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Background Focused ultrasound (FUS) is rapidly redefining non-invasive oncology by depositing precise, non-ionizing acoustic energy deep within solid tumors. 1 2 In the thermally ablative regime (T-FUS), it can simultaneously cytoreduce tumor burden and ignite local immune activation, making it an attractive backbone for antibody-based immunotherapies. Yet therapeutic success depends on hitting a ‘Goldilocks’ thermal window: too little heat fails to provoke immunogenic remodeling, while excessive ablation collapses the vascular pathways antibodies need for tumor entry. To quantify this balance, we utilize a theranostic immunoPET strategy to chart the spatiotemporal kinetics of a model tumor-targeted checkpoint antibody (αCD47) following partial T-FUS. By defining the thermal dose that maximizes both tumor disruption and antibody penetrance, our study provides a quantitative roadmap for rational combination regimens.Methods Solid tumor (4T1)-bearing mice underwent T-FUS with a custom ultrasound-guided FUS system ( figure 1A). Shear wave elastography (SWE) was performed before and after T-FUS, and on days 2 and 5 post-treatment using a Philips EPIQ scanner. Following T-FUS, mice received intravenous (i.v.) injection of [18F]-Fludeoxyglucose(FDG), followed one day later by a single i.v. injection of [89Zr]-αCD47 and serial PET/CT imaging. Terminal ex-vivo biodistribution analysis was performed and quantified via automated gamma counter (figure 1C).Results B-mode ultrasound imaging revealed hyperechoic signatures, a common qualitative indicator of ablation, immediately following T-FUS ( figure 1B). This was corroborated by FDG-PET scans revealing a significant reduction in tumor metabolic activity acutely following ablation (figure 1D). T-FUS induced an acute drop in tumor stiffness, as measured SWE, which normalized to sham levels within 48 hours – a transient mechanical softening that may define a therapeutic window for enhanced antibody delivery prior to microenvironmental reconstitution(figure 1E-F). [89Zr]-αCD47 PET revealed modestly reduced antibody uptake in ablated tumors at early time points, with gradual recovery by Day 3 post-ablation(figure 2A-B). At this time point, ex-vivo biodistribution confirmed significantly lower intratumoral antibody levels in the T-FUS group (figure 2C-D), with unchanged peripheral organ distribution(figure 2E-K). These results suggest that T-FUS does not preclude tumor access for CD47 blockade and underscore the importance of optimizing treatment timing to exploit a reversible delivery window.Conclusions A theranostic PET approach revealed that T-FUS can impact but is not deleterious to tumor-targeted antibody penetrance, herein demonstrated with [89Zr]-αCD47. Interestingly, a steady increase in intratumoral [89Zr]-αCD47 accumulation was noted over days subsequent to T-FUS exposure, provoking future consideration of windows for optimal tumor-drug exposure in T-FUS paradigms. Future studies will expand findings to other solid tumor and immunotherapy settings.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.References Demir ZEF, Sheybani ND. Therapeutic ultrasound for multimodal cancer treatment: a spotlight on breast cancer. Annual Review of Biomedical Engineering 2025;27:371–402.DeWitt M, Demir ZEF, Sherlock T, Brenin DR, Sheybani ND. MR imaging-guided focused ultrasound for breast tumors. Magnetic Resonance Imaging Clinics 2024;32:593–613.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.Abstract 655 Figure 1T-FUS reduces tumor metabolic activity and stiffness as determined by PET/CT imaging with [18F]-FDG imaging and shear wave elastographyAbstract 655 Figure 2ImmunoPET reveals differences in intratumoral [89Zr]-aCD47 accumulation over time. Ex vivo biodistribution analysis corroborates a modest local decrease in [89Zr]-aCD47 in ablated tumors, with no appreciable changes in off-target peripheral organs