Protocadherin Gamma C3 (PCDHGC3) Is Strongly Expressed in Glioblastoma and Its High Expression Is Associated with Longer Progression-Free Survival of Patients.

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, ß, 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.

Effects of Long-Term Temozolomide Treatment on Glioblastoma and Astrocytoma WHO Grade 4 Stem-like Cells.

Glioblastoma leads to a fatal course within two years in more than two thirds of patients. An essential cornerstone of therapy is chemotherapy with temozolomide (TMZ). The effect of TMZ is counteracted by the cellular repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). The MGMT promoter methylation, the main regulator of MGMT expression, can change from primary tumor to recurrence, and TMZ may play a significant role in this process. To identify the potential mechanisms involved, three primary stem-like cell lines (one astrocytoma with the mutation of the isocitrate dehydrogenase (IDH), CNS WHO grade 4 (HGA)), and two glioblastoma (IDH-wildtype, CNS WHO grade 4) were treated with TMZ. The MGMT promoter methylation, migration, proliferation, and TMZ-response of the tumor cells were examined at different time points. The strong effects of TMZ treatment on the MGMT methylated cells were observed. Furthermore, TMZ led to a loss of the MGMT promoter hypermethylation and induced migratory rather than proliferative behavior. Cells with the unmethylated MGMT promoter showed more aggressive behavior after treatment, while HGA cells reacted heterogenously. Our study provides further evidence to consider the potential adverse effects of TMZ chemotherapy and a rationale for investigating potential relationships between TMZ treatment and change in the MGMT promoter methylation during relapse.

Senescence and associated blood-brain barrier alterations in vitro.

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.

iClick Reactions of Square-Planar Palladium(II) and Platinum(II) Azido Complexes with Electron-Poor Alkynes: Metal-Dependent Preference for N1 vs N2 Triazolate Coordination and Kinetic Studies with 1H and 19F NMR Spectroscopy.

Two square-planar palladium(II) and platinum(II) azido complexes [M(N3)(L)] with L = N-phenyl-2-[1-(2-pyridinyl)ethylidene]hydrazine carbothioamide reacted with four different electron-poor alkynes R-C≡C-R' with R = R' = COOCH3, COOEt, COOCH2CH2OCH3 or R = CF3, R' = COOEt in a [3 + 2] cycloaddition "iClick" reaction. The resulting triazolate complexes [M(triazolateR,R')(L)] were isolated by simple precipitation and/or washing in high purity and good yield. Six out of the eight new compounds feature the triazolate ligand coordinated to the metal center via the N2 nitrogen atom, but fortuitous solubility properties allowed isolation of the N1 isomer in two cases from acetone. When the solvent was changed to DMSO, the N1 → N2 isomerization could be studied by NMR spectroscopy and took several days to complete. 19F NMR studies of the iClick reaction with F3C-C≡C-COOEt led to identification of a putative early linear intermediate in addition to the N1 and N2 isomers, however with the latter as the final product. Rate constants determined by 1H or 19F NMR spectroscopy increased in the order Pd > Pt and CF3/COOEt > COOR/COOR with R = CH3, Et, CH2CH2OCH3. The second-order rate constant k2 > 3.7 M-1 s-1 determined for the reaction of [Pd(N3)(L)] with F3C-C≡C-COOEt is the fastest observed for an iClick reaction so far and compares favorably with that of the most evolved strained alkynes reported for the SPAAC (strain-promoted azide-alkyne cycloaddition) to date. Selected title compounds were evaluated for their anticancer activity on the GaMG human glioblastoma brain cancer cell line and gave EC50 values in the low micromolar range (2-16 µM). The potency of the Pd(II) complexes increased with the chain length of the substituents in the 4- and 5-positions of the triazolate ligand.

High-grade glioma associated immunosuppression does not prevent immune responses induced by therapeutic vaccines in combination with Treg depletion.

