Natasha Diba Sheybani

The blood‐brain and blood‐tumor barriers impede therapeutic delivery to glioblastoma (GBM). Furthermore, dysfunctional endothelial cells in GBM enable an immunosuppressive tumor microenvironment (TME) and enhance therapeutic resistance. However, the significance of endothelial dysfunction for GBM progression also positions the endothelium as a rich target for gene therapy. Here, we leveraged the presence of exofacial thiols (SH) on cells within the TME and developed polymeric, plasmid‐bearing, densely PEGylated, polyethyleneimine nanoparticles (NPs) functionalized with free thiol groups to facilitate targeting and transfection (SH‐NPs). Delivering SH‐NPs to GBM with focused ultrasound (FUS) elicited exceptionally high tropism for GBM endothelium (>70% efficiency), significantly surpassing that achieved via convection‐enhanced delivery (CED). “Sonoselective,” endothelial cell‐specific, transfection was then achieved by incorporating the CD144 promoter. Using this system, we delivered a CXCL9 plasmid to GBM endothelium. This intervention enhanced CD8 ⁺ effector and CD4 ⁺ helper T cell representation in the TME and significantly improved responsiveness of the tumor to aPD1 checkpoint inhibition. Broadly considered, this sonoselective non‐viral transfection platform may be adapted to multiple therapeutic strategies. Further, the approach holds an advantage over AAVs with tropism for brain endothelial cells due to the ability of FUS to provide locoregional targeting specifically to brain tumor endothelium.

Background: Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we investigated whether subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across immunologically and molecularly distinct murine BC models. Methods: Subtotal ultrasound-guided T-FUS was applied to E0771, BRPKP110, EMT6, and 4T1 tumors in mice treated with systemic agonistic αCD40. Histology, ATP bioluminescence imaging, and multispectral flow cytometry were used to define acute tumor injury and immune remodeling. Therapeutic efficacy was evaluated by tumor growth, survival, T-cell depletion, and contralateral tumor rechallenge. Results: Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4+ and CD8+ T cells. When layered onto this immunogenic milieu, αCD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 30% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Combination treatment also extended survival across three of four models. Efficacy required both CD4+ and CD8+ T cells, as depletion of either compartment abrogated tumor control. Complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T-cell activation. Conclusions: Subtotal T-FUS cooperates with CD40 agonism to drive durable, T cell-dependent BC regression and immunologic memory. These findings establish T-FUS as an immune-potentiating partner for CD40 agonism and position this combination as a clinically scalable, in situ vaccination-like strategy with potential to broaden immunotherapy benefit across breast cancer subtypes, including luminal tumors that remain largely refractory to immune checkpoint blockade.

Background: The blood brain barrier (BBB) limits effective treatment for cerebellar diseases. Region-specific parameterization and safety data for MRI-guided focused ultrasound (MRgFUS) BBB opening (BBBO) in the cerebellum are scarce. The goal of this study is to establish safety and feasibility of cerebellar BBBO for antibody delivery. Methods: Mice received i.v. AF680 IgG and microbubbles immediately prior to sonication at 0.30 or 0.45 MPa peak negative pressure (1.15 MHz, 10 ms bursts, 2 s burst period, 60 bursts). BBBO was quantified by contrast-enhanced T1-weighted MRI (9.4T) and assessed for recovery at 12 h. Radiologic safety was evaluated by T2-weighted MRI. Acoustic emissions were analyzed via real-time passive cavitation detection, and acoustic simulations estimated the derated pressure caused by skull incidence angle. IgG delivery was quantified by ex vivo IVIS epifluorescence imaging and immunofluorescence analysis. Safety assessments included cardio-respiratory monitoring, body weight, motor function, and neuropathology assessment of endpoint H&E. Results: Both regimens yielded cerebellar BBBO without radiologic evidence of injury. BBB integrity was restored by 12 h. While the higher pressure increased MRI and cavitation signals, IgG delivery was similarly enhanced (> 2-fold vs. sham) by FUS, irrespective of peak negative pressure. Heart rate, respiratory rate, body weight, and motor function were unchanged by FUS, and neuropathologic evaluation revealed no hemorrhage, inflammation, vacuolation, or architectural abnormalities. Conclusions: FUS enables safe, transient BBBO in the cerebellum and significantly enhances antibody delivery without measurable physiologic, functional, radiologic, or histologic injury. These findings establish a foundation for expansion of pharmacologic and theranostic delivery strategies directed to the cerebellum.

