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Simple Summary Anti-hormonal therapie regimes are well established in oncological treatments in breast cancer. In contrast there is limited knowledge of their effects on metastatic brain metastases in advanced breast cancer and their ability to cross the blood brain-barrier. In this review, we point out the usual antihormonal therapy options in the primary disease, but also in metastatic breast cancer. In addition, we explain the epidemiological facts of brain metastases, as well as the basics of the blood-brain barrier and how this is overcome by metastase. Last but not least, we deal with the known anti-hormonal therapy options and present clinical studies on their intracerebral effect, as well as the known basics of their blood-brain barrier penetration. Not all common anti-hormonal therapeutics are able to penetrate the CNS. It is therefore important for the treating oncologists to use substances that have been proven to cross the BBB, despite the limited data available. Aromataseinhibitors, especially letrozole, probably also tamoxifen, everolimus and CDK4/6 inhibitors, especially abemaciclib, appear to act intracerebrally by overcoming the blood-brain barrier. Nevertheless, further data must be obtained in basic research, but also health care research in relation to patients with brain metastases. Abstract The molecular receptor status of breast cancer has implications for prognosis and long-term metastasis. Although metastatic luminal B-like, hormone-receptor-positive, HER2−negative, breast cancer causes brain metastases less frequently than other subtypes, though tumor metastases in the brain are increasingly being detected of this patient group. Despite the many years of tried and tested use of a wide variety of anti-hormonal therapeutic agents, there is insufficient data on their intracerebral effectiveness and their ability to cross the blood-brain barrier. In this review, we therefore summarize the current state of knowledge on anti-hormonal therapy and its intracerebral impact and effects on the blood-brain barrier in breast cancer.

Chronic stress, even stress of a moderate intensity related to daily life, is widely acknowledged to be a predisposing or precipitating factor in neuropsychiatric diseases. There is a clear relationship between disturbances induced by stressful stimuli, especially long-lasting stimuli, and cognitive deficits in rodent models of affective disorders. Regular physical activity has a positive effect on the central nervous system (CNS) functions, contributes to an improvement in mood and of cognitive abilities (including memory and learning), and is correlated with an increase in the expression of the neurotrophic factors and markers of synaptic plasticity as well as a reduction in the inflammatory factors. Studies published so far show that the energy challenge caused by physical exercise can affect the CNS by improving cellular bioenergetics, stimulating the processes responsible for the removal of damaged organelles and molecules, and attenuating inflammation processes. Regular physical activity brings another important benefit: increased stress robustness. The evidence from animal studies is that a sedentary lifestyle is associated with stress vulnerability, whereas a physically active lifestyle is associated with stress resilience. Here, we have performed a comprehensive PubMed Search Strategy for accomplishing an exhaustive literature review. In this review, we discuss the findings from experimental studies on the molecular and neurobiological mechanisms underlying the impact of exercise on brain resilience. A thorough understanding of the mechanisms underlying the neuroprotective potential of preconditioning exercise and of the role of exercise in stress resilience, among other things, may open further options for prevention and therapy in the treatment of CNS diseases.

Protocadherins (PCDHs) belong to the cadherin superfamily and represent the largest subgroup of calcium-dependent adhesion molecules. In the genome, most PCDHs are arranged in three clusters, α, β, and γ on chromosome 5q31. PCDHs are highly expressed in the central nervous system (CNS). Several PCDHs have tumor suppressor functions, but their individual role in primary brain tumors has not yet been elucidated. Here, we examined the mRNA expression of PCDHGC3, a member of the PCDHγ cluster, in non-cancerous brain tissue and in gliomas of different World Health Organization (WHO) grades and correlated it with the clinical data of the patients. We generated a PCDHGC3 knockout U343 cell line and examined its growth rate and migration in a wound healing assay. We showed that PCDHGC3 mRNA and protein were significantly overexpressed in glioma tissue compared to a non-cancerous brain specimen. This could be confirmed in glioma cell lines. High PCDHGC3 mRNA expression correlated with longer progression-free survival (PFS) in glioma patients. PCDHGC3 knockout in U343 resulted in a slower growth rate but a significantly faster migration rate in the wound healing assay and decreased the expression of several genes involved in WNT signaling. PCDHGC3 expression should therefore be further investigated as a PFS-marker in gliomas. However, more studies are needed to elucidate the molecular mechanisms underlying the PCDHGC3 effects.

Progressive deterioration of the central nervous system (CNS) is commonly associated with aging. An important component of the neurovasculature is the blood–brain barrier (BBB), majorly made up of endothelial cells joined together by intercellular junctions. The relationship between senescence and changes in the BBB has not yet been thoroughly explored. Moreover, the lack of in vitro models for the study of the mechanisms involved in those changes impede further and more in-depth investigations in the field. For this reason, we herein present an in vitro model of the senescent BBB and an initial attempt to identify senescence-associated alterations within.

Brain metastases are the most severe tumorous spread during breast cancer disease. They are associated with a limited quality of life and a very poor overall survival. A subtype of extracellular vesicles, exosomes, are sequestered by all kinds of cells, including tumor cells, and play a role in cell-cell communication. Exosomes contain, among others, microRNAs (miRs). Exosomes can be taken up by other cells in the body, and their active molecules can affect the cellular process in target cells. Tumor-secreted exosomes can affect the integrity of the blood-brain barrier (BBB) and have an impact on brain metastases forming. Serum samples from healthy donors, breast cancer patients with primary tumors, or with brain, bone, or visceral metastases were used to isolate exosomes and exosomal miRs. Exosomes expressed exosomal markers CD63 and CD9, and their amount did not vary significantly between groups, as shown by Western blot and ELISA. The selected 48 miRs were detected using real-time PCR. Area under the receiver-operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. We identified two miRs with the potential to serve as prognostic markers for brain metastases. Hsa-miR-576-3p was significantly upregulated, and hsa-miR-130a-3p was significantly downregulated in exosomes from breast cancer patients with cerebral metastases with AUC: 0.705 and 0.699, respectively. Furthermore, correlation of miR levels with tumor markers revealed that hsa-miR-340-5p levels were significantly correlated with the percentage of Ki67-positive tumor cells, while hsa-miR-342-3p levels were inversely correlated with tumor staging. Analysis of the expression levels of miRs in serum exosomes from breast cancer patients has the potential to identify new, non-invasive, blood-borne prognostic molecular markers to predict the potential for brain metastasis in breast cancer. Additional functional analyzes and careful validation of the identified markers are required before their potential future diagnostic use.

Defects in the endothelial cell barrier accompany diverse malfunctions of the central nervous system such as neurodegenerative diseases, stroke, traumatic brain injury, and systemic diseases such as sepsis, viral and bacterial infections, and cancer. Compromised endothelial sealing leads to leaking blood vessels, followed by vasogenic edema. Brain edema as the most common complication caused by stroke and traumatic brain injury is the leading cause of death. Brain microvascular endothelial cells, together with astrocytes, pericytes, microglia, and neurons form a selective barrier, the so-called blood-brain barrier, which regulates the movement of molecules inside and outside of the brain. Mechanisms that regulate blood-brain barrier permeability in health and disease are complex and not fully understood. Several newly discovered molecules that are involved in the regulation of cellular processes in brain microvascular endothelial cells have been described in the literature in recent years. One of these molecules that are highly expressed in brain microvascular endothelial cells is protocadherin gamma C3. In this review, we discuss recent evidence that protocadherin gamma C3 is a newly identified key player involved in the regulation of vascular barrier function.

Objective Plasminogen activator inhibitor-1 (PAI-1) is the key endogenous inhibitor of fibrinolysis, and enhances clot formation after injury. In traumatic brain injury, dysregulation of fibrinolysis may lead to sustained microthrombosis and accelerated lesion expansion. In the present study, we hypothesized that PAI-1 mediates post-traumatic malfunction of coagulation, with inhibition or genetic depletion of PAI-1 attenuating clot formation and lesion expansion after brain trauma. Methods We evaluated PAI-1 as a possible new target in a mouse controlled cortical impact (CCI) model of traumatic brain injury. We performed the pharmacological inhibition of PAI-1 with PAI-039 and stimulation by tranexamic acid, and we confirmed our results in PAI-1-deficient animals. Results PAI-1 mRNA was time-dependently upregulated, with a 305-fold peak 12 hours after CCI, which effectively counteracted the 2- to 3-fold increase in cerebral tissue-type/urokinase plasminogen activator expression. PAI-039 reduced brain lesion volume by 26% at 24 hours and 43% at 5 days after insult. This treatment also attenuated neuronal apoptosis and improved neurofunctional outcome. Moreover, intravital microscopy demonstrated reduced post-traumatic thrombus formation in the pericontusional cortical microvasculature. In PAI-1-deficient mice, the therapeutic effect of PAI-039 was absent. These mice also displayed 13% reduced brain damage compared with wild type. In contrast, inhibition of fibrinolysis with tranexamic acid increased lesion volume by 25% compared with vehicle. Interpretation This study identifies impaired fibrinolysis as a critical process in post-traumatic secondary brain damage and suggests that PAI-1 may be a central endogenous inhibitor of the fibrinolytic pathway, promoting a procoagulatory state and clot formation in the cerebral microvasculature. Ann Neurol 2019;85:667-680.