High-grade gliomas (HGG) exert systemic immunosuppression, which is of particular importance as immunotherapeutic strategies such as therapeutic vaccines are increasingly used to treat HGGs. In a first cohort of 61 HGG patients we evaluated a panel of 30 hematological and 34 plasma biomarkers. Then, we investigated in a second cohort of 11 relapsed HGG patients receiving immunomodulation with metronomic cyclophosphamide upfront to a DC-based vaccine whether immune abnormalities persisted and whether they hampered induction of IFNγ+ T-cell responses. HGG patients from the first cohort showed increased numbers of leukocytes, neutrophils and MDSCs and in parallel reduced numbers of CD4+/CD8+ T-cells, plasmacytoid and conventional DC2s. MDSCs and T-cell alterations were more profound in WHO IV° glioma patients. Moreover, levels of MDSCs and epidermal growth factor were negatively associated with survival. Serum levels of IL-2, IL-4, IL-5 and IL-10 were altered in HGG patients, however, without any impact on clinical outcome. In the immunotherapy cohort, 6-month overall survival was 100%. Metronomic cyclophosphamide led to > 40% reduction of regulatory T cells (Treg). In parallel to Treg-depletion, MDSCs and DC subsets became indistinguishable from healthy controls, whereas T-lymphopenia persisted. Despite low T-cells, IFNγ-responses could be induced in 9/10 analyzed cases. Importantly, frequency of CD8+VLA-4+ T-cells with CNS-homing properties, but not of CD4+ VLA-4+ T-cells, increased during vaccination. Our study identifies several features of systemic immunosuppression in HGGs. Metronomic cyclophosphamide in combination with an active immunization alleviates the latter and the combined treatment allows induction of a high rate of anti-glioma immune responses.

Study protocol for a multicentre randomised controlled trial evaluating the efficacy of an online yoga intervention in high-grade glioma patients and their caregivers: the YINOTA-O-trial.

INTRODUCTION: High-grade glioma patients and their caregivers often suffer from distress and a lower quality of life. Results from studies with patients with mixed cancer entities suggest that yoga can be an effective support. However, it is unclear whether this also applies to high-grade glioma patients and their caregivers. This study aims to investigate the effects of mindfulness-based online yoga for patients and their caregivers on emotional distress, quality of life and stress-associated physiological parameters compared with a waiting control group (WCG). METHODS & ANALYSIS: The study is designed as a multicentre randomised controlled trial. Adult glioma patients (central nervous system WHO grades 3 and 4) and their caregivers will be recruited. Examined yoga instructors deliver the intervention (1 hour per week) in a synchronous format over 8 weeks via video conferencing. The WCG will receive standard care during the 8-week waiting period. Data will be collected before and after the end of the intervention and another 3 months later using questionnaires as well as blood serum and hair samples to evaluate biochemical stress parameters. Primary outcome is self-reported generalised anxiety and secondary outcomes are self-reported fear of progression, depression and quality of life as well as brain-derived neurotrophic factor (BDNF), dehydroepiandrosterone (DHEA)/dehydroepiandrosterone sulfate (DHEAS), ferritin and hair cortisol. We hypothesise better outcomes in the intervention group compared with the WCG at all measurement points. 70 patients and 70 caregivers will be recruited consecutively. Primary endpoints are significant effect detections in the Generalised Anxiety Disorder scale-7 of patients and caregivers at the end of the intervention. Analyses of covariance will be performed to analyse the treatment effects. ETHICS AND DISSEMINATION: The Ethics Committee of the University of Würzburg approved the YINOTA-O (Yoga-Intervention bei Neuroonkologischen Tumorpatienten und deren Angehörigen - Online) study on 26 October 2021 (No.185/18-me). Results will be presented at conferences and published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: German Clinical Trials Register No. DRKS00029554.

Development of a vestibular schwannoma tumor slice model for pharmacological testing.

BACKGROUND: Our goal was to develop a 3D tumor slice model, replicating the individual tumor microenvironment and for individual pharmaceutical testing in vestibular schwannomas with and without relation to NF2. METHODS: Tissue samples from 16 VS patients (14 sporadic, 2 NF2-related) were prospectively analyzed. Slices of 350 µm thickness were cultured in vitro, and the 3D tumor slice model underwent thorough evaluation for culturing time, microenvironment characteristics, morphology, apoptosis, and proliferation rates. Common drugs - Lapatinib (10 µM), Nilotinib (20 µM), and Bevacizumab (10 µg/ml) - known for their responses in VS were used for treatment. Treatment responses were assessed using CC3 as an apoptosis marker and Ki67 as a proliferation marker. Standard 2D cell culture models of the same tumors served as controls. RESULTS: The 3D tumor slice model accurately mimicked VS ex vivo, maintaining stability for three months. Cell count within the model was approximately tenfold higher than in standard cell culture, and the tumor microenvironment remained stable for 46 days. Pharmacological testing was feasible for up to three weeks, revealing interindividual differences in treatment response to Lapatinib and intraindividual variability in response to Lapatinib and Nilotinib. The observed effects were less pronounced in tumor slices than in standard cell culture, indicating the model's proximity to in vivo tumor biology and enhanced realism. Bevacizumab had limited impact in both models. CONCLUSION: This study introduces a 3D tumor slice model for sporadic and NF2-related VS, demonstrating stability for up to 3 months, replication of the schwannoma microenvironment, and utility for individualized pharmacological testing.

