and increased apoptosis and DNA damage (p <0.05) in the radiotherapy-resistant background compared with Conv-RT. Furthermore, transcriptome sequencing indicated that FLASH-RT significantly upregulated immune-regulatory molecules and pathways and downregulated DNA damage repair-related pathways (especially homologous recombination [HR] repair pathway) in radioresistant tumors (p_adj <0.05). The IF analyses also confirmed that the proportion of CD8+ T cells and ?-H2AX-positive cells was significantly increased in the radioresistant group (p <0.05), suggesting activation of the TIME and down-regulation of DNA damage repair.
Conclusion: This study demonstrates the significant efficacy of FLASH-RT in reversing radiotherapy resistance in HNSCC. The mechanism may involve upregulation of immune-related pathways, enhancement of antigen presentation, T cell activation, and activation of the TIME, while downregulating tumor DNA damage repair pathways, particularly the HR repair pathway. These findings provide experimental and theoretical support for the application of X-ray-based FLASH-RT in the treatment of radioresistant HNSCC.