Blood-brain barrier (BBB) integrity is one of the important elements of central nervous system (CNS) homeostasis. MicroRNAs (miRs) have been demonstrated to play a role in many CNS disorders such as stroke and traumatic brain injury. MiR-212/132 are highly expressed in the CNS but their role at the BBB has not been characterized yet. Thus, we analyzed the expression of miR-212/132 in hypoxic mouse and human brain microvascular endothelial cells (BMEC) as well as in posttraumatic mouse and human brain tissue and serum exosomes. MiR-212/132 expression was detected in brain capillaries by in situ hybridization and was increased up to ten times in hypoxic BMEC. Over-expression of pre-miR-212/132 in BMEC decreased barrier properties and reduced migration of BMEC in the wound healing assay. We identified and validated tight junction proteins claudin-1 (Cldn1), junctional adhesion molecule 3 (Jam3), and tight junction-associated protein 1 (Tjap1) as potential miR-212/132 targets. Over-expression of miRs led to a decrease in mRNA and protein expression of Cldn1, Jam3, and Tjap1, which could be rescued by a respective anti-miR. In conclusion, our study identifies miR-212/132 as critical players at the hypoxic BBB. In addition, we propose three new direct miR-212/132 targets to be involved in miR-212/132-mediated effects on BBB properties. Electronic supplementary material The online version of this article (10.1007/s12975-018-0683-2) contains supplementary material, which is available to authorized users.

The clinical translatability of novel drug delivery systems begins with basic scientific breakthroughs. Our recent discovery of the ability of Tumor Treating Fields (TTFields) to potentially and transiently disrupt the blood brain barrier (BBB) using our murine in vitro and in vivo models, led us to validate our findings in a human 3D in vitro model established in our lab. The model consists of primary brain microvascular endothelial cells co-cultured with immortalized perciytes in a transwell system. TTFields are alternating electric fields of low intensity (1-3V/cm) and intermediate frequency (100-300kHz), which are effective and approved for the treatment of glioblastoma (GBM) using 200kHz frequency. Our murine data point out that TTFields could disrupt the BBB optimally at 100kHz. To investigate if TTFields exhibit similar effects in the human cell-based in vitro model, it was subjected to TTFields at various frequencies for 24-96h. Cells were afterwards made to recover for 24-96h. To assess BBB integrity and compromise, transendothelial electrical resistance (TEER) was measured before start of TTFields, immediately after end of TTFields, as well as 24-96h after TTFields. In addition, a permeability assay was performed. Finally, immunofluorescence (IF) staining visualized the effects of TTFields on tight junction protein claudin-5 localization. TTFields application of all investigated frequencies significantly decreased TEER. However, the strongest effects were observed with 100kHz after 72h. IF staining revealed delocalization of claudin-5 from the cell boundaries to the cytoplasm. Restoration of cell integrity was already evident as early as 24h, with complete recovery after 48h. Results using our human 3D in vitro model validated our previous observations from murine in vitro and in vivo models that TTFields could transiently disrupt the BBB. These findings provide fundamental pre-clinical data for translation from bench to bedside. Accordingly, TTFields demonstrate to be a promising novel approach in opening the BBB to facilitate drug delivery for improved treatment of central nervous system diseases including devastating brain tumors such as GBM. Citation Format: Ellaine Salvador, Almuth F. Kessler, Theresa Köppl, Sebastian Schönhärl, Malgorzata Burek, Catherine Tempel Brami, Tali Voloshin, Moshe Giladi, Ralf-Ingo Ernestus, Mario Löhr, Carola Y. Förster, Carsten Hagemann. Blood brain barrier (BBB) disruption by tumor treating fields (TTFields) in a human 3D in vitro model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 387.

Extracellular vesicles transport variable content and have crucial functions in cell–cell communication. The role of extracellular vesicles in cancer is a current hot topic, and no bibliometric study has ever analyzed research production regarding their role in breast cancer and indicated the trends in the field. In this way, we aimed to investigate the trends in breast cancer management involved with extracellular vesicle research. Articles were retrieved from Scopus, including all the documents published concerning breast cancer and extracellular vesicles. We analyzed authors, journals, citations, affiliations, and keywords, besides other bibliometric analyses, using R Studio version 3.6.2. and VOSviewer version 1.6.0. A total of 1151 articles were retrieved, and as the main result, our analysis revealed trending topics on biomarkers of liquid biopsy, drug delivery, chemotherapy, autophagy, and microRNA. Additionally, research related to extracellular vesicles in breast cancer has been focused on diagnosis, treatment, and mechanisms of action of breast tumor-derived vesicles. Future studies are expected to explore the role of extracellular vesicles on autophagy and microRNA, besides investigating the application of extracellular vesicles from liquid biopsies for biomarkers and drug delivery, enabling the development and validation of therapeutic strategies for specific cancers.

Recent studies have suggested a pathogenetic link between ischemic stroke and Takotsubo cardiomyopathy (TCM) with poor outcome, when occurring simultaneously. Increased catecholamine (CAT) levels as well as elevated inflammatory mediators (INF) are found in the blood of patients with ischemic stroke concomitant with Takotsubo syndrome (TTS). On molecular level, the impact of these stressors combined with hypoxemia could compromise the integrity of the blood brain barrier (BBB) resulting in poor outcomes. As a first step in the direction of investigating possible molecular mechanisms, an in vitro model of the described pathological constellation was designed. An immortalized murine microvascular endothelial cell line from the cerebral cortex (cEND) was used as an established in vitro model of the BBB. cEND cells were treated with supraphysiological concentrations of CAT (dopamine, norepinephrine, epinephrine) and INF (TNF-α and Interleukin-6). Simultaneously, cells were exposed to oxygen glucose deprivation (OGD) as an established in vitro model of ischemic stroke with/without subsequent reoxygenation. We investigated the impact on cell morphology and cell number by immunofluorescence staining. Furthermore, alterations of selected tight and adherens junction proteins forming paracellular barrier as well as integrins mediating cell-matrix adhesion were determined by RT-PCR and/or Western Blot technique. Especially by choosing this wide range of targets, we give a detailed overview of molecular changes leading to compromised barrier properties. Our data show that the proteins forming the BBB and the cell count are clearly influenced by CAT and INF applied under OGD conditions. Most of the investigated proteins are downregulated, so a negative impact on barrier integrity can be assumed. The structures affected by treatment with CAT and INF are potential targets for future therapies in ischemic stroke and TTS.

Moyamoya disease (MMD) is a rare steno-occlusive cerebrovascular disorder. Mechanisms driving the formation of aberrant MMD vessels remain elusive. We collected serum and vessel specimens from MMD and atherosclerotic cerebrovascular disease (ACVD) patients serving as controls due to the same hypoxic stimulus but substantial differences in terms of vascular features. Based on patient material and an in vitro model mimicking ACVD and MMD conditions, matrix metalloproteinase-9 (MMP-9) and vascular-endothelial growth factor (VEGF) were tested for their potential involvement in cerebrovascular disintegration. While serum concentration of both molecules did not significantly differ in both patient groups, excessive collagenase activity and lowered collagen IV protein amount in MMD vessels pointed to a focal MMP-9 activity at the affected vessel sites. We observed overexpressed and autocrinely secreted MMP-9 and VEGF along with disturbances of EC–matrix interactions in MMD but not ACVD serum-treated cEND cells. These seemingly brain-specific effects were partially attenuated by VEGF signaling inhibition suggesting its role in the MMD etiology. In conclusion, our findings support the understanding of the high incidence of hemorrhagic and ischemic events in MMD and provide the basis for novel therapeutic strategies stopping or slowing the development of fragile cerebrovasculature or micro-bleeds characterizing the disease.

Introduction Alternating electric fields of intermediate frequency and low intensity, known as Tumor Treating Fields (TTFields), are an effective and clinically approved approach for treatment of glioblastoma (GBM). The optimal frequency for treatment of glioma cells based on the cytotoxic response is at 200 kHz. Combination of TTFields with chemotherapy appears to be synergistic with further increase in overall survival of patients with GBM, beyond that with chemotherapy alone. The blood brain barrier (BBB) limits delivery of a majority of drugs to the brain thus limiting treatment options for GBM patients. Recent in vitro studies suggest that TTFields applied at 100 kHz can disturb the BBB. In this study, we investigated the potential use of TTFields to transiently disturb the BBB in animal models. Methods BBB permeation was tested in healthy rats subsequently to 100 kHz TTFields or sham (heat) application to the rat head. BBB permeability was analyzed by several staining agents: (1) Evans Blue (EB) that was quantified at 610 nm in brain homogenates; (2) 4 kDa TRITC-dextran (TD) that was quantified based on fluorescence intensity in brain cryosections; and (3) the MRI contrast agent Gd-DTPA. Accumulation and clearance of Gd-DTPA were tracked by serial dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). In addition, brain sections were stained for claudin-5, occludin, PECAM-1 and immunoglobulin G (IgG). BBB permeation induced by TTFields was further evaluated in rats orthotopically bearing F98 glioma cells and treated with TTFields at 100 kHz in combination with the chemotherapeutic drug paclitaxel (PTX) for a duration of 72 h. Tumor cell proliferation was assessed by Ki67 staining and the tumor volume was measured by T2 weighted MRI. Results BBB permeation of EB and TD staining agents was observed in the brains of healthy rats after TTFields application. Moreover, brain cryosections displayed delocalization of claudin-5 and occludin, but not of PECAM-1. Accumulation of IgG in the brain parenchyma was also noted. Confirming these observations, DCE-MRI post-TTFields treatment showed accumulation of Gd in the brain. Return to normal BBB integrity was detected 96 h after TTFields treatment cessation, indicating the effect was transient and reversible. In GBM-induced rats, the combination of PTX (a drug which normally does not cross the BBB) with TTFields significantly decreased tumor cell proliferation and tumor volume compared to animals treated with TTFields alone, sham alone, or sham combined with PTX. Conclusions Administration of 100 kHz TTFields to the brain of rats led to transient alterations in BBB integrity and permeability, allowing increased uptake of combination chemotherapy. These data indicate that TTFields treatment may be a feasible, novel clinical strategy for transient opening of the BBB to allow for enhanced and more effective delivery of permeable and non-permeable anticancer drugs to the brain. Citation Format: Catherine Tempel Brami, Ellaine Salvador, Almuth F. Kessler, Malgorzata Burek, Tali Voloshin, Moshe Giladi, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Transient opening of the blood brain barrier by Tumor Treating Fields (TTFields) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 279.

