Profiling of the immune landscape in murine glioblastoma following blood brain/tumor barrier disruption with MR image-guided focused ultrasound.

PURPOSE: Glioblastoma (GB) poses formidable challenges to systemic immunotherapy approaches owing to the paucity of immune infiltration and presence of the blood brain/tumor barriers (BBB/BTB). We hypothesize that BBB/BTB disruption (BBB/BTB-D) with focused ultrasound (FUS) and microbubbles (MB) increases immune infiltration in GB. As a prelude to rational combination of FUS with ITx, we herein investigate the impact of localized BBB/BTB-D on innate and adaptive immune responses in an orthotopic murine GB model. METHODS: Mice with GL261 gliomas received i.v. MB and underwent FUS BBB/BTB-D (1.1 MHz, 0.5 Hz pulse repetition frequency, 10 ms bursts, 0.4-0.6 MPa). Brains, meninges, and peripheral lymphoid organs were excised and examined by flow cytometry 1-2 weeks following FUS. RESULTS: The number of dendritic cells (DC) was significantly elevated in GL261 tumors and draining cervical LN in response to sonication. CD86 + DC frequency was also upregulated with 0.6 MPa FUS, suggesting increased maturity. While FUS did not significantly alter CD8 + T cell frequency across evaluated organs, these cells upregulated checkpoint molecules at 1 week post-FUS, suggesting increased activation. By 2 weeks post-FUS, we noted emergence of adaptive resistance mechanisms, including upregulation of TIGIT on CD4 + T cells and CD155 on non-immune tumor and stromal cells. CONCLUSIONS: FUS BBB/BTB-D exerts mild, transient inflammatory effects in gliomas-suggesting that its combination with adjunct therapeutic strategies targeting adaptive resistance may improve outcomes. The potential for FUS-mediated BBB/BTB-D to modify immunological signatures is a timely and important consideration for ongoing clinical trials investigating this regimen in GB.

Pediatric ocular nanomedicines: Challenges and opportunities.

The eye is a highly complex, yet readily accessible organ within the human body. As such, the eye is an appealing candidate target for a vast array of drug therapies. Despite advances in ocular drug therapy research, the focus on pediatric ocular drug delivery continues to be highly underrepresented due to the limited number of degenerative ocular diseases with childhood onset. In this review, we explore more deeply the reasons underlying the disparity between ocular therapies available for children and for adults by highlighting diseases that most commonly afflict children (with focus on the anterior eye) and existing prognoses, recent developments in ocular drug delivery systems and nanomedicines for children, and barriers to use for pediatric patients.

MR Imaging-Guided Focused Ultrasound for Breast Tumors.

Breast tumors remain a complex and prevalent health burden impacting millions of individuals worldwide. Challenges in treatment arise from the invasive nature of traditional surgery and, in malignancies, the complexity of treating metastatic disease. The development of noninvasive treatment alternatives is critical for improving patient outcomes and quality of life. This review aims to explore the advancements and applications of focused ultrasound (FUS) technology over the past 2 decades. FUS offers a promising noninvasive, nonionizing intervention strategy in breast tumors including primary breast cancer, fibroadenomas, and metastatic breast cancer.

Barriers to immune cell infiltration in tumors.

Increased immune cell infiltration into tumors is associated with improved patient survival and predicts response to immune therapies. Thus, identification of factors that determine the extent of immune infiltration is crucial, so that methods to intervene on these targets can be developed. T cells enter tumor tissues through the vasculature, and under control of interactions between homing receptors on the T cells and homing receptor ligands (HRLs) expressed by tumor vascular endothelium and tumor cell nests. HRLs are often deficient in tumors, and there also may be active barriers to infiltration. These remain understudied but may be crucial for enhancing immune-mediated cancer control. Multiple intratumoral and systemic therapeutic approaches show promise to enhance T cell infiltration, including both approved therapies and experimental therapies. This review highlights the intracellular and extracellular determinants of immune cell infiltration into tumors, barriers to infiltration, and approaches for intervention to enhance infiltration and response to immune therapies.

Guidelines for immunological analyses following focused ultrasound treatment.

Focused ultrasound (FUS) is a powerful emerging tool for non-invasive, non-ionizing targeted destruction of tumors. The last two decades have seen a growing body of preclinical and clinical literature supporting the capacity of FUS to increase nascent immune responses to tumors and to potentiate cancer immunotherapies (e.g. checkpoint inhibitors) through a variety of means, including immune modulation and drug delivery. With the rapid acceleration of this field and a multitude of FUS immunotherapy clinical trials having now been deployed worldwide, there is a need to streamline and standardize the methodology for immunological analyses field-wide. Recently, the Focused Ultrasound Foundation and Cancer Research Institute partnered to convene a group of over 85 leaders to discuss the nexus of FUS and immuno-oncology. The guidelines documented herein were assembled in response to recommendations that emerged from this discussion, emphasizing the urgent need for heightened accessibility of immune analysis methods and standardized protocols unique to the field. These guidelines are designated for existing stakeholders in the FUS immuno-oncology domain or those newly entering the field, to provide guidance on collection, storage, and immunological profiling of tissue or blood specimens in the context of FUS immunotherapy studies, and additionally offer templates for standardized deployment of these methods based on collective experience gained within the field to date. These guidelines are tumor-agnostic and provide evidence-based, consensus-based recommendations for both preclinical and clinical immune analysis of tissue and blood specimens.

Solid Tumor Treatment via Augmentation of Bioactive C6 Ceramide Levels with Thermally Ablative Focused Ultrasound.

Sparse scan partial thermal ablation (TA) with focused ultrasound (FUS) may be deployed to treat solid tumors and increase delivery of systemically administered therapeutics. Further, C6-ceramide-loaded nanoliposomes (CNLs), which rely upon the enhanced permeation and retention (EPR) effect for delivery, have shown promise for treating solid tumors and are being tested in clinical trials. Here, our objective was to determine whether CNLs synergize with TA in the control of 4T1 breast tumors. CNL-monotherapy of 4T1 tumors yielded significant intratumoral bioactive C6 accumulation by the EPR effect, but tumor growth was not controlled. TA increased bioactive C6 accumulation by ∼12.5-fold over the EPR effect. In addition, TA+CNL caused shifts in long-chain to very-long-chain ceramide ratios (i.e., C16/24 and C18/C24) that could potentially contribute to tumor control. Nonetheless, these changes in intratumoral ceramide levels were still insufficient to confer tumor growth control beyond that achieved when combining with TA with control "ghost" nanoliposomes (GNL). While this lack of synergy could be due to increased "pro-tumor" sphingosine-1-phosphate (S1P) levels, this is unlikely because S1P levels exhibited only a moderate and statistically insignificant increase with TA+CNL. In vitro studies showed that 4T1 cells are highly resistant to C6, offering the most likely explanation for the inability of TA to synergize with CNL. Thus, while our results show that sparse scan TA is a powerful approach for markedly enhancing CNL delivery and generating "anti-tumor" shifts in long-chain to very-long-chain ceramide ratios, resistance of the tumor to C6 can still be a rate-limiting factor for some solid tumor types.

Solid tumor treatment via augmentation of bioactive C6 ceramide levels with thermally ablative focused ultrasound.

Sparse scan partial thermal ablation (TA) with focused ultrasound (FUS) may be deployed to treat solid tumors and increase delivery of systemically administered therapeutics. Furthermore, C6-ceramide-loaded nanoliposomes (CNLs), which rely upon the enhanced-permeation and retention (EPR) effect for delivery, have shown promise for treating solid tumors and are being tested in clinical trials. Here, our objective was to determine whether CNLs synergize with TA in the control of 4T1 breast tumors. CNL monotherapy of 4T1 tumors yielded significant intratumoral bioactive C6 accumulation by the EPR effect, but tumor growth was not controlled. TA increased bioactive C6 accumulation by ~ 12.5-fold over the EPR effect. In addition, TA + CNL caused shifts in long-chain to very-long-chain ceramide ratios (i.e., C16/24 and C18/C24) that could potentially contribute to tumor control. Nonetheless, these changes in intratumoral ceramide levels were still insufficient to confer tumor growth control beyond that achieved when combining with TA with control "ghost" nanoliposomes (GNL). While this lack of synergy could be due to increased "pro-tumor" sphingosine-1-phosphate (S1P) levels, this is unlikely because S1P levels exhibited only a moderate and statistically insignificant increase with TA + CNL. In vitro studies showed that 4T1 cells are highly resistant to C6, offering the most likely explanation for the inability of TA to synergize with CNL. Thus, while our results show that sparse scan TA is a powerful approach for markedly enhancing CNL delivery and generating "anti-tumor" shifts in long-chain to very-long-chain ceramide ratios, resistance of the tumor to C6 can still be a rate-limiting factor for some solid tumor types.

Determinants of resistance to engineered T cell therapies targeting CD19 in large B cell lymphomas.

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

Clinical Role of Positron Emission Tomography/Computed Tomography Imaging in Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) imaging is nearly synonymous with positron emission tomography (PET) scans. Many of the nearly 60,000 newly diagnosed patients with HNSCC in the US-and 900,000 worldwide-will undergo a PET scan, if not multiple, throughout the course of their care. In this review, we describe the clinical utility of PET scans in HNSCC, emphasizing whereby their input is most impactful in improving patient outcomes as well as scenarios whereby PET/CT scans should be avoided. We also describe important considerations for capturing and processing PET scans with a special focus on the important role of tumor volume segmentation, scan timing relative to therapy, and concurrent conditions (eg, COVID-19). In addition, we will illustrate the latest innovations in the management of HNSCC. This article also will delve to exhibit novel potential biomarkers in the management of HNSCC. Finally, we describe future directions for PET imaging, including the advent of novel PET radiotracers as an alternative to 18F-fluorodeoxyglucose (18F-FDG).

Applications of focused ultrasound-mediated blood-brain barrier opening.

The blood brain barrier (BBB) plays a critically important role in the regulation of central nervous system (CNS) homeostasis, but also represents a major limitation to treatments of brain pathologies. In recent years, focused ultrasound (FUS) in conjunction with gas-filled microbubble contrast agents has emerged as a powerful tool for transiently and non-invasively disrupting the BBB in a targeted and image-guided manner, allowing for localized delivery of drugs, genes, or other therapeutic agents. Beyond the delivery of known therapeutics, FUS-mediated BBB opening also demonstrates the potential for use in neuromodulation and the stimulation of a range of cell- and tissue-level physiological responses that may prove beneficial in disease contexts. Clinical trials investigating the safety and efficacy of FUS-mediated BBB opening are well underway, and offer promising non-surgical approaches to treatment of devastating pathologies. This article reviews a range of pre-clinical and clinical studies demonstrating the tremendous potential of FUS to fundamentally change the paradigm of treatment for CNS diseases.

Functional intersections between extracellular vesicles and oncolytic therapies.

Minimally invasive focal therapies for nonviral oncolysis are a cornerstone of cancer therapeutics. Our ability to optimally deploy oncolytic therapies and identify synergistic combination approaches requires a deeper understanding of elicited biological responses. Extracellular vesicles (EV), which orchestrate a variety of pathophysiological processes and have a critical role in the evolution of primary and disseminated tumors, are now known to be potently modulated by oncolytic focal therapies, such as radiotherapy, photodynamic therapy (PDT), and therapeutic ultrasound (TUS). In this review, we summarize the diverse impacts of the aforementioned therapeutic modalities on EV biology, and highlight the most recent advances in EV-based drug delivery systems leveraging these modalities.

Intracranial Sonodynamic Therapy With 5-Aminolevulinic Acid and Sodium Fluorescein: Safety Study in a Porcine Model.

BACKGROUND: Sonodynamic therapy (SDT) is an emerging ultrasound-based treatment modality for malignant gliomas which combines ultrasound with sonosensitizers to produce a localized cytotoxic and modulatory effect. Tumor-specificity of the treatment is achieved by the selective extravasation and accumulation of sonosensitizers in the tumor-bearing regions. The aim of this study is to demonstrate the safety of low-intensity ultrasonic irradiation of healthy brain tissue after the administration of FDA-approved sonosensitizers used for SDT in experimental studies in an in vivo large animal model. METHODS: In vivo safety of fluorescein (Na-Fl)- and 5 aminolevulinic acid (5-ALA)-mediated low-intensity ultrasound irradiation of healthy brain parenchyma was assessed in two sets of four healthy swine brains, using the magnetic resonance imaging (MRI)-guided Insightec ExAblate 4000 220 kHz system. After administration of the sonosensitizers, a wide fronto-parietal craniotomy was performed in pig skulls to allow transmission of ultrasonic beams. Sonication was performed on different spots within the thalamus and periventricular white matter with continuous thermal monitoring. Sonication-related effects were investigated with MRI and histological analysis. RESULTS: Post-treatment MRI images acquired within one hour following the last sonication, on day one, and day seven did not visualize any sign of brain damage. On histopathology, no signs of necrosis or apoptosis attributable to the ultrasonic treatments were shown in target areas. CONCLUSIONS: The results of the present study suggest that either Na-FL or 5-ALA-mediated sonodynamic therapies under MRI-guidance with the current acoustic parameters are safe towards healthy brain tissue in a large in vivo model. These results further support growing interest in clinical translation of sonodynamic therapy for intracranial gliomas and other brain tumors.

Transcriptomic response of brain tissue to focused ultrasound-mediated blood-brain barrier disruption depends strongly on anesthesia.

Focused ultrasound (FUS) mediated blood-brain barrier disruption (BBBD) targets the delivery of systemically-administered therapeutics to the central nervous system. Preclinical investigations of BBBD have been performed on different anesthetic backgrounds; however, the influence of the choice of anesthetic on the molecular response to BBBD is unknown, despite its potential to critically affect interpretation of experimental therapeutic outcomes. Here, using bulk RNA sequencing, we comprehensively examined the transcriptomic response of both normal brain tissue and brain tissue exposed to FUS-induced BBBD in mice anesthetized with either isoflurane with medical air (Iso) or ketamine/dexmedetomidine (KD). In normal murine brain tissue, Iso alone elicited minimal differential gene expression (DGE) and repressed pathways associated with neuronal signaling. KD alone, however, led to massive DGE and enrichment of pathways associated with protein synthesis. In brain tissue exposed to BBBD (1 MHz, 0.5 Hz pulse repetition frequency, 0.4 MPa peak-negative pressure), we systematically evaluated the relative effects of anesthesia, microbubbles, and FUS on the transcriptome. Of particular interest, we observed that gene sets associated with sterile inflammatory responses and cell-cell junctional activity were induced by BBBD, regardless of the choice of anesthesia. Meanwhile, gene sets associated with metabolism, platelet activity, tissue repair, and signaling pathways, were differentially affected by BBBD, with a strong dependence on the anesthetic. We conclude that the underlying transcriptomic response to FUS-mediated BBBD may be powerfully influenced by anesthesia. These findings raise considerations for the translation of FUS-BBBD delivery approaches that impact, in particular, metabolism, tissue repair, and intracellular signaling.

ImmunoPET-informed sequence for focused ultrasound-targeted mCD47 blockade controls glioma.

Phagocytic immunotherapies such as CD47 blockade have emerged as promising strategies for glioblastoma (GB) therapy, but the blood brain/tumor barriers (BBB/BTB) pose a persistent challenge for mCD47 delivery that can be overcome by focused ultrasound (FUS)-mediated BBB/BTB disruption. We here leverage immuno-PET imaging to determine how timing of [89Zr]-mCD47 injection relative to FUS impacts antibody penetrance into orthotopic murine gliomas. We then design and implement a rational paradigm for combining FUS and mCD47 for glioma therapy. We demonstrate that timing of antibody injection relative to FUS BBB/BTB disruption is a critical determinant of mCD47 access, with post-FUS injection conferring superlative antibody delivery to gliomas. We also show that mCD47 delivery across the BBB/BTB with repeat sessions of FUS can significantly constrain tumor outgrowth and extend survival in glioma-bearing mice. This study generates provocative insights for ongoing pre-clinical and clinical evaluations of FUS-mediated antibody delivery to brain tumors. Moreover, our results confirm that mCD47 delivery with FUS is a promising therapeutic strategy for GB therapy.

Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity.

BACKGROUND: Triple-negative breast cancer (TNBC) remains recalcitrant to most targeted therapy approaches. However, recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy. We therefore tested a strategy for immune sensitization of murine TNBC (4T1 tumors) through combination of focused ultrasound (FUS) thermal ablation and a chemotherapy, gemcitabine (GEM), known to attenuate myeloid-derived suppressor cells (MDSCs). METHODS: We applied a sparse-scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM. We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity. We also tested this combination in Rag1-/- mice or T cell-depleted wild-type mice to determine the essentiality of adaptive immunity. Further, we layered Programmed cell death protein 1 (PD-1) blockade onto this combination to evaluate its impact on tumor outgrowth and survival. RESULTS: The immune-modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1, consistent with the dominant MDSC-driven immunosuppression evident in this model. The combination of FUS+GEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice. Tumor control correlated with increased circulating antigen-experienced T cells and was entirely dependent on T cell-mediated immunity. The ability of FUS+GEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti-PD-1. CONCLUSION: Thermally ablative FUS in combination with GEM restricts primary tumor outgrowth, improves survival and enhances immunogenicity in a murine metastatic TNBC model. This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluation. TRIAL REGISTRATION NUMBERS: NCT03237572 and NCT04116320.

Focused Ultrasound Hyperthermia Augments Release of Glioma-derived Extracellular Vesicles with Differential Immunomodulatory Capacity.

Background: Increasing evidence points to the critical role of extracellular vesicles (EVs) as molecular parcels that carry a diverse array of bioactive payloads for coordination of complex intracellular signaling. Focused ultrasound (FUS) hyperthermia is a technique for non-invasive, non-ionizing sublethal heating of cells in a near-instantaneous manner; while it has been shown to improve drug delivery and immunological recognition of tumors, its impact on EVs has not been explored to date. The goal of this study was to determine whether FUS impacts the release, proteomic profile, and immune-activating properties of tumor-derived EVs. Methods: Monolayered murine glioma cells were seeded within acoustically transparent cell culture chambers, and FUS hyperthermia was applied to achieve complete coverage of the chamber. Glioma-derived EVs (GEVs) were isolated for characterization by Nanoparticle Tracking Analysis, cryo-electron microscopy and mass spectrometry. An in vitro experimental setup was designed to further dissect the impact of GEVs on innate inflammation; immortalized murine dendritic cells (DCs) were pulsed with GEVs (either naïve or FUS hyperthermia-exposed) and assayed for production of IL-12p70, an important regulator of DC maturation and T helper cell polarization toward the interferon-γ-producing type 1 phenotype. Results: We confirmed that FUS hyperthermia significantly augments GEV release (by ~46%) as well as shifts the proteomic profile of these GEVs. Such shifts included enrichment of common EV-associated markers, downregulation of markers associated with cancer progression and resistance and modulation of inflammation-associated markers. When DCs were pulsed with GEVs, we noted that naïve GEVs suppressed IL-12p70 production by DCs in a GEV dose-dependent manner. In contrast, GEVs from cells exposed to FUS hyperthermia promoted a significant upregulation in IL-12p70 production by DCs, consistent with a pro-inflammatory stimulus. Conclusion: FUS hyperthermia triggers release of proteomically distinct GEVs that are capable of facilitating an important component of innate immune activation, lending both to a potential mechanism by which FUS interfaces with the tumor-immune landscape and to a role for GEV-associated biomarkers in monitoring response to FUS.

Deconvolution of the immunological contexture of mouse tumors with multiplexed immunohistochemistry.

In a variety of solid tumors, the presence of higher densities of tumor-infiltrating lymphocytes or tertiary lymphoid structures (TLS) are correlated with prolonged patient survival. Murine studies are usually required to define mechanisms that govern immunologic infiltrate in tumors. However, few methods have been described that could enable a more comprehensive understanding of the functionality of intratumoral immune infiltrate and TLS in solid murine cancers. In this chapter, we describe multiplex immunohistochemistry and microscopy approaches for identifying, characterizing, and quantifying intratumoral immune infiltrate and TLS in murine tumor models.