Analysis of tumor microenvironment composition in vestibular schwannomas: insights into NF2-associated and sporadic variations and their clinical correlations.

Objective: Vestibular schwannomas (VS), benign tumors stemming from the eighth cranial nerve's Schwann cells, are associated with Merlin gene mutations, inflammation, and the tumor microenvironment (TME), influencing tumor initiation, maintenance, and potential neural dysfunction. Understanding TME composition holds promise for systemic therapeutic interventions, particularly for NF2-related schwannomatosis. Methodology: A retrospective analysis of paraffin-embedded tissue from 40 patients (2013-2020), evenly divided by neurofibromatosis type 2 status, with further stratification based on magnetic resonance imaging (MRI) progression and hearing function. Immunohistochemistry assessed TME components, including T-cell markers (CD4, CD8, CD25), NK cells (CD7), and macrophages (CD14, CD68, CD163, CCR2). Fiji software facilitated image analysis. Results: T-cell markers (CD4, CD8, CD7) exhibited low expression in VS, with no significant NF2-associated vs. sporadic distinctions. Macrophage-related markers (CD14, CD68, CD163, CCR2) showed significantly higher expression (CD14: p = 0.0187, CD68: p < 0.0001, CD163: p = 0.0006, CCR2: p < 0.0001). CCR2 and CD163 significantly differed between NF2-associated and sporadic VS. iNOS, an M1-macrophage marker, was downregulated. CD25, a regulatory T-cell marker, correlated significantly with tumor growth dynamics (p = 0.016). Discussion: Immune cells, notably monocytes and macrophages, crucially contribute to VS pathogenesis in both NF2-associated and sporadic cases. Significant differences in CCR2 and CD163 expression suggest distinct immune responses. Regulatory T-cells may serve as growth dynamic markers. These findings highlight immune cells as potential biomarkers and therapeutic targets for managing VS.

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper.

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing realistic ex vivo models remain challenging. Patient-derived 3-dimensional organoid (PDO) models offer innovative platforms that capture the phenotypic and molecular heterogeneity of GBM, while preserving key characteristics of the original tumors. However, manual dissection for PDO generation is time-consuming, expensive and can result in a number of irregular and unevenly sized PDOs. This study presents an innovative method for PDO production using an automated tissue chopper. Tumor samples from four GBM and one astrocytoma, IDH-mutant, CNS WHO grade 2 patients were processed manually as well as using the tissue chopper. In the manual approach, the tumor material was dissected using scalpels under microscopic control, while the tissue chopper was employed at three different angles. Following culture on an orbital shaker at 37 °C, morphological changes were evaluated using bright field microscopy, while proliferation (Ki67) and apoptosis (CC3) were assessed by immunofluorescence after 6 weeks. The tissue chopper method reduced almost 70% of the manufacturing time and resulted in a significantly higher PDOs mean count compared to the manually processed tissue from the second week onwards (week 2: 801 vs. 601, P = 0.018; week 3: 1105 vs. 771, P = 0.032; and week 4:1195 vs. 784, P < 0.01). Quality assessment revealed similar rates of tumor-cell apoptosis and proliferation for both manufacturing methods. Therefore, the automated tissue chopper method offers a more efficient approach in terms of time and PDO yield. This method holds promise for drug- or immunotherapy-screening of GBM patients.

Hypoxia induced CA9 inhibitory targeting by two different sulfonamide derivatives including acetazolamide in human glioblastoma.