OBJECTIVE For glioblastoma patients Tumor Treating Fields (TTFields) have been established as adjuvant therapy. The blood brain barrier (BBB) tightly controls the influx of the majority of compounds from blood to brain. Therefore, the BBB may block delivery of drugs for treatment of brain tumors. Here, the influence of TTFields on BBB permeability was assessed in vivo. METHODS Rats were treated with 100 kHz TTFields for 72 h and thereupon i.v. injected with Evan’s Blue (EB) which directly binds to Albumin. To evaluate effects on BBB, EB was extracted after brain homogenization and quantified. In addition, cryosections of rat brains were prepared following TTFields application. The sections were stained for tight junction proteins Claudin-5 and Occludin and for immunoglobulin G (IgG) to assess vessel structure. Furthermore, serial dynamic contrast-enhanced DCE-MRI with Gadolinium contrast agent was performed before and after TTFields application. RESULTS TTFields application significantly increased the EB accumulation in the rat brain. In TTFields-treated rats, the vessel structure became diffuse compared to control cryosections of rat brains; Claudin 5 and Occludin were delocalized and IgG was found throughout the brain tissue. Serial DCE-MRI demonstrated significantly increased accumulation of Gadolinium in the brain, observed directly after 72 h of TTFields application. The effect of TTFields on the BBB disappeared 96 h after end of treatment and no difference in contrast enhancement between controls and TTFields treated animals was detectable. CONCLUSION By altering BBB integrity and permeability, application of TTFields at 100 kHz may have the potential to deliver drugs to the brain, which are unable to cross the BBB. Utilizing TTFields to open the BBB and its subsequent recovery could be a clinical approach of drug delivery for treatment of brain tumors and other diseases of the central nervous system. These results will be further validated in clinical Trials.

In order to prevent cerebral vasospasm after a subarachnoid hemorrhage (SAH), the so-called triple H-therapy (hypertension, hypervolemia, hemodilution) could be applied. In these cases, colloidal solutions containing Hydroxyethylstarch (HES) are used to induce hypervolemia. The administration of HES is very much under debate for the mentioned use, because in general the application of HES for the treatment of critical ill patients has been reduced tremendously in the last years due to its nephrotoxic effects. In this context, there are limited data investigating the influence of HES on the blood-brain barrier. These data might help to assess if a transient administration of HES is possibly justifiable to prevent cerebral ischemia during vasospasm despite the risk of an acute kidney injury. To address this question, a mouse blood-brain barrier in vitro model based on cell line cerebEND was exposed to different HES concentrations and compared to NaCl-containing control solutions. In order to assess the effects of HES on blood-brain barrier properties, cell viability, transendothelial electrical resistance, permeability of carboxyfluorescein, mRNA and protein expression and localization of tight junction proteins were determined. In summary, 1.5–4% HES attenuated cell viability in a mild, concentration dependent manner compared to the NaCl control solution (0% HES). At the mRNA level 1% and 4% HES significantly increased the expression of tight junction associated proteins (ZO-1 and occludin) and the glucose transporter Glut-1 (Slc2a1). In correspondence to this, 4% HES inhibited breakdown of the paracellular barrier in comparison to the control NaCl group (0% HES) shown by transendothelial electrical resistance values and the permeability of the paracellular marker carboxyfluorescein. These effects at the functional level were confirmed by immunofluorescence microscopic images of junctional proteins. The obtained in vitro data showed a potential for HES to counteract blood-brain barrier damage. Future studies are needed to reveal the applicability of HES as a blood-brain barrier stabilizing agent.

Microvascular endothelial cells are an essential part of many biological barriers, such as the blood–brain barrier (BBB) and the endothelium of the arteries and veins. A reversible opening strategy to increase the permeability of drugs across the BBB could lead to improved therapies due to enhanced drug bioavailability. Vanilloids, such as capsaicin, are known to reversibly open tight junctions of epithelial and endothelial cells. In this study, we used several in vitro assays with the murine endothelial capillary brain cells (line cEND) as a BBB model to characterize the interaction between capsaicin and endothelial tight junctions.

Infusion of the colloid hydroxyethylstarch has been used for volume substitution to maintain hemodynamics and microcirculation after e.g., severe blood loss. In the last decade it was revealed that hydroxyethylstarch can aggravate acute kidney injury, especially in septic patients. Because of the serious risk for critically ill patients, the administration of hydroxyethylstarch was restricted for clinical use. Animal studies and recently published in vitro experiments showed that hydroxyethylstarch might exert protective effects on the blood-brain barrier. Since the prevention of blood-brain barrier disruption was shown to go along with the reduction of brain damage after several kinds of insults, we revisit the topic hydroxyethylstarch and discuss a possible niche for the application of hydroxyethylstarch in acute brain injury treatment.

