{"doi":"10.1101/2025.10.21.683770","title":"FLASH Radiotherapy Mitigates Radiation-Induced Lymphopenia and Prevents Immunosuppression via Chk1-STAT3 Axis Modulation in a Preclinical Thoracic Irradiation Model","abstract":"Abstract Background and Aims Radiation-induced lymphopenia (RIL) is a frequent side effect of conventional radiation therapy (CONV RT), due to the high radiosensitivity of circulating lymphocytes. Ultra-high dose rate “FLASH” RT may preferentially spare normal tissue while maintaining tumor control. This study evaluates the impact of single-fraction and multi-fraction thoracic FLASH RT on lymphocyte preservation, apoptosis, and immunosuppressive signaling in mice. Methods We compared the immunological impact of thoracic FLASH RT and CONV RT in C57BL/6 mice using single-fraction (17 Gy) and multi-fraction (2 Gy × 5) regimens using the Mobetron (IntraOp). Longitudinal blood sampling was performed at multiple time-points post-irradiation through facial vein bleed with flow cytometry analysis for CD4+, CD8+, CD19+, and NK cells to assess lymphocyte counts, apoptotic lymphocytes through Annexin V staining, and immune suppression by examining regulatory T cells (Tregs) and PD-1/PD-L1 expression. Mechanistic studies included immunofluorescence and Western blot analyses of splenic tissues to evaluate Chk1 and STAT3 signaling pathways. Results In single-fraction RT, FLASH significantly reduced lung and heart fibrosis (p < 0.0001) at 28 weeks post-RT. The FLASH effect was also seen acutely on circulating immune cells, with significantly reduced lymphocyte apoptosis and accelerated recovery of CD4 ⁺ , CD8 ⁺ , CD3 ⁺ , NK, and B cell populations compared to CONV RT in both single-fraction and multi-fraction regimens. Conversely, CONV RT induced long-lasting increases in Tregs and sustained PD-1 and PD-L1 expression on T- and B-cells at 2- and 5-months post-irradiation in both fractionation regimens. Within the spleen, we also found CONV RT induced sustained activation of the Chk1–STAT3 pathway in CD45+ immune cells, which correlates with increased PD-1/PD-L1 expression. Conclusion FLASH RT mitigates RIL, reduces lymphocyte apoptosis, and prevents long-term immunosuppression by reduced activation of the Chk1–STAT3 pathway. These findings suggest FLASH RT may confer immunological advantages over CONV RT to enhance therapeutic efficacy.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":578900,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9613,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":560488,"name":"Kevin Liu","orcid":"0000-0002-0942-6513","position":1,"is_corresponding":false},{"id":1326335,"name":"Edgardo Aguilar","orcid":null,"position":2,"is_corresponding":false},{"id":1488753,"name":"Min Wang","orcid":"0000-0003-2052-199X","position":3,"is_corresponding":false},{"id":1361064,"name":"Sadhna Aggarwal","orcid":"0000-0002-4646-9352","position":4,"is_corresponding":false},{"id":1054681,"name":"Denae Neill","orcid":null,"position":5,"is_corresponding":false},{"id":1052494,"name":"Brett Velasquez","orcid":null,"position":6,"is_corresponding":false},{"id":587254,"name":"Sam Beddar","orcid":"0000-0001-5825-110X","position":7,"is_corresponding":false},{"id":1489152,"name":"Albert C Koong","orcid":null,"position":8,"is_corresponding":false},{"id":409471,"name":"Radhe Mohan","orcid":"0000-0003-3746-4557","position":9,"is_corresponding":false},{"id":487622,"name":"Emil Schüler","orcid":null,"position":10,"is_corresponding":false},{"id":236019,"name":"Steven H. Lin","orcid":"0000-0003-4411-0634","position":11,"is_corresponding":false},{"id":1489151,"name":"Rong-Hua Tao","orcid":null,"position":0,"is_corresponding":true}],"reference_count":73,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:58:24.957414Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}