HIF-1α regulated genes are mainly responsible for tumour resistance to radiation- and chemo-therapy. Among these genes, carbonic anhydrase isoform IX (CA9) is highly over expressed in many types of cancer especially in high grade brain cancer like Glioblastoma (GBM). Inhibition of the enzymatic activity by application of specific chemical CA9 inhibitor sulphonamides (CAI) like Acetazolamide (Aza.), the new sulfonamide derivative carbonic anhydrase inhibitor (SU.D2) or indirect inhibitors like the HIF-1α inhibitor Chetomin or molecular inhibitors like CA9-siRNA are leading to an inhibition of the functional role of CA9 during tumorigenesis. Human GBM cells were treated with in vitro hypoxia (1, 6, or 24 h at 0.1%, O2). Aza. application was at a range between 250 and 8000 nM and the HIF-1α inhibitor Chetomin at a concentration range of 150-500 nM. Cell culture plates were incubated for 24 h under hypoxia (0.1% O2). Further, CA9-siRNA constructs were transiently transfected into GBM cells exposed to extreme hypoxic aeration conditions. CA9 protein expression level was detectable in a cell-type specific manner under normoxic conditions. Whereas U87-MG exhibited a strong aerobic expression, U251 and U373 displayed moderate and GaMG very weak normoxic CA9 protein bands. Aza. as well as SU.D2 displayed inhibitory characteristics to hypoxia induced CA9 expression in the four GBM cell lines for 24 h of hypoxia (0.1% O2) at concentrations between 3500 and 8000 nM, on both the protein and mRNA level. Parallel experiments using CA9-siRNA confirmed these results. Application of 150-500 nM of the glycolysis inhibitor Chetomin under similar oxygenation conditions led to a sharply reduced expression of both CA IX protein and CA9 mRNA levels, indicating a clear glucose availability involvement for the hypoxic HIF-1α and CA9 expression in GBM cells. Hypoxia significantly influences the behaviour of human tumour cells by activation of genes involved in the adaptation to hypoxic stress. The main objective in malignant GBM therapy is either to eradicate the tumour or to convert it into a controlled, quiescent chronic disease. Aza., SU.D2, Chetomin or CA9-siRNA possesses functional CA9 inhibitory characteristics when applied against human cancers with hypoxic regions like GBM. They may be used as alternative or in conjunction with other direct inhibitors possessing similar functionality, thereby rendering them as potential optimal tools for the development of an optimized therapy in human brain cancer treatment.

Tumor Treating Fields (TTFields) Therapy Concomitant with Taxanes for Cancer Treatment.

Non-small cell lung cancer, ovarian cancer, and pancreatic cancer all present with high morbidity and mortality. Systemic chemotherapies have historically been the cornerstone of standard of care (SOC) regimens for many cancers, but are associated with systemic toxicity. Multimodal treatment combinations can help improve patient outcomes; however, implementation is limited by additive toxicities and potential drug-drug interactions. As such, there is a high unmet need to develop additional therapies to enhance the efficacy of SOC treatments without increasing toxicity. Tumor Treating Fields (TTFields) are electric fields that exert physical forces to disrupt cellular processes critical for cancer cell viability and tumor progression. The therapy is locoregional and is delivered noninvasively to the tumor site via a portable medical device that consists of field generator and arrays that are placed on the patient's skin. As a noninvasive treatment modality, TTFields therapy-related adverse events mainly consist of localized skin reactions, which are manageable with effective acute and prophylactic treatments. TTFields selectively target cancer cells through a multi-mechanistic approach without affecting healthy cells and tissues. Therefore, the application of TTFields therapy concomitant with other cancer treatments may lead to enhanced efficacy, with low risk of further systemic toxicity. In this review, we explore TTFields therapy concomitant with taxanes in both preclinical and clinical settings. The summarized data suggest that TTFields therapy concomitant with taxanes may be beneficial in the treatment of certain cancers.

Tumor Treating Fields (TTFields) Induce Cell Junction Alterations in a Human 3D In Vitro Model of the Blood-Brain Barrier.

In a recent study, we showed in an in vitro murine cerebellar microvascular endothelial cell (cerebEND) model as well as in vivo in rats that Tumor-Treating Fields (TTFields) reversibly open the blood-brain barrier (BBB). This process is facilitated by delocalizing tight junction proteins such as claudin-5 from the membrane to the cytoplasm. In investigating the possibility that the same effects could be observed in human-derived cells, a 3D co-culture model of the BBB was established consisting of primary microvascular brain endothelial cells (HBMVEC) and immortalized pericytes, both of human origin. The TTFields at a frequency of 100 kHz administered for 72 h increased the permeability of our human-derived BBB model. The integrity of the BBB had already recovered 48 h post-TTFields, which is earlier than that observed in cerebEND. The data presented herein validate the previously observed effects of TTFields in murine models. Moreover, due to the fact that human cell-based in vitro models more closely resemble patient-derived entities, our findings are highly relevant for pre-clinical studies.

Metastasis Associated in Colorectal Cancer 1 (MACC1) mRNA Expression Is Enhanced in Sporadic Vestibular Schwannoma and Correlates to Deafness.

Vestibular schwannoma (VS) are benign cranial nerve sheath tumors of the vestibulocochlear nerve. Their incidence is mostly sporadic, but they can also be associated with NF2-related schwannomatosis (NF2), a hereditary tumor syndrome. Metastasis associated in colon cancer 1 (MACC1) is known to contribute to angiogenesis, cell growth, invasiveness, cell motility and metastasis of solid malignant cancers. In addition, MACC1 may be associated with nonsyndromic hearing impairment. Therefore, we evaluated whether MACC1 may be involved in the pathogenesis of VS. Sporadic VS, recurrent sporadic VS, NF2-associated VS, recurrent NF2-associated VS and healthy vestibular nerves were analyzed for MACC1 mRNA and protein expression by quantitative polymerase chain reaction and immunohistochemistry. MACC1 expression levels were correlated with the patients' clinical course and symptoms. MACC1 mRNA expression was significantly higher in sporadic VS compared to NF2-associated VS (p < 0.001). The latter expressed similar MACC1 concentrations as healthy vestibular nerves. Recurrent tumors resembled the MACC1 expression of the primary tumors. MACC1 mRNA expression was significantly correlated with deafness in sporadic VS patients (p = 0.034). Therefore, MACC1 might be a new molecular marker involved in VS pathogenesis.

Telemedicine in Neuro-Oncology-An Evaluation of Remote Consultations during the COVID-19 Pandemic.

In order to minimize the risk of infections during the COVID-19 pandemic, remote video consultations (VC) experienced an upswing in most medical fields. However, telemedicine in neuro-oncology comprises unique challenges and opportunities. So far, evidence-based insights to evaluate and potentially customize current concepts are scarce. To fill this gap, we analyzed >3700 neuro-oncological consultations, of which >300 were conducted as VC per patients' preference, in order to detect how both patient collectives distinguished from one another. Additionally, we examined patients' reasons, suitable/less suitable encounters, VC's benefits and disadvantages and future opportunities with an anonymized survey. Patients that participated in VC had a worse clinical condition, higher grade of malignancy, were more often diagnosed with glioblastoma and had a longer travel distance (all p < 0.01). VC were considered a fully adequate alternative to face-to-face consultations for almost all encounters that patients chose to participate in (>70%) except initial consultations. Most participants preferred to alternate between both modalities rather than participate in one alone but preferred VC over telephone consultation. VC made patients feel safer, and participants expressed interest in implementing other telemedicine modalities (e.g., apps) into neuro-oncology. VC are a promising addition to patient care in neuro-oncology. However, patients and encounters should be selected individually.

BRMS1 in Gliomas-An Expression Analysis.

The metastatic suppressor BRMS1 interacts with critical steps of the metastatic cascade in many cancer entities. As gliomas rarely metastasize, BRMS1 has mainly been neglected in glioma research. However, its interaction partners, such as NFκB, VEGF, or MMPs, are old acquaintances in neurooncology. The steps regulated by BRMS1, such as invasion, migration, and apoptosis, are commonly dysregulated in gliomas. Therefore, BRMS1 shows potential as a regulator of glioma behavior. By bioinformatic analysis, in addition to our cohort of 118 specimens, we determined BRMS1 mRNA and protein expression as well as its correlation with the clinical course in astrocytomas IDH mutant, CNS WHO grade 2/3, and glioblastoma IDH wild-type, CNS WHO grade 4. Interestingly, we found BRMS1 protein expression to be significantly decreased in the aforementioned gliomas, while BRMS1 mRNA appeared to be overexpressed throughout. This dysregulation was independent of patients' characteristics or survival. The protein and mRNA expression differences cannot be finally explained at this stage. However, they suggest a post-transcriptional dysregulation that has been previously described in other cancer entities. Our analyses present the first data on BRMS1 expression in gliomas that can provide a starting point for further investigations.

Characterization and Optimization of the Tumor Microenvironment in Patient-Derived Organotypic Slices and Organoid Models of Glioblastoma.

While glioblastoma (GBM) is still challenging to treat, novel immunotherapeutic approaches have shown promising effects in preclinical settings. However, their clinical breakthrough is hampered by complex interactions of GBM with the tumor microenvironment (TME). Here, we present an analysis of TME composition in a patient-derived organoid model (PDO) as well as in organotypic slice cultures (OSC). To obtain a more realistic model for immunotherapeutic testing, we introduce an enhanced PDO model. We manufactured PDOs and OSCs from fresh tissue of GBM patients and analyzed the TME. Enhanced PDOs (ePDOs) were obtained via co-culture with PBMCs (peripheral blood mononuclear cells) and compared to normal PDOs (nPDOs) and PT (primary tissue). At first, we showed that TME was not sustained in PDOs after a short time of culture. In contrast, TME was largely maintained in OSCs. Unfortunately, OSCs can only be cultured for up to 9 days. Thus, we enhanced the TME in PDOs by co-culturing PDOs and PBMCs from healthy donors. These cellular TME patterns could be preserved until day 21. The ePDO approach could mirror the interaction of GBM, TME and immunotherapeutic agents and may consequently represent a realistic model for individual immunotherapeutic drug testing in the future.

The Mechanisms of Action of Tumor Treating Fields.

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.

Tumor Treating Fields (TTFields) Reversibly Permeabilize the Blood-Brain Barrier In Vitro and In Vivo.

Despite the availability of numerous therapeutic substances that could potentially target CNS disorders, an inability of these agents to cross the restrictive blood-brain barrier (BBB) limits their clinical utility. Novel strategies to overcome the BBB are therefore needed to improve drug delivery. We report, for the first time, how Tumor Treating Fields (TTFields), approved for glioblastoma (GBM), affect the BBB's integrity and permeability. Here, we treated murine microvascular cerebellar endothelial cells (cerebEND) with 100-300 kHz TTFields for up to 72 h and analyzed the expression of barrier proteins by immunofluorescence staining and Western blot. In vivo, compounds normally unable to cross the BBB were traced in healthy rat brain following TTFields administration at 100 kHz. The effects were analyzed via MRI and immunohistochemical staining of tight-junction proteins. Furthermore, GBM tumor-bearing rats were treated with paclitaxel (PTX), a chemotherapeutic normally restricted by the BBB combined with TTFields at 100 kHz. The tumor volume was reduced with TTFields plus PTX, relative to either treatment alone. In vitro, we demonstrate that TTFields transiently disrupted BBB function at 100 kHz through a Rho kinase-mediated tight junction claudin-5 phosphorylation pathway. Altogether, if translated into clinical use, TTFields could represent a novel CNS drug delivery strategy.

Glioblastoma-Derived Three-Dimensional Ex Vivo Models to Evaluate Effects and Efficacy of Tumor Treating Fields (TTFields).

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.

Human decidua and invasive trophoblasts are rich sources of nearly all human matrix metalloproteinases.

Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. As for all invasive cell types, the ability of CTB to infiltrate the uterine wall is facilitated by the activity of matrix metalloproteinases (MMPs), which is regulated by tissue inhibitors of MMPs (TIMPs). There is evidence for the expression of several MMPs and TIMPs in decidua. However, published data are limited. Therefore, to set a foundation for future research, we screened a panel of healthy human deciduas obtained during first, second and third trimester of pregnancy in addition to isolated decidual cell populations for the expression of all known human MMPs and TIMPs by RT-PCR, western blot and immunohistochemistry. In the decidual samples, we detected almost all MMPs and all four TIMPs at mRNA level. While the expression of proMMP-3 and active MMP-13 and -23 was down-regulated in the course of pregnancy, the pro forms of MMP-8, -19 and -23, active MMP-9, -10, -12, -15, -16, -26 and -28, and pro- and active MMP-14 increased towards the end of gestation. All MMPs and TIMPs were expressed in uterine natural killer cells, decidual fibroblasts and/or trophoblasts, with the exception of MMP-20 and -25. In summary, a remarkably broad spectrum of MMPs was expressed at the human feto-maternal interface, reflecting the highly invasive and remodelling effect on placenta formation. It can be speculated that expression of MMPs correlates with the invasive potential of CTBs together with a crucial role in activation of labour at term.