Introduction: Although a number of effective drugs are available to treat central nervous system (CNS) disorders, their ability to breach the tight regulation of the blood brain barrier (BBB) still remains a major challenge. Recently, the use of tumor treating fields (TTFields) has become an effective treatment approach for glioblastoma. Furthermore, its combination with chemotherapy significantly improved overall patient survival. Nonetheless, how TTFields could affect the BBB has not yet been studied. Our recent findings exhibit the potential of TTFields administration to open up the BBB in vitro with an optimal frequency of 100 kHz. Consequently, in this study, we therefore aimed to validate our data in vivo. Experimental procedures: Subsequent to 100 kHz TTFields or heat treatment for 72 h, rats were i.v. injected with Evan´s Blue (EB). Next, they were sacrificed to extract and quantify EB from the brain. In the same manner, rats were injected with TRITC-dextran (TD), after which permeation was visualized in sectioned brains. Cryosections of rat brains were also prepared post-TTFields treatment. These were stained for intercellular junction proteins claudin-5, occludin and PECAM-1 as well as immunoglobulin G (IgG) to assess vessel structure. Finally, serial dynamic contrast-enhanced (DCE) MRI with gadolinium (Gd) contrast agent was performed before and after TTFields administration. Results: Permeation of both EB and TD was observed in the brain after TTFields application. Moreover, brain cryosections displayed claudin-5 and occludin delocalization but not PECAM-1. Accumulation of IgG in the brain parenchyma was also noted. Confirming these observations, increased Gd in the brain was shown by DCE-MRI post TTFields treatment. A reversion to normal conditions was, however, detected 96 h after end of treatment demonstrated by no difference in contrast enhancement between control and TTFields-treated rats. Conclusions: Administration of 100 kHz TTFields in rats led to alterations in BBB integrity and permeability, which signal its opening. The subsequent recovery of the BBB at the end of treatment demonstrates transient effects, hence presenting TTFields as a possible novel clinical strategy to open the BBB for enhanced and more effective drug delivery strategy for CNS disorders. Citation Format: Ellaine Salvador, Almuth F. Kessler, Julia Hörmann, Malgorzata Burek, Catherine T. Brami, Tali V. Sela, Moshe Giladi, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Blood brain barrier opening by TTFields: a future CNS drug delivery strategy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6251.

2551 Background: The greatest hurdle, which even potent and effective drugs targeting central nervous system (CNS) tumors and other disorders face, is the blood brain barrier (BBB). The inability to cross the tight regulatory mechanism renders these drugs futile. Of late, administration of tumor treating fields (TTFields) as part of a combined treatment modality for glioblastoma demonstrated increased overall patient survival. Still, the effects of TTFields on the BBB have not yet been investigated. Here, we report the potential of TTFields application to open up the BBB. Methods: Murine brain endothelial cells were treated with 100-300 kHz TTFields for 24-96 h. Cells were also allowed to recover from 24-96 h after treatment. Subsequently, changes in cell morphology, integrity, and permeability were observed via staining of intercellular junction proteins (IJP) as well as transendothelial electrical resistance (TEER)and permeability assays. In vivo, rats were treated with 100 kHz TTFields or heat for 72 h after which they were IV injected with Evan´s Blue (EB)/ TRITC-dextran (TD) which was later quantified from the brain. Rat brain cryosections were also stained for IJPs as well as immunoglobulin G (IgG) to assess vessel structure. Finally, serial dynamic contrast-enhanced (DCE) MRI with gadolinium (Gd) contrast agent was performed pre- and post- TTFields. Results: Upon TTFields application, IJPs such as claudin-5 were delocalized from the cell membrane to the cytoplasm with maximal effects at 100 kHz. In addition, BBB integrity was significantly reduced and permeability for 4 kDa molecules was significantly increased. Cell morphology recovery was first observed at 48 h post-treatment and completely restored to normal after 96 h, indicating a reversibility of the TTFields effect on the BBB. In addition, EB and TD permeated the rat brain post-TTFields treatment. Brain cryosections displayed IJPs delocalization as well as IgG accumulation in the brain parenchyma. Confirming these observations, increased Gd in the brain was shown by DCE-MRI post-TTFields application. A reversion to normal conditions was detected 96 h after end of treatment, which was demonstrated by no difference in contrast enhancement between control and treated rats. Conclusions: TTFields application both in vitro and in vivo points towards its ability to transiently open the BBB. This presents TTFields as a novel aid for drug delivery geared towards treatment of CNS tumors and other related diseases. Hence, it is indicative of the possibility of an enhanced and more effective combinatorial therapeutic strategy.

BACKGROUND: Drug application for the treatment of malignant brain tumors, in particular glioblastoma multiforme (GBM), can be impeded by the blood–brain barrier (BBB). Recently, low intensity, intermediate frequency alternating electric fields called Tumor Treating Fields (TTFields) have been established as novel adjuvant treatment modality for GBM. Here, the effect of TTFields on BBB permeability is inspected. MATERIAL AND METHODS: Immortalized murine brain capillary endothelial cells (cerebEND) that were grown on coverslips and transwell inserts, were treated with TTFields at a frequency of 100–300 kHz for up to 72 h. Immunofluorescence staining of the tight-junction proteins Claudin 5 and ZO-1 was utilized to analyze cell morphology. BBB integrity was determined using transendothelial resistance (TEER) and BBB permeability was checked with flow cytometry analysis applying fluorescein isothiocyanate (FITC). In rats, Evans Blue (EB) was utilized to quantify the increase in vessel permeability during TTFields application to the brain (100 kHz, 72 h). RESULTS: TTFields application disturbs the BBB by delocalization of tight junction proteins from the cell boundaries to the cytoplasm with most dramatic effects at 100 kHz. The BBB integrity was significantly reduced by 65% and the BBB permeability for 4 kDa large molecules was significantly increased upon TTFields application. The cell morphology started to recover 48 h and was completed 96 h after treatment end pointing to a reversibility of the TTFields-effect on the BBB. TTFields application to the rat head significantly increased the average accumulation of EB in the brain. CONCLUSION: As TTFields at a frequency of 100 kHz may potentially permeabilize the BBB, they could be utilized to deliver drugs generally unable to cross the BBB to the central nervous system. The presented in vitro and in vivo data may lead to a phase I clinical trial and clinical application in the future.

Traumatic brain injury (TBI) is a significant problem causing high mortality globally. Methods to increase possibilities for treatment and prevention of secondary injuries resulting from the initial physical insult are thus much needed. TBI affects the central nervous system (CNS) and the neurovascular unit as a whole in numerous ways but one of the primarily compromised components is the blood-brain barrier (BBB).In this chapter, we present a detailed procedure on how stretch injury and oxygen-glucose deprivation (OGD) are applied to brain microvascular endothelial cells of the BBB in order to replicate the actual impact they receive during TBI.

Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood–brain barrier.

Claudin-5 is an integral membrane protein and a critical component of endothelial tight junctions that control paracellular permeability. Claudin-5 is expressed at high levels in the brain vascular endothelium. Estrogens have multiple effects on vascular physiology and function. The biological actions of estrogens are mediated by two different estrogen receptor (ER) subtypes, ER alpha and ER beta. Estrogens have beneficial effects in several vascular disorders. Recently we have cloned and characterized a murine claudin-5 promoter and demonstrated 17beta-estradiol (E2)-mediated regulation of claudin-5 in brain and heart microvascular endothelium on promoter, mRNA and protein level. Sequence analysis revealed a putative estrogen response element (ERE) and a putative Sp1 transcription factor binding site in the claudin-5 promoter. The aim of the present study was to further characterize the estrogen-responsive elements of claudin-5 promoter. First, we introduced point mutations in ERE or Sp1 site in -500/+111 or in Sp1 site of -268/+111 claudin-5 promoter construct, respectively. Basal and E2-mediated transcriptional activation of mutated constructs was abrogated in the luciferase reporter gene assay. Next, we examined whether estrogen receptor subtypes bind to the claudin-5 promoter region. For this purpose we performed chromatin immunoprecipitation assays using anti-estrogen receptor antibodies and cellular lysates of E2-treated endothelial cells followed by quantitative PCR analysis. We show enrichment of claudin-5 promoter fragments containing the ERE- and Sp1-binding site in immunoprecipitates after E2 treatment. Finally, in a gel mobility shift assay, we demonstrated DNA-protein interaction of both ER subtypes at ERE. In summary, this study provides evidence that both a non-consensus ERE and a Sp1 site in the claudin-5 promoter are functional and necessary for the basal and E2-mediated activation of the promoter.

Background Information Glucocorticoids (GCs), including the synthetic GC derivate dexamethasone, are widely used as immunomodulators. One of the numerous side effects of dexamethasone therapy is hypertension arising from reduced release of the endothelium-derived vasodilator nitric oxide (NO). Results Herein, we described the role of dexamethasone and its glucocorticoid receptor (GR) in the regulation of NO synthesis in vitro using the mouse myocardial microvascular endothelial cell line, MyEND. GC treatment caused a firm decrease of extracellular NO levels, whereas the expression of endothelial NO synthase (eNOS) was not affected. However, GC application induced an impairment of tetrahydrobiopterin (BH4) concentrations as well as GTP cyclohydrolase-1 (GTPCH-1) expression, both essential factors for NO production upstream of eNOS. Moreover, dexamethasone stimulation resulted in a substantially decreased GR gene and protein expression in MyEND cells. Importantly, inhibition of proteasome-mediated proteolysis of the GR or overexpression of an ubiquitination-defective GR construct improved the bioavailability of BH4 and strengthened GTPCH-1 expression and eNOS activity. Conclusions Summarising our results, we propose a new mechanism involved in the regulation of NO signalling by GCs in myocardial endothelial cells. We suggest that a sufficient GR protein expression plays a crucial role for the management of GC-induced harmful adverse effects, including deregulations of vasorelaxation arising from disturbed NO biosynthesis.

