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Tumor treating fields (TTFields), a new modality of cancer treatment, are electric fields transmitted transdermally to tumors. The FDA has approved TTFields for the treatment of glioblastoma multiforme and mesothelioma, and they are currently under study in many other cancer types. While antimitotic effects were the first recognized biological anticancer activity of TTFields, data have shown that tumor treating fields achieve their anticancer effects through multiple mechanisms of action. TTFields therefore have the ability to be useful for many cancer types in combination with many different treatment modalities. Here, we review the current understanding of TTFields and their mechanisms of action.

Supplementary Figure from The Mechanisms of Action of Tumor Treating Fields

Both nerve injury and complex regional pain syndrome (CRPS) can result in chronic pain. In traumatic neuropathy, the blood nerve barrier (BNB) shielding the nerve is impaired—partly due to dysregulated microRNAs (miRNAs). Upregulation of microRNA-21-5p (miR-21) has previously been documented in neuropathic pain, predominantly due to its proinflammatory features. However, little is known about other functions. Here, we characterized miR-21 in neuropathic pain and its impact on the BNB in a human-murine back translational approach. MiR-21 expression was elevated in plasma of patients with CRPS as well as in nerves of mice after transient and persistent nerve injury. Mice presented with BNB leakage, as well as loss of claudin-1 in both injured and spared nerves. Moreover, the putative miR-21 target RECK was decreased and downstream Mmp9 upregulated, as was Tgfb. In vitro experiments in human epithelial cells confirmed a downregulation of CLDN1 by miR-21 mimics via inhibition of the RECK/MMP9 pathway but not TGFB. Perineurial miR-21 mimic application in mice elicited mechanical hypersensitivity, while local inhibition of miR-21 after nerve injury reversed it. In summary, the data support a novel role for miR-21, independent of prior inflammation, in elicitation of pain and impairment of the BNB via RECK/MMP9.

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.

TTFields are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), which are effective and approved for the treatment of glioblastoma (GBM) using 200 kHz frequency. However, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Therefore, we adapted patient-derived three-dimensional GBM tissue culture models to be compatible with TTFields application and recently published the feasibility of such an approach (Nickl, et al., 2022, doi: 10.3390/cancers14215177). Here, we applied one of those models, i.e. tumor-organoids cultured as microtumors on murine organotypic hippocampal slice cultures (OHSCs), to additional brain tumor entities, namely a sample of an anaplastic ependymoma (AE) patient and an oligodendroglioma patient. Organoids were generated from fresh intra-operatively obtained tumor tissue and cultured for 2 weeks. OHSCs were prepared by slicing the brains of mice 5-8 days postpartum to sections with a thickness of 350 µm using a vibratome, and culturing them for 2 weeks as well. Subsequently, organoids were placed onto the OHSCs. The inovitro™ laboratory research system was used for TTFields administration at 200 kHz and 1.5 V/cm for 72 h. Microtumor growth was evaluated on fluorescence images. Viable organoids formed from the GBM, AE and oligodendroglioma sample and grew to microtumors when placed onto OHSCs. Application of TTFields at 200 kHz led to a significant decrease of microtumor size of the GBM and AE (both p Citation Format: Vera Nickl, Ellina Schulz, Ellaine Salvador, Laureen Trautmann, Leopold Diener, Almuth F. Kessler, Camelia M. Monoranu, Ralf-Ingo Ernestus, Mario Löhr, Carsten Hagemann. Evaluation of tumor treating fields (TTFields) effects at 200 kHz on a glioblastoma, an anaplastic ependymoma and an oligodendroglioma sample in a patient-derived ex vivo organoid model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4573.

Tumor treating fields (TTFields), a new modality of cancer treatment, are electric fields transmitted transdermally to tumors. The FDA has approved TTFields for the treatment of glioblastoma multiforme and mesothelioma, and they are currently under study in many other cancer types. While antimitotic effects were the first recognized biological anticancer activity of TTFields, data have shown that tumor treating fields achieve their anticancer effects through multiple mechanisms of action. TTFields therefore have the ability to be useful for many cancer types in combination with many different treatment modalities. Here, we review the current understanding of TTFields and their mechanisms of action.

