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Background Advances in immunotherapies offer a promising therapeutic strategy for cancer, yet the lack of a ubiquitous response in breast cancer(BC) remains a problem—particularly concerning given the rising incidence among younger populations. Toward improved immunological control of BC with minimal trade-off in safety or toxicity, focused ultrasound (FUS) is a uniquely disruptive tool for non-invasive, non-ionizing acoustic energy deposition into tumors with submillimeter precision. 1 FUS-mediated thermal ablation(T-FUS) presents a multi-pronged, tunable strategy for tandem partial non-surgical tumor debulking and immunomodulation.2–4 We here investigate combination of T-FUS with CD40 agonism(αCD40) in preclinical BC models and probe mechanisms of T cell-mediated immunity and complete response therein.Methods An αCD40 priming regimen was initiated one week prior to T-FUS in murine mammary carcinomas (E0771, BRPKP110, EMT-6, 4T1). Subtotal T-FUS was performed with a custom ultrasound-guided FUS system ( figure 1A). Tumor outgrowth and survival were monitored, yielding stratifications of progressive disease, stable disease, and complete response. Complete responders (CRs) underwent contralateral tumor rechallenge alongside age-matched, naïve wild-type controls. Flow cytometry was performed on tissue and serial blood samples.Results Qualitatively, tumors exhibited evidence of increased cleaved caspase 3, poly(ADP-ribose) polymerase(PARP), and heat shock protein 70(HSP70) expression one day post-T-FUS( figure 1B-E). Furthermore, in-vivo assessment of ATP release revealed a sustained increase following T-FUS, as evaluated by bioluminescence signal (figure 1F-G). By the time point of T-FUS intervention, αCD40 priming increased intratumoral T cells and their respective expression of CD40L (figure 1H-K), as well as representation of antigen-presenting cells in the tumor-draining lymph node(TDLN; figure 1L-O). Combination of T-FUS+αCD40 elicited superlative growth control and survival benefits compared to controls-yielding 33% complete response rate in E0771-treated tumors (figure 2A-H). By CD8 and/or CD4 antibody depletion, this protective effect was determined to be dependent on T cells (figure 2I-J). TDLN of CRs exhibited a distinct T cell architecture in comparison to non-responders, with an increased percentage of CD4+ and CD8+ T cells (figure 2K-M). CRs remained protected upon contralateral primary tumor rechallenge (figure 2N), suggestive of immune-mediated that was probed further via serial blood sampling. CRs displayed an elevation in circulating CD44+,CD62L− subset of CD4+ T cells following rechallenge (figure 2O).Conclusions T-FUS+αCD40 represents a novel paradigm for immunotherapy of BC-capable of conferring complete responses underscored by evidence of immunological memory. This study, utilizing a clinically ripe therapeutic platform, offers timely insights toward translation of T-FUS for breast immuno-oncology applications. Ongoing studies include immunoPET imaging and blood biomarker assessments for forecasting responder/non-responder stratifications in the T-FUS+αCD40 setting.Acknowledgements This work was supported by NIH DP5OD031846 and T32CA009109, DOD BCRP Era of Hope Scholar Award, Wallace H. Coulter Foundation for Translational Research, UVA Victor Orphan Endowed Fellowship, and the UVA Cancer Center Trainee Fellowship.References Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomatarm I, Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA A Cancer J Clinicians 2024;74:229–263.Arnold M, Morgan E, Rumgay H, Mafra A, Singh D, Laversanne M, Vignat J, Gralow JR, Cardoso F, Siesling S, Soerjomatarm I. Current and future burden of breast cancer: global statistics for 2020 and 2040. The Breast 2022;66:15–23.Yedjou CG, Sims JN, Miele L, Noubissi F, Lowe L, Fonseca DD, Alo RA, Payton M, Tchounwou PB. Health and racial disparity in breast cancer. Advances in Experimental Medicine and Biology 2019;1152:31–49.Trayes KP, Cokenakes SE. Breast cancer treatment. American Family Physician 2021;104:171–178.Ethics Approval All mouse experiences were conducted in accordance with the guidelines and regulations of the University of Virginia and approved by the University of Virginia Animal Care and Use Committee.Abstract 656 Figure 1Subtotal T-FUS regime in BC yields clear ablative transition zones and immunogenic signatures, while αCD40 priming promotes local and systemic immunityAbstract 656 Figure 2T-FUS combined with CD40 agonism shows efficacy across multiple preclinical BC models and protection is T cell-dependent. Complete responders (CRs) from the T-FUS+αCD40 group remain protected following rechallenge