BACKGROUND Alternating electric fields with intermediate frequency (100 - 300 kHz) and low intensity (1 - 3 V/cm), known as Tumor Treating Fields (TTFields), have been established as a novel adjuvant therapy for glioblastoma (GBM) patients. The blood brain barrier (BBB) tightly controls the influx of the majority of compounds from blood to brain. Due to this regulation, the BBB may block delivery of drugs for treatment of brain tumors, in particular GBM. In this study, we investigated the influence of TTFields on BBB permeability in vivo. MATERIAL AND METHODS For determination of BBB permeability, rats were treated with 100 kHz TTFields for 72 h. At the end of treatment, rats were i.v. injected with Evan′s Blue (EB), which binds Albumin (~70 kDa) upon injection to the blood. EB was extracted after brain homogenization and quantified at 610 nm. In addition, cryosections of rat brains were prepared following TTFields application at 100 kHz for 72 h, and sections were stained for Claudin 5, Occludin and immunoglobulin G (IgG) to assess vessel structure. Moreover, serial dynamic contrast-enhanced DCE-MRI with Gadolinium contrast agent (Gd) was performed before and after TTFields application. RESULTS In vivo, the EB accumulation in the brain was significantly increased by application of TTFields to the rat head. Claudin 5 and Occludin staining was visible in vessel endothelial cells and localized at the cells’ edges in control cryosections of rat brains. In TTFields-treated rats, the vessel structure became diffuse; Claudin 5 and Occludin were delocalized and IgG was found throughout the brain tissue and not solely inside the vessels, as it is normally the case. Serial DCE-MRI demonstrated significantly increased accumulation of Gd in the brain, detected directly after 72 h of TTFields application. 96 h after end of TTFields treatment the effect on the BBB disappeared and no difference in contrast enhancement between controls and TTFields treated animals was observable. CONCLUSION Application of TTFields at 100 kHz could have the potential to deliver drugs to the brain, which normally are unable to cross the BBB by altering BBB integrity and permeability. Utilizing TTFields to open the BBB and its subsequent recovery, as demonstrated by the data presented herein, could lead to a clinical approach of drug delivery for treatment of malignant brain tumors and other diseases of the central nervous system. These results will be further validated in clinical trials.

Background: The blood-brain barrier (BBB) may impede application of drugs to the brain for treatment of malignant brain tumors, in particular glioblastoma multiforme (GBM). Alternating electric fields with intermediate frequency and low intensity, called Tumor Treating Fields (TTFields), are an established novel adjuvant treatment modality for GBM. Here, the effect of TTFields on BBB permeability is analyzed. Material and Methods: After TTFields treatment with a frequency of 100-300 kHz for up to 72 h, immortalized murine brain capillary endothelial cells (cerebEND) grown on cover slips and transwell inserts were stained for immunofluorescent assessment of the tight junction proteins Claudin-5 and ZO-1. Transendothelial electrical resistance (TEER) was applied to investigate BBB integrity. Moreover, BBB permeability was determined by fluorescein isothiocyanate (FITC) staining followed by flow cytometry. For in vivo analysis, the increase in vessel permeability was quantified by utilizing i.v. injected Evans Blue (EB) in rats during TTFields application to the brain (100 kHz, 72 h). Results: The BBB was disturbed by treatment with TTFields as tight junction proteins were delocalized from the cell boundaries to the cytoplasm with maximal effects at 100 kHz. TTFields application significantly reduced the BBB integrity by 65% and significantly increased the BBB permeability for 4 kDa large molecules. Initial recovery of the cell morphology was observed 48 h post-treatment and a complete recovery could be detected after 96 h, indicating a reversibility of the TTFields effect on the BBB. Average accumulation of EB in the rat brain was significantly increased by TTFields application to the rats head. Conclusion: In the future, TTFields could be utilized to deliver drugs generally unable to cross the BBB to the central nervous system as TTFields at a frequency of 100 kHz are potentially able to disrupt the BBB. The data presented on in vitro and in vivo application of TTFields to permeabilize the BBB may be a rationale for a phase I clinical trial and clinical application in the future. Citation Format: Almuth F. Kessler, Clara M. Schaeffer, Malgorzata Burek, Ursula Ruschig, Catherine Tempel-Brami, Tali Voloshin, Moshe Giladi, Ellaine Salvador, Ralf-Ingo Ernestus, Mario Löhr, Carola Förster, Carsten Hagemann. Tumor treating fields (TTFields) affect blood brain barrier (BBB) integrity in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 252.