Simple Summary In glioblastoma, tumor recurrence is inevitable and the prognosis of patients is poor, despite multidisciplinary treatment approaches involving surgical resection, radiotherapy and chemotherapy. Recently, Tumor Treating Fields (TTFields) have been added to the therapeutic set-up. These alternating electric fields are applied to glioblastoma at 200 kHz frequency via arrays placed on the shaved scalp of patients. Patients show varying response to this therapy. Molecular effects of TTFields have been investigated largely in cell cultures and animal models, but not in patient tissue samples. Acquisition of matched treatment-naïve and recurrent patient tissues is a challenge. Therefore, we suggest three reliable patient-derived three-dimensional ex vivo models (primary cells grown as microtumors on murine organotypic hippocampal slices, organoids and tumor slice cultures) which may facilitate prediction of patients’ treatment responses and provide important insights into clinically relevant cellular and molecular alterations under TTFields. Abstract Glioblastoma (GBM) displays a wide range of inter- and intra-tumoral heterogeneity contributing to therapeutic resistance and relapse. Although Tumor Treating Fields (TTFields) are effective for the treatment of GBM, there is a lack of ex vivo models to evaluate effects on patients’ tumor biology or to screen patients for treatment efficacy. Thus, we adapted patient-derived three-dimensional tissue culture models to be compatible with TTFields application to tissue culture. Patient-derived primary cells (PDPC) were seeded onto murine organotypic hippocampal slice cultures (OHSC), and microtumor development with and without TTFields at 200 kHz was observed. In addition, organoids were generated from acute material cultured on OHSC and treated with TTFields. Lastly, the effect of TTFields on expression of the Ki67 proliferation marker was evaluated on cultured GBM slices. Microtumors exhibited increased sensitivity towards TTFields compared to monolayer cell cultures. TTFields affected tumor growth and viability, as the size of microtumors and the percentage of Ki67-positive cells decreased after treatment. Nevertheless, variability in the extent of the response was preserved between different patient samples. Therefore, these pre-clinical GBM models could provide snapshots of the tumor to simulate patient treatment response and to investigate molecular mechanisms of response and resistance.

Blood nerve barrier disruption and edema are common in neuropathic pain as well as in complex regional pain syndrome (CRPS). MicroRNAs (miRNA) are epigenetic multitarget switches controlling neuronal and non-neuronal cells in pain. The miR-183 complex attenuates hyperexcitability in nociceptors, but additional non-neuronal effects via transcription factors could contribute as well. This study explored exosomal miR-183 in CRPS and murine neuropathy, its effect on the microvascular barrier via transcription factor FoxO1 and tight junction protein claudin-5, and its antihyperalgesic potential. Sciatic miR-183 decreased after CCI. Substitution with perineural miR-183 mimic attenuated mechanical hypersensitivity and restored blood nerve barrier function. In vitro, serum from CCI mice und CRPS patients weakened the microvascular barrier of murine cerebellar endothelial cells, increased active FoxO1 and reduced claudin-5, concomitant with a lack of exosomal miR-183 in CRPS patients. Cellular stress also compromised the microvascular barrier which was rescued either by miR-183 mimic via FoxO1 repression or by prior silencing of Foxo1. PERSPECTIVE: Low miR-183 leading to barrier impairment via FoxO1 and subsequent claudin-5 suppression is a new aspect in the pathophysiology of CRPS and neuropathic pain. This pathway might help untangle the wide symptomatic range of CRPS and nurture further research into miRNA mimics or FoxO1 inhibitors.

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.

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 this study, we investigated the cytotoxic effects of unmodified α-cyclodextrin (α-CD) and modified cyclodextrins, including trimethyl-β-cyclodextrin (TRIMEB) and hydroxypropyl-β-cyclodextrin (HPβCD), on immortalized murine microvascular endothelial (cEND) cells of the blood-brain barrier (BBB). A CellTiter-Glo viability test, performed on the cEND cells showed significant differences among the different cyclodextrins. After 24 hr of incubation, TRIMEB was the most cytotoxic, and HPβCD was non-toxic. α-CD and TRIMEB exhibited greater cytotoxicity in the Dulbecco's modified Eagle's medium than in heat-inactivated human serum indicating protective properties of the human serum. The predicted dynamic toxicity profiles (Td) for α-CD and TRIMEB indicated higher cytotoxicity for these cyclodextrins compared to the reference compound (dimethylsulfoxide). Molecular dynamics simulation of cholesterol binding to the CDs suggested that not just cholesterol but phospholipids extraction might be involved in the cytotoxicity. Overall, the results demonstrate that HPβCD has the potential to be used as a candidate for drug delivery vector development and signify a correlation between the in vitro cytotoxic effect and cholesterol binding of cyclodextrins.

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.

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.

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.

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.

Glucocorticoids (GCs) are steroid hormones that have inflammatory and immunosuppressive effects on a wide variety of cells. They are used as therapy for inflammatory disease and as a common agent against edema. The blood brain barrier (BBB), comprising microvascular endothelial cells, serves as a permeability screen between the blood and the brain. As such, it maintains homeostasis of the central nervous system (CNS). In many CNS disorders, BBB integrity is compromised. GC treatment has been demonstrated to improve the tightness of the BBB. The responses and effects of GCs are mediated by the ubiquitous GC receptor (GR). Ligand-bound GR recognizes and binds to the GC response element located within the promoter region of target genes. Transactivation of certain target genes leads to improved barrier properties of endothelial cells. In this review, we deal with the role of GCs in endothelial cell barrier function. First, we describe the mechanisms of GC action at the molecular level. Next, we discuss the regulation of the BBB by GCs, with emphasis on genes targeted by GCs such as occludin, claudins and VE-cadherin. Finally, we present currently available GC therapeutic strategies and their limitations.

In this study, we investigated the ability of the general anesthetic propofol (PR) to form inclusion complexes with modified β-cyclodextrins, including sulfobutylether-β-cyclodextrin (SBEβCD) and hydroxypropyl-β-cyclodextrin (HPβCD). The PR/SBEβCD and PR/HPβCD complexes were prepared and characterized, and the blood–brain barrier (BBB) permeation potential of the formulated PR was examined in vivo for the purpose of controlled drug delivery. The PR/SBEβCD complex was found to be more stable in solution with a minimal degradation constant of 0.25 h–1, a t1/2 of 2.82 h, and a Kc of 5.19 × 103 M–1 and revealed higher BBB permeability rates compared with the reference substance (PR-LIPURO) considering the calculated brain-to-blood concentration ratio (logBB) values. Additionally, the diminished PR binding affinity to SBEβCD was confirmed in molecular dynamics simulations by a maximal Gibbs free energy of binding (ΔGbind = −18.44 kcal·mol–1), indicating the more rapid PR/SBEβCD dissociation. Overall, the resul...

The blood–brain barrier (BBB) maintains brain homoeostasis and is a physical and enzymatic barrier between blood circulation and brain parenchyma. Many substances such as glucose or amino acids are transported into the brain by specific transporter molecules expressed at the luminal side of brain vessels. In vitro BBB models have become a standard tool for estimating the ability of drugs to pass the BBB at the early stage of drug development. The simplest in vitro BBB models are endothelial cell–based monocultures in Transwell systems. Such models can be optimized by co-culturing endothelial cells with astrocytes and/or pericytes or using a cell culture medium containing astrocyte/pericyte-derived growth factors. This chapter summarizes cell-based in vitro BBB models described in the literature, gives an overview of strategies to optimize the in vitro BBB model, and briefly describes how to measure drug permeability in vitro.

The blood-brain barrier (BBB), made up of endothelial cells of capillaries in the brain, maintains the microenvironment of the central nervous system. During ischemia and traumatic brain injury (TBI), cellular disruption leading to mechanical insult results to the BBB being compromised. Oxygen glucose deprivation (OGD) is the most commonly used in vitro model for ischemia. On the other hand, stretch injury is currently being used to model TBI in vitro. In this paper, the two methods are used alone or in combination, to assess their effects on cerebrovascular endothelial cells cEND in the presence or absence of astrocytic factors. Applying severe stretch and/or OGD to cEND cells in our experiments resulted to cell swelling and distortion. Damage to the cells induced release of lactate dehydrogenase enzyme (LDH) and nitric oxide (NO) into the cell culture medium. In addition, mRNA expression of inflammatory markers interleukin (I L)-6, IL-1\(\alpha\) chemokine (C-C motif) ligand 2 (CCL2) and tumor necrosis factor (TNF)-\(\alpha\) also increased. These events could lead to the opening of calcium ion channels resulting to excitotoxicity. This could be demonstrated by increased calcium level in OGD-subjected cEND cells incubated with astrocyte-conditioned medium. Furthermore, reduction of cell membrane integrity decreased tight junction proteins claudin-5 and occludin expression. In addition, permeability of the endothelial cell monolayer increased. Also, since cell damage requires an increased uptake of glucose, expression of glucose transporter glut1 was found to increase at the mRNA level after OGD. Overall, the effects of OGD on cEND cells appear to be more prominent than that of stretch with regards to TJ proteins, NO, glutl expression, and calcium level. Astrocytes potentiate these effects on calcium level in cEND cells. Combining both methods to model TBI in vitro shows a promising improvement to currently available models.

Sergey Shityakov,1 Ellaine Salvador,1 Giorgia Pastorin,2 Carola Förster1 1Department of Anaesthesia and Critical Care, University of Würzburg, Würzburg, Germany; 2Department of Pharmacy, National University ofSingapore, Singapore Abstract: In this study, the ability of a multiwalled carbon nanotube functionalized with fluorescein isothiocyanate (MWCNT–FITC) was assessed as a prospective central nervous system-targeting drug delivery system to permeate the blood–brain barrier. The results indicated that the MWCNT–FITC conjugate is able to penetrate microvascular cerebral endothelial monolayers; its concentrations in the Transwell® system were fully equilibrated after 48 hours. Cell viability test, together with phase-contrast and fluorescence microscopies, did not detect any signs of MWCNT–FITC toxicity on the cerebral endothelial cells. These microscopic techniques also revealed presumably the intracellular localization of fluorescent MWCNT–FITCs apart from their massive nonfluorescent accumulation on the cellular surface due to nanotube lipophilic properties. In addition, the 1,000 ps molecular dynamics simulation in vacuo discovered the phenomenon of carbon nanotube aggregation driven by van der Waals forces via MWCNT–FITC rapid dissociation as an intermediate phase. Keywords: blood–brain barrier, multiwalled carbon nanotube, fluorescein isothiocyanate, Transwell® system, aggregation, fluorescence microscopy, molecular dynamics

Asymptomatic bacteriuria established by intravesical inoculation of Escherichia coli 83972 is protective in patients with recurrent urinary tract infections. In this randomized, controlled crossover study a total of 3 symptomatic urinary tract infection episodes developed in 2 patients while they carried E. coli 83972. We examined whether virulence reacquisition by symptom isolates may account for the switch from asymptomatic bacteriuria to symptomatic urinary tract infection.We used E. coli 83972 re-isolates from 2 patients in a prospective study and from another 2 in whom symptoms developed after study completion. We phylogenetically classified the re-isolates, and identified the genomic restriction patterns and gene expression profiles as well as virulence gene structure and phenotypes. In vivo virulence was examined in the murine urinary tract infection model.The fim, pap, foc, hlyA, fyuA, iuc, iroN, kpsMT K5 and malX genotypes of the symptomatic re-isolates remained unchanged. Bacterial gene expression profiles of flagellated symptomatic re-isolates were unique to each host, providing no evidence of common deregulation. Symptomatic isolates did not differ in virulence from the wild-type strain, as defined in the murine urinary tract infection model by persistence, symptoms or innate immune activation.The switch from asymptomatic E. coli 83972 carriage to symptomatic urinary tract infection was not explained by reversion to a functional virulence gene repertoire.

Introduction The existence of the blood–brain barrier in the human body leads to the insufficiency in delivering therapeutic compounds into the brain for the effective treatment of various neurological disorders. In order to determine the possibility of such agents to penetrate through the blood–brain barrier, different in silico, in vitro and in vivo methods may be implemented. Some of them are often provided with unreliable results while others are not feasible in high-throughput screening environment. The goal of this review was to characterise the latest state-of-the-art methods that have been developed and used in the pharmaceutical research in the last few decades to assess the permeation of novel therapeutic entities across the blood–brain barrier. We carried out a literature research and study selection by searching for published biomedical articles in the PubMed archive. Conclusion

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. 1 ). Human tissue samples are available only in a restricted number of laboratories or companies 2,3 . While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest. Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND 4 (from cerebral cortex) and cerebEND 5 (from cerebellar cortex), were isolated according to this procedure in the Forster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- 4,6-9 and estrogen-treatment 10 as well as to pro-infammatory mediators, such as TNFalpha 5,8 . Moreover, we have studied the pathology of multiple sclerosis 11 and hypoxia 12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.

