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Methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter has emerged as strong prognostic factor in the therapy of glioblastoma multiforme. It is associated with an improved response to chemotherapy with temozolomide and longer overall survival. MGMT promoter methylation has implications for the clinical course of patients. In recent years, there have been observations of patients changing their MGMT promoter methylation from primary tumor to relapse. Still, data on this topic are scarce. Studies often consist of only few patients and provide rather contrasting results, making it hard to draw a clear conclusion on clinical implications. Here, we summarize the previous publications on this topic, add new cases of changing MGMT status in relapse and finally combine all reports of more than ten patients in a statistical analysis based on the Wilson score interval. MGMT promoter methylation changes are seen in 115 of 476 analyzed patients (24%; CI: 0.21–0.28). We discuss potential reasons like technical issues, intratumoral heterogeneity and selective pressure of therapy. The clinical implications are still ambiguous and do not yet support a change in clinical practice. However, retesting MGMT methylation might be useful for future treatment decisions and we encourage clinical studies to address this topic
Despite its significant overexpression in several malignant neoplasms, the expression of RPS27 in the central nervous system (CNS) is widely unknown. We identified the cell types expressing RPS27 in the CNS under normal and disease conditions. We acquired specimens of healthy brain (NB), adult pilocytic astrocytoma (PA) World Health Organization (WHO) grade I, anaplastic PA WHO grade III, gliomas WHO grade II/III with or without isocitrate dehydrogenase (IDH) mutation, and glioblastoma multiforme (GBM). RPS27 protein expression was examined by immunohistochemistry and double-fluorescence staining and its mRNA expression quantified by RT-PCR. Patients’ clinical and tumor characteristics were collected retrospectively. RPS27 protein was specifically expressed in tumor cells and neurons, but not in healthy astrocytes. In tumor tissue, most macrophages were positive, while this was rarely the case in inflamed tissue. Compared to NB, RPS27 mRNA was in mean 6.2- and 8.8-fold enhanced in gliomas WHO grade II/III with (p < 0.01) and without IDH mutation (p = 0.01), respectively. GBM displayed a 4.6-fold increased mean expression (p = 0.02). Although RPS27 expression levels did not affect the patients’ survival, their association with tumor cells and tumor-associated macrophages provides a rationale for a future investigation of a potential function during gliomagenesis and tumor immune response.
Background
The most threatening metastases in breast cancer are brain metastases, which correlate with a very poor overall survival, but also a limited quality of life. A key event for the metastatic progression of breast cancer into the brain is the migration of cancer cells across the blood-brain barrier (BBB).
Methods
We adapted and validated the CD34\(^+\) cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) to analyse the effects of patient serum on BBB properties. We collected serum samples from healthy donors, breast cancer patients with primary cancer, and breast cancer patients with, bone, visceral or cerebral metastases. We analysed cytokine levels in these sera utilizing immunoassays and correlated them with clