Focused Ultrasound Impels the Delivery and Penetration of Model Therapeutics into Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular neoplasms in the brain that can cause debilitating symptoms. Current treatments pose significant risks to some patients, motivating the development of new nonsurgical options. We recently discovered that focused ultrasound-mediated blood-brain barrier opening (FUS) arrests CCM formation and growth. Here, we build on this discovery and assess the ability of FUS to deliver model therapeutics into CCMs. METHODS: Quantitative T1 mapping MRI sequences were used with 1 kDa (MultiHance; MH) and 17 kDa (GadoSpin D; GDS) contrast agents to assess the FUS-mediated delivery and penetration of model small molecule drugs and biologics, respectively, into CCMs of Krit1 mutant mice. RESULTS: FUS elevated the rate of MH delivery to both the lesion core (4.6-fold) and perilesional space (6.7-fold). Total MH delivery more than doubled in the lesion core and tripled in the perilesional space when FUS was applied immediately prior to MH injection. For the model biologic drug (i.e. GDS), FUS was of greater relative benefit, resulting in 21.7-fold and 3.8-fold delivery increases to the intralesional and perilesional spaces, respectively. CONCLUSIONS: FUS is capable of impelling the delivery and penetration of therapeutics into the complex and disorganized CCM microenvironment. Benefits to small molecule drug delivery are more evident in the perilesional space, while benefits to biologic delivery are more evident in CCM cores. These findings, when combined with ability of FUS alone to control CCMs, highlight the potential of FUS to serve as a powerful non-invasive therapeutic platform for CCM.

Tissue- and Temporal-Dependent Dynamics of Myeloablation in Response to Gemcitabine Chemotherapy.

For triple-negative breast cancer (TNBC), the most aggressive subset of breast cancer, immune cell infiltrates have prognostic implications. The presence of myeloid-derived suppressor cells supports tumor progression, while tumor-infiltrating lymphocytes (TILs) correlate with improved survival and responsiveness to immunotherapy. Manipulating the abundance of these populations may enhance tumor immunity. Gemcitabine (GEM), a clinically employed chemotherapeutic, is reported to be systemically myeloablative, and thus it is a potentially useful adjunct therapy for promoting anti-tumor immunity. However, knowledge about the immunological effects of GEM intratumorally is limited. Thus, we directly compared the impact of systemic GEM on immune cell presence and functionality in the tumor microenvironment (TME) to its effects in the periphery. We found that GEM is not myeloablative in the TME; rather, we observed sustained, significant reductions in TILs and dendritic cells-crucial components in initiating an adaptive immune response. We also performed bulk-RNA sequencing to identify immunological alterations transcriptionally induced by GEM. While we found evidence of upregulation in the interferon-gamma (IFN-γ) response pathway, we determined that GEM-mediated growth control is not dependent on IFN-γ. Overall, our findings yield new insights into the tissue- and temporal-dependent immune ablative effects of GEM, contrasting the paradigm that this therapy is specifically myeloablative.

Brain Nucleic Acid Delivery and Genome Editing via Focused Ultrasound-Mediated Blood-Brain Barrier Opening and Long-Circulating Nanoparticles.

We introduce a two-pronged strategy comprising focused ultrasound (FUS)-mediated blood-brain barrier (BBB) opening and long-circulating biodegradable nanoparticles (NPs) for systemic delivery of nucleic acids to the brain. Biodegradable poly(ß-amino ester) polymer-based NPs were engineered to stably package various types of nucleic acid payloads and enable prolonged systemic circulation while retaining excellent serum stability. FUS was applied to a predetermined coordinate within the brain to transiently open the BBB, thereby allowing the systemically administered long-circulating NPs to traverse the BBB and accumulate in the FUS-treated brain region, where plasmid DNA or mRNA payloads produced reporter proteins in astrocytes and neurons. In contrast, poorly circulating and/or serum-unstable NPs, including the lipid NP analogous to a platform used in clinic, were unable to provide efficient nucleic acid delivery to the brain regardless of the BBB-opening FUS. The marriage of FUS-mediated BBB opening and the long-circulating NPs engineered to copackage mRNA encoding CRISPR-associated protein 9 and single-guide RNA resulted in genome editing in astrocytes and neurons precisely in the FUS-treated brain region. The combined delivery strategy provides a versatile means to achieve efficient and site-specific therapeutic nucleic acid delivery to and genome editing in the brain via a systemic route.