Focused ultrasound (FUS) combined with micro/nanobubbles enables transient, localized blood brain barrier opening (BBBO) for targeted drug delivery to the brain. Although imaging-guided FUS methods provide accurate targeting, they require specialized infrastructure that increases cost and workflow complexity. Commercial stereotactic FUS platforms such as the RK-50 offer an accessible alternative, but standard procedures rely on a scalp incision to identify bregma for atlas registration, introducing tissue injury and potential confounds for repeat-BBBO studies. Here, we describe and validate a fully non-invasive targeting approach for FUS BBBO in mice using the RK-50 platform. Our novel targeting method achieved accurate, reproducible targeting without surgical exposure. Evans blue extravasation and atlas co-registration demonstrated sub-millimeter targeting accuracy at bregma and confirmed reliable BBBO in the cortex, hippocampus, and cerebellum. Across these anatomically distinct targets, passive cavitation detection showed broadly comparable acoustic emissions profiles. We further compared this non-invasive workflow with the standard incisional approach in a repeat-BBBO paradigm and observed similar acoustic emissions and comparable delivery of both small molecule and large molecule (antibody) reporters. Together, these findings establish a high-fidelity, incision-free targeting strategy that preserves the accessibility of stereotactic FUS while reducing procedural burden and improving suitability for longitudinal preclinical BBBO studies.

Background: Thermally ablative focused ultrasound (T-FUS) offers a noninvasive, spatially precise strategy for local tumor destruction, with the added potential to remodel tumor architecture and immune dynamics in ways that influence downstream therapeutic delivery and efficacy. Despite promising preclinical and clinical findings, the T-FUS parameters that best balance tumor debulking with preservation of local biologic, e.g. immunotherapy, penetrance remain unclear. Thermal dose, defined by the relationship between tissue heating, exposure duration, and biological effect, is likely a critical determinant of this balance. Excessive thermal dose may eliminate the vascular and stromal features needed to support immunotherapy access, whereas insufficient thermal dose may fail to achieve meaningful cytoreduction. Here, we deploy multimodal PET, contrast-enhanced ultrasound, and tissue profiling to define a “Goldilocks Zone” for T-FUS that balances bulk tumor destruction with immunotherapy delivery. Method: Subtotal T-FUS was applied to 4T1 tumors using three thermal dose regimens resolved by in silico modeling. Ablation was quantified by H&E and TTC staining. Post-ablative perfusion and microvascular coverage were assessed by contrast-enhanced ultrasound and immunofluorescence, respectively. Tumor oxygenation was measured by intravenous hypoxyprobe labeling. After T-FUS, mice underwent dynamic [ ¹⁸ F]-FDG PET and immunoPET with a model tumor-targeted antibody, [ ⁸⁹ Zr]-αCD47, to relate cytoreduction to antibody penetrance. ImmunoPET findings were further evaluated by ex vivo biodistribution analysis. Results: In silico modeling established three T-FUS regimens that generated distinct thermal dose profiles and were deployed in vivo in a solid breast tumor model. Histopathology, perfusion imaging, and hypoxia analysis revealed dose-dependent and dose-divergent biological effects that informed a candidate Goldilocks thermal window. Low thermal dose produced measurable but limited tumor debulking, whereas high thermal dose caused disproportionate functional perfusion collapse. An intermediate thermal dose achieved robust partial ablation, broad hypoxia relief, and preservation of residual tumor physiology sufficient to support antibody access. Dynamic [ ¹⁸ F]-FDG PET confirmed a marked reduction in metabolically active tumor burden after Goldilocks T-FUS. Serial [ ⁸⁹ Zr]-αCD47 immunoPET showed that bulk antibody signal was maintained after ablation, and integration of immunoPET with matched [ ¹⁸ F]-FDG PET revealed approximately 3-fold enrichment of antibody exposure within the residual viable tumor compartment of ablated tumors. These findings demonstrate that appropriately tuned thermal ablation can debulk tumor while preserving, and potentially concentrating, immunotherapy access within the remaining targetable tumor niche. Conclusion: This study identifies thermal dose as a critical consideration for T-FUS immunotherapy combinations and establishes a PET-informed framework for balancing cytoreduction with therapeutic delivery. Rather than functioning solely as a local debulking modality, we demonstrate that T-FUS can be tuned to yield a post-ablation tumor state that remains accessible to large biologics. These findings provide timely, translationally relevant guidance for tailoring T-FUS regimens to achieve local tumor destruction while preserving an immunotherapy-permissive niche for combination treatment.