Immunomodulation of intracranial melanoma in response to blood-tumor barrier opening with focused ultrasound.

Background: Focused ultrasound (FUS) activation of microbubbles (MBs) for blood-brain (BBB) and blood-tumor barrier (BTB) opening permits targeted therapeutic delivery. While the effects of FUS+MBs mediated BBB opening have been investigated for normal brain tissue, no such studies exist for intracranial tumors. As this technology advances into clinical immunotherapy trials, it will be crucial to understand how FUS+MBs modulates the tumor immune microenvironment. Methods and Results: Bulk RNA sequencing revealed that FUS+MBs BTB/BBB opening (1 MHz, 0.5 MPa peak-negative pressure) of intracranial B16F1cOVA tumors increases the expression of genes related to proinflammatory cytokine and chemokine signaling, pattern recognition receptor signaling, and antigen processing and presentation. Flow cytometry revealed increased maturation (i.e. CD86) of dendritic cells (DCs) in the meninges and altered antigen loading of DCs in both the tumor and meninges. For DCs in tumor draining lymph nodes, FUS+MBs had no effect on maturation and elicited only a trend towards increased presentation of tumor-derived peptide by MHC. Neither tumor endothelial cell adhesion molecule expression nor homing of activated T cells was affected by FUS+MBs. Conclusion: FUS+MBs-mediated BTB/BBB opening elicits signatures of inflammation; however, the response is mild, transient, and unlikely to elicit a systemic response independent of administration of immune adjuvants.

Perspectives on Recent Progress in Focused Ultrasound Immunotherapy.

Immunotherapy holds tremendous promise as a strategy for eradicating solid tumors. However, poor T cell infiltration and persistence within most solid tumor microenvironments, as well as mechanisms of adaptive resistance, continue to severely limit the accessibility of most immunotherapies to a broad patient population. This limitation perpetuates the demand for allied therapeutic strategies. Among such strategies is focused ultrasound (FUS), a non-invasive, non-ionizing technique for precisely targeted acoustic energy deposition into tissues. FUS has gained remarkable attention over recent years as a modality for elicitation of immune mechanisms in cancer and other pathologies. In 2017, we published a comprehensive review paper detailing existing evidence for immune modulation and therapy with FUS, as well as impending challenges and opportunities of consideration for the field. Over the last two years, a multitude of clinical trials have come online to explore safety, feasibility, and efficacy of FUS for cancers of the brain and periphery - including the first clinical trial to combine FUS with immunotherapy. Moreover, the last two years have seen a surge in FUS immunotherapy presentations at therapeutic ultrasound scientific meetings. Given the burst of activity in this field, we submit that an update on FUS immunotherapy progress is timely. In this review, we offer an updated overview and perspectives on scientific and clinical development in the FUS immunotherapy domain.

Listening in on the Microbubble Crowd: Advanced Acoustic Monitoring for Improved Control of Blood-Brain Barrier Opening with Focused Ultrasound.

Non-invasive drug and gene delivery to the brain to treat central nervous system pathologies has long been inhibited by the blood-brain barrier. The activation of microbubbles with focused ultrasound has emerged as a promising non-invasive approach to circumvent this obstacle, by transiently disrupting the blood-brain barrier and permitting passage of systemically administered therapeutics into the tissue. Clinical trials are underway to evaluate the safety of this technique; however, concerns remain regarding the potential for the treatment to induce sterile inflammation or petechiae. In this issue of Theranostics, Jones et al.[1] address these concerns through the development of an advanced three-dimensional imaging system for monitoring acoustic emissions from oscillating microbubbles. When subharmonic emissions are detected with this system, focused ultrasound pressure is reduced by 50% for the remainder of the treatment. This serves to transiently open the blood-brain barrier without generating adverse effects. While the ideal configuration of the transducer array for treatment and monitoring still presents an area for further optimization, the approach indicates that the acoustic signature of microbubble behavior within the skull can be used to ensure safe and effective blood-brain barrier opening using focused ultrasound.