Exploring the dynamics of adult Axin2 cell lineage integration into dentate gyrus granule neurons.

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/ß-catenin signaling, making this gene a reliable indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2+ lineage in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with the induction of Axin2 fate mapping by tamoxifen, we marked the dividing cells with 5-ethynyl-2'-deoxyuridine (EdU). Tamoxifen induction led to a significant increase in labeled dentate gyrus granule cells three months later. However, none of these neurons showed any EdU signal. Conversely, six months after the pulse-chase labeling with tamoxifen/EdU, we identified granule neurons that were positive for both EdU and tdTomato lineage tracer in each animal. Our data indicates that Axin2 is expressed at multiple stages of adult granule neuron differentiation. Furthermore, these findings suggest that the integration process of adult-born neurons from specific cell lineages may require more time than previously thought.

Focused Ultrasound Blood-Brain Barrier Opening Arrests the Growth and Formation of Cerebral Cavernous Malformations.

BACKGROUND: Cerebral cavernous malformations (CCM) are vascular lesions within the central nervous system, consisting of dilated and hemorrhage-prone capillaries. CCMs can cause debilitating neurological symptoms, and surgical excision or stereotactic radiosurgery are the only current treatment options. Meanwhile, transient blood-brain barrier opening (BBBO) with focused ultrasound (FUS) and microbubbles is now understood to exert potentially beneficial bioeffects, such as stimulation of neurogenesis and clearance of amyloid-ß. Here, we tested whether FUS BBBO could be deployed therapeutically to control CCM formation and progression in a clinically-representative murine model. METHODS: CCMs were induced in mice by postnatal, endothelial-specific Krit1 ablation. FUS was applied for BBBO with fixed peak-negative pressures (PNPs; 0.2-0.6 MPa) or passive cavitation detection-modulated PNPs. Magnetic resonance imaging (MRI) was used to target FUS treatments, evaluate safety, and measure longitudinal changes in CCM growth after BBBO. RESULTS: FUS BBBO elicited gadolinium accumulation primarily at the perilesional boundaries of CCMs, rather than lesion cores. Passive cavitation detection and gadolinium contrast enhancement were comparable in CCM and wild-type mice, indicating that Krit1 ablation does not confer differential sensitivity to FUS BBBO. Acutely, CCMs exposed to FUS BBBO remained structurally stable, with no signs of hemorrhage. Longitudinal MRI revealed that FUS BBBO halted the growth of 94% of CCMs treated in the study. At 1 month, FUS BBBO-treated lesions lost, on average, 9% of their pre-sonication volume. In contrast, non-sonicated control lesions grew to 670% of their initial volume. Lesion control with FUS BBBO was accompanied by a marked reduction in the area and mesenchymal appearance of Krit mutant endothelium. Strikingly, in mice receiving multiple BBBO treatments with fixed PNPs, de novo CCM formation was significantly reduced by 81%. Mock treatment plans on MRIs of patients with surgically inaccessible lesions revealed their lesions are amenable to FUS BBBO with current clinical technology. CONCLUSIONS: Our results establish FUS BBBO as a novel, non-invasive modality that can safely arrest murine CCM growth and prevent their de novo formation. As an incisionless, MR image-guided therapy with the ability to target eloquent brain locations, FUS BBBO offers an unparalleled potential to revolutionize the therapeutic experience and enhance the accessibility of treatments for CCM patients.