Combining positron emission tomography (PET) with magnetic resonance imaging (MRI) and focused ultrasound (FUS) has emerged as a promising hybrid technique in the medical imaging field. Despite the potential benefits of using simultaneous PET and MRI acquisitions to monitor therapeutic effects and ensure precision and safety during FUS applications, no commercial or academic PET/MRI-guided FUS system (referred in the following as trimodal PET-MRI-FUS) exists. This work presents the design and evaluation of a preclinical PET insert for simultaneous operation with MRI and FUS systems. The proposed PET insert is based on monolithic LYSO crystals arranged in two octagonal rings that define inner and outer diameters of 72 and 114 mm, respectively, with an axial length of 67 mm. The system performance was evaluated according to the NEMA NU 4 2008 protocol. It achieves a uniform spatial resolution of ~0.9 mm along the whole PET Field of View (FOV) when enabling Depth of Interaction (DOI) capabilities. Moreover, we found a sensitivity peak of 3.8% for an energy window of 255–766 keV and a peak noise equivalent count rate peak of 80 kcps at an activity of 19 MBq. The compatibility of the PET insert with both low- and high-field MRI systems, as well as with custom and commercially available FUS devices, was studied using phantoms and in vivo experiments, respectively. The results of the trimodal tests have proven that the PET insert works simultaneously with both MRI and FUS systems.

Cancers of the brain remain notoriously difficult to treat because of the unique and complex barriers imposed by the central nervous system and tumor microenvironment. Current therapeutic paradigms remain largely insufficient to achieve durable responses or cures. Cellular immunotherapies (CIs), including chimeric antigen receptor (CAR) T-cells and natural killer cell therapies, represent a new class of “living drugs” that harness endogenous or adoptively transferred immune cells to target and destroy cancer. Although transformative in hematologic malignancies, CIs have faced substantial barriers in solid tumors, particularly in the brain. Focused ultrasound (FUS), a noninvasive, nonionizing, and highly tunable platform for transcranial acoustic intervention, may help address these challenges through blood-brain barrier opening, vascular activation, immunologic reprogramming, and sonogenetic control. In this review, we systematically survey the current literature at the intersection of FUS and CIs in cancer, with emphasis on brain tumors and highlight key evidence supporting combinatorial strategies, immuno-imaging approaches for CI surveillance, and emerging translational opportunities—including a first-in-human clinical investigation in glioblastoma. We conclude with perspectives on how FUS may help enable safer, more effective, and more precise CI paradigms in neuro-oncology.

Focused ultrasound thermal ablation (T-FUS) is a clinically accessible, non-invasive modality capable of inducing rapid tumor cytoreduction while mobilizing early immunologic danger signals. However, its capacity to synergize with potent co-stimulatory immunotherapies in breast cancer (BC) remains undefined. Here, we demonstrate that subtotal T-FUS cooperates with CD40 agonism to elicit durable, T cell-dependent tumor control across four immunologically and hormonally distinct murine BC models. Partial thermal ablation triggered canonical immunogenic cell-death signatures and acute remodeling of intratumoral myeloid populations, while expanding circulating CD4 ⁺ and CD8 ⁺ T cells. When layered onto this immunogenic milieu, αCD40 markedly constrained tumor outgrowth, yielding significant reductions in tumor burden across all models and complete tumor eradication in 33% of E0771 tumors, with additional complete responses in BRPKP110 and EMT6. Efficacy required both CD4 ⁺ and CD8 ⁺ T cells, and complete responders mounted robust systemic immunity, rejecting contralateral tumor rechallenge with 100% protection and displaying persistent effector-memory T cell activation. Together, these findings establish T-FUS as an immune-potentiating partner for CD40 agonism, capable of driving durable, robust BC regression and immunological memory. This work positions T-FUS+CD40 agonism as a clinically scalable, in situ vaccination-like strategy with potential to benefit breast cancers, including luminal subtypes, that remain largely refractory to immune checkpoint blockade.

Cancer remains a leading cause of mortality worldwide, and the demand for improved efficacy, precision, and safety of management options has never been greater. Focused ultrasound (FUS) is a rapidly emerging strategy for nonionizing, noninvasive intervention that holds promise for the multimodal treatment of solid cancers. Owing to its versatile array of bioeffects, this technology is now being evaluated across preclinical and clinical oncology trials for tumor ablation, therapeutic delivery, radiosensitization, sonodynamic therapy, and enhancement of tumor-specific immune responses. Given the breadth of this burgeoning domain, this review places a spotlight on recent advancements in breast cancer care to exemplify the multifaceted role of FUS technology for oncology indications—outlining physical principles of FUS-mediated thermal and mechanical bioeffects, giving an overview of results from recent preclinical and clinical studies investigating FUS with and without adjunct therapeutics in primary or disseminated breast cancer settings, and offering perspectives on the future of the field.