Bacterial pathogens with the ability to cause persistent infection have different strategies to withstand the induction of host immune responses and to successfully establish long-term colonization. In case of asymptomatic bacteriuria and other persistent infections, prolonged growth in the host is accompanied with genomic alterations that result in e.g., bacterial attenuation thus contributing to bacterial adaptation to their host niche and a reduced activation of host immune responses. The accumulating amount of information regarding bacterial adaptation during persistent infection helps to increase our understanding of driving forces of bacterial adaptation in vivo as well as of factors that contribute to symptomatic infection.

The mucosal immune system identifies and fights invading pathogens, while allowing non-pathogenic organisms to persist. Mechanisms of pathogen/non-pathogen discrimination are poorly understood, as is the contribution of human genetic variation in disease susceptibility. We describe here a new, IRF3-dependent signaling pathway that is critical for distinguishing pathogens from normal flora at the mucosal barrier. Following uropathogenic E. coli infection, Irf3−/− mice showed a pathogen-specific increase in acute mortality, bacterial burden, abscess formation and renal damage compared to wild type mice. TLR4 signaling was initiated after ceramide release from glycosphingolipid receptors, through TRAM, CREB, Fos and Jun phosphorylation and p38 MAPK-dependent mechanisms, resulting in nuclear translocation of IRF3 and activation of IRF3/IFNβ-dependent antibacterial effector mechanisms. This TLR4/IRF3 pathway of pathogen discrimination was activated by ceramide and by P-fimbriated E. coli, which use ceramide-anchored glycosphingolipid receptors. Relevance of this pathway for human disease was supported by polymorphic IRF3 promoter sequences, differing between children with severe, symptomatic kidney infection and children who were asymptomatic bacterial carriers. IRF3 promoter activity was reduced by the disease-associated genotype, consistent with the pathology in Irf3−/− mice. Host susceptibility to common infections like UTI may thus be strongly influenced by single gene modifications affecting the innate immune response., Author Summary The host immune system must identify pathogens and defeat them through TLR-dependent signaling pathway activation, while distinguishing them from commensal flora. Contrary to current dogma, the host cannot solely use “pattern recognition” since the microbial molecules involved in such recognition are present on pathogens and commensals alike. We identify here a pathogen-specific mechanism of TLR4 activation and signaling intermediates in this pathway, leading to IRF3-dependent transcription of innate immune response genes. We show in knockout mice that Irf3 deficiency causes severe tissue pathology and that effector functions controlled by IFNβ are involved. Finally, in highly disease-prone pyelonephritis patients we found a high frequency of IRF3 promoter polymorphism compared to asymptomatic bacterial carriers or controls. The polymorphisms influenced promoter activity in reporter assays, suggesting that they are functionally important. Urinary tract infections are among the most common bacterial infections in man, and are a major cause of morbidity and mortality. A subset of disease-prone individuals is at risk for recurrent disease, severe renal dysfunction and end-stage renal disease. At present, there is no method to identify disease-prone infants and to prevent future morbidity and renal damage. The genetic and functional studies described here indicate that genetic variation in IRF3 influences individual susceptibility to kidney infection and might serve as a new tool for future risk assessment in this patient group.

Purpose of review Tight junctions (TJs) are specialized differentiations of epithelial and endothelial cell membranes. TJs play an important role in the adhesion of cells and their interaction with each other. Most cancers originate from epithelial cells. Thus, it is of significance to examine the role of TJs in the tumor microenvironment (TME) and how they affect cancer metastasis. Recent findings In epithelium-derived cancers, intactness of the primary tumor mass is influenced by intercellular structures as well as cell-to-cell adhesion. Irregularities of these factors may lead to tumor dissociation and subsequent metastasis. Low expression of TJs is observed among highly metastatic cancer cells. Summary In this review, we summarized findings from current literature in consideration of the role of TJs in relation to the TME and cancer. Deeper understanding of the mechanisms leading to TJ dysregulation is needed to facilitate the design and conceptualization of new and better therapeutic strategies for cancer.