Focused ultrasound ablation of melanoma with boiling histotripsy yields abscopal tumor control and antigen-dependent dendritic cell activation.

Background: Boiling histotripsy (BH), a mechanical focused ultrasound ablation strategy, can elicit intriguing signatures of anti-tumor immunity. However, the influence of BH on dendritic cell function is unknown, compromising our ability to optimally combine BH with immunotherapies to control metastatic disease. Methods: BH was applied using a sparse scan (1 mm spacing between sonications) protocol to B16F10-ZsGreen melanoma in bilateral and unilateral settings. Ipsilateral and contralateral tumor growth was measured. Flow cytometry was used to track ZsGreen antigen and assess how BH drives dendritic cell behavior. Results: BH monotherapy elicited ipsilateral and abscopal tumor control in this highly aggressive model. Tumor antigen presence in immune cells in the tumor-draining lymph nodes (TDLNs) was ~3-fold greater at 24h after BH, but this abated by 96h. B cells, macrophages, monocytes, granulocytes, and both conventional dendritic cell subsets (i.e. cDC1s and cDC2s) acquired markedly more antigen with BH. BH drove activation of both cDC subsets, with activation being dependent upon tumor antigen acquisition. Our data also suggest that BH-liberated tumor antigen is complexed with damage-associated molecular patterns (DAMPs) and that cDCs do not traffic to the TDLN with antigen. Rather, they acquire antigen as it flows through afferent lymph vessels into the TDLN. Conclusion: When applied with a sparse scan protocol, BH monotherapy elicits abscopal melanoma control and shapes dendritic cell function through several previously unappreciated mechanisms. These results offer new insight into how to best combine BH with immunotherapies for the treatment of metastatic melanoma.

Focused ultrasound ablation of melanoma with boiling histotripsy yields abscopal tumor control and antigen-dependent dendritic cell activation.

Background: Boiling histotripsy (BH), a mechanical focused ultrasound ablation strategy, can elicit intriguing signatures of anti-tumor immunity. However, the influence of BH on dendritic cell function is unknown, compromising our ability to optimally combine BH with immunotherapies to control metastatic disease. Methods: BH was applied using a sparse scan (1 mm spacing between sonications) protocol to B16F10-ZsGreen melanoma in bilateral and unilateral settings. Ipsilateral and contralateral tumor growth was measured. Flow cytometry was used to track ZsGreen antigen and assess how BH drives dendritic cell behavior. Results: BH monotherapy elicited ipsilateral and abscopal tumor control in this highly aggressive model. Tumor antigen presence in immune cells in the tumor-draining lymph nodes (TDLNs) was ~3-fold greater at 24h after BH, but this abated by 96h. B cells, macrophages, monocytes, granulocytes, and both conventional dendritic cell subsets (i.e. cDC1s and cDC2s) acquired markedly more antigen with BH. BH drove activation of both cDC subsets, with activation being dependent upon tumor antigen acquisition. Our data also suggest that BH-liberated tumor antigen is complexed with damage-associated molecular patterns (DAMPs) and that cDCs do not traffic to the TDLN with antigen. Rather, they acquire antigen as it flows through afferent lymph vessels into the TDLN. Conclusion: When applied with a sparse scan protocol, BH monotherapy elicits abscopal melanoma control and shapes dendritic cell function through several previously unappreciated mechanisms. These results offer new insight into how to best combine BH with immunotherapies for the treatment of metastatic melanoma.

Dynamics of Adult Axin2 Cell Lineage Integration in Granule Neurons of the Dentate Gyrus.

The Wnt pathway plays critical roles in neurogenesis. The expression of Axin2 is induced by Wnt/ß-catenin signaling, making this gene a sensitive indicator of canonical Wnt activity. We employed pulse-chase genetic lineage tracing with the Axin2-CreERT2 allele to follow the fate of Axin2 -positive cells in the adult hippocampal formation. We found Axin2 expressed in astrocytes, neurons and endothelial cells, as well as in the choroid plexus epithelia. Simultaneously with tamoxifen induction of Axin2 fate mapping, the dividing cells were marked with 5-ethynyl-2'-deoxyuridine (EdU). Tamoxifen induction resulted in significant increase of dentate gyrus granule cells three months later; however, none of these neurons contained EdU signal. Conversely, six months after the tamoxifen/EdU pulse-chase labeling, EdU-positive granule neurons were identified in each animal. Our data imply that Axin2 is expressed at several different stages of adult granule neuron differentiation and suggest that the process of integration of the adult-born neurons from certain cell lineages may take longer than previously thought.

Emerging roles for ITAM and ITIM receptor signaling in microglial biology and Alzheimer's disease-related amyloidosis.

Microglia are critical responders to amyloid beta (Aß) plaques in Alzheimer's disease (AD). Therefore, the therapeutic targeting of microglia in AD is of high clinical interest. While previous investigation has focused on the innate immune receptors governing microglial functions in response to Aß plaques, how microglial innate immune responses are regulated is not well understood. Interestingly, many of these microglial innate immune receptors contain unique cytoplasmic motifs, termed immunoreceptor tyrosine-based activating and inhibitory motifs (ITAM/ITIM), that are commonly known to regulate immune activation and inhibition in the periphery. In this review, we summarize the diverse functions employed by microglia in response to Aß plaques and also discuss the innate immune receptors and intracellular signaling players that guide these functions. Specifically, we focus on the role of ITAM and ITIM signaling cascades in regulating microglia innate immune responses. A better understanding of how microglial innate immune responses are regulated in AD may provide novel therapeutic avenues to tune the microglial innate immune response in AD pathology.

Intraarterial Transplantation of Mitochondria After Ischemic Stroke Reduces Cerebral Infarction.

Background-: Transplantation of autologous mitochondria into ischemic tissue may mitigate injury caused by ischemia and reperfusion. Methods-: Using murine stroke models of middle cerebral artery occlusion, we sought to evaluate feasibility of delivery of viable mitochondria to ischemic brain parenchyma. We evaluated the effects of concurrent focused ultrasound activation of microbubbles, which serves to open the blood-brain barrier, on efficacy of delivery of mitochondria. Results-: Following intra-arterial delivery, mitochondria distribute through the stroked hemisphere and integrate into neural and glial cells in the brain parenchyma. Consistent with functional integration in the ischemic tissue, the transplanted mitochondria elevate concentration of adenosine triphosphate in the stroked hemisphere, reduce infarct volume and increase cell viability. Additional of focused ultrasound leads to improved blood brain barrier opening without hemorrhagic complications. Conclusions-: Our results have implications for the development of interventional strategies after ischemic stroke and suggest a novel potential modality of therapy after mechanical thrombectomy.

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.

Magnetic Resonance Imaging of Mouse Cerebral Cavernomas Reveal Differential Lesion Progression and Variable Permeability to Gadolinium.

BACKGROUND: Cerebral cavernous malformations, also known as cavernous angiomas, are blood vessel abnormalities comprised of clusters of grossly enlarged and hemorrhage-prone capillaries. The prevalence in the general population, including asymptomatic cases, is estimated to be 0.5%. Some patients develop severe symptoms, including seizures and focal neurological deficits, whereas others remain asymptomatic. The causes of this remarkable presentation heterogeneity within a primarily monogenic disease remain poorly understood. METHODS: We established a chronic mouse model of cerebral cavernous malformations, induced by postnatal ablation of Krit1 with Pdgfb-CreERT2, and examined lesion progression in these mice with T2-weighted 7T magnetic resonance imaging (MRI). We also established a modified protocol for dynamic contrast-enhanced MRI and produced quantitative maps of gadolinium tracer gadobenate dimeglumine. After terminal imaging, brain slices were stained with antibodies against microglia, astrocytes, and endothelial cells. RESULTS: These mice develop cerebral cavernous malformations lesions gradually over 4 to 5 months of age throughout the brain. Precise volumetric analysis of individual lesions revealed nonmonotonous behavior, with some lesions temporarily growing smaller. However, the cumulative lesional volume invariably increased over time and after about 2 months followed a power trend. Using dynamic contrast-enhanced MRI, we produced quantitative maps of gadolinium in the lesions, indicating a high degree of heterogeneity in lesional permeability. MRI properties of the lesions were correlated with cellular markers for endothelial cells, astrocytes, and microglia. Multivariate comparisons of MRI properties of the lesions with cellular markers for endothelial and glial cells revealed that increased cell density surrounding lesions correlates with stability, whereas denser vasculature within and surrounding the lesions may correlate with high permeability. CONCLUSIONS: Our results lay a foundation for better understanding individual lesion properties and provide a comprehensive preclinical platform for testing new drug and gene therapies for controlling cerebral cavernous malformations.

The Alzheimer's disease risk factor INPP5D restricts neuroprotective microglial responses in amyloid beta-mediated pathology.

INTRODUCTION: Mutations in INPP5D, which encodes for the SH2-domain-containing inositol phosphatase SHIP-1, have recently been linked to an increased risk of developing late-onset Alzheimer's disease. While INPP5D expression is almost exclusively restricted to microglia in the brain, little is known regarding how SHIP-1 affects neurobiology or neurodegenerative disease pathogenesis. METHODS: We generated and investigated 5xFAD Inpp5dfl/fl Cx3cr1Ert2Cre mice to ascertain the function of microglial SHIP-1 signaling in response to amyloid beta (Aß)-mediated pathology. RESULTS: SHIP-1 deletion in microglia led to substantially enhanced recruitment of microglia to Aß plaques, altered microglial gene expression, and marked improvements in neuronal health. Further, SHIP-1 loss enhanced microglial plaque containment and Aß engulfment when compared to microglia from Cre-negative 5xFAD Inpp5dfl/fl littermate controls. DISCUSSION: These results define SHIP-1 as a pivotal regulator of microglial responses during Aß-driven neurological disease and suggest that targeting SHIP-1 may offer a promising strategy to treat Alzheimer's disease. HIGHLIGHTS: Inpp5d deficiency in microglia increases plaque-associated microglia numbers. Loss of Inpp5d induces activation and phagocytosis transcriptional pathways. Plaque encapsulation and engulfment by microglia are enhanced with Inpp5d deletion. Genetic ablation of Inpp5d protects against plaque-induced neuronal dystrophy.

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.

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.

Sonodynamic therapy: Rapid progress and new opportunities for non-invasive tumor cell killing with sound.

Solid tumor treatment relies heavily upon chemotherapies, radiation, surgical resection, and/or immunotherapies. Although many alternative non-invasive solid tumor therapies have been proposed through the years and continue to be tested in various contexts, tumor cell eradication remains a daunting task for the current cancer armamentarium. Indeed, solid tumors exhibit physically and biochemically heterogenous microenvironments, allowing them to easily acquire resistance mechanisms. Progress in sonodynamic therapy (SDT), a treatment modality capable of controlling tumor growth while limiting off-target effects and toxicities, has accelerated in recent years. SDT combines "sonosensitizing" agents with the non-invasive application of focused acoustic energy [i.e. focused ultrasound (FUS)] to drive highly localized formation of tumor cell-killing reactive oxygen species (ROS). Sonosensitizers selectively accumulate in tumor cells, after which FUS radiation eliminates the tumor by forcing the tumor cells to undergo cell death. In this article, we comprehensively review recent studies wherein SDT has been applied to treat primary and metastatic tumors. We discuss sonosensitizers, combination therapies with SDT, developments in defining the mechanism of SDT-induced cell cytotoxicity, and the promise SDT offers as a modulator of anti-tumor immunity.

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.

Computational model of brain endothelial cell signaling pathways predicts therapeutic targets for cerebral pathologies.

Brain endothelial cells serve many critical homeostatic functions. In addition to sensing and regulating blood flow, they maintain blood-brain barrier function, including precise control of nutrient exchange and efflux of xenobiotics. Many signaling pathways in brain endothelial cells have been implicated in both health and disease; however, our understanding of how these signaling pathways functionally integrate is limited. A model capable of integrating these signaling pathways could both advance our understanding of brain endothelial cell signaling networks and potentially identify promising molecular targets for endothelial cell-based drug or gene therapies. To this end, we developed a large-scale computational model, wherein brain endothelial cell signaling pathways were reconstructed from the literature and converted into a network of logic-based differential equations. The model integrates 63 nodes (including proteins, mRNA, small molecules, and cell phenotypes) and 82 reactions connecting these nodes. Specifically, our model combines signaling pathways relating to VEGF-A, BDNF, NGF, and Wnt signaling, in addition to incorporating pathways relating to focused ultrasound as a therapeutic delivery tool. To validate the model, independently established relationships between selected inputs and outputs were simulated, with the model yielding correct predictions 73% of the time. We identified influential and sensitive nodes under different physiological or pathological contexts, including altered brain endothelial cell conditions during glioma, Alzheimer's disease, and ischemic stroke. Nodes with the greatest influence over combinations of desired model outputs were identified as potential druggable targets for these disease conditions. For example, the model predicts therapeutic benefits from inhibiting AKT, Hif-1α, or cathepsin D in the context of glioma - each of which are currently being studied in clinical or pre-clinical trials. Notably, the model also permits testing multiple combinations of node alterations for their effects on the network and the desired outputs (such as inhibiting AKT and overexpressing the P75 neurotrophin receptor simultaneously in the context of glioma), allowing for the prediction of optimal combination therapies. In all, our approach integrates results from over 100 past studies into a coherent and powerful model, capable of both revealing network interactions unapparent from studying any one pathway in isolation and predicting therapeutic targets for treating devastating brain pathologies.

Multiple regression analysis of a comprehensive transcriptomic data assembly elucidates mechanically- and biochemically-driven responses to focused ultrasound blood-brain barrier disruption.

Background: Focused ultrasound (FUS) blood brain barrier disruption (BBBD) permits the noninvasive, targeted, and repeatable delivery of drugs to the brain. FUS BBBD also elicits secondary responses capable of augmenting immunotherapies, clearing amyloid-ß and hyperphosphorylated tau, and driving neurogenesis. Leveraging these secondary effects will benefit from an understanding of how they correlate to the magnitude of FUS BBBD and are differentially affected by the mechanical and biochemical stimuli imparted during FUS BBBD. Methods: We aggregated 75 murine transcriptomes in a multiple regression framework to identify genes expressed in proportion to biochemical (i.e. contrast MR image enhancement (CE)) or mechanical (i.e. harmonic acoustic emissions from MB-activation (MBA)) stimuli associated with FUS BBBD. Models were constructed to control for potential confounders, such as sex, anesthesia, and sequencing batch. Results: MBA and CE differentially predicted expression of 1,124 genes 6 h or 24 h later. While there existed overlap in the transcripts correlated with MBA vs CE, MBA was principally predictive of expression of genes associated with endothelial reactivity while CE chiefly predicted sterile inflammation gene sets. Over-representation analysis identified transcripts not previously linked to BBBD, including actin filament organization, which is likely important for BBB recovery. Transcripts and pathways associated with neurogenesis, microglial activation, and amyloid-ß clearance were significantly correlated to BBBD metrics. Conclusions: The secondary effects of BBBD may have the potential to be tuned by modulating FUS parameters during BBBD, and MBA and CE may serve as independent predictors of transcriptional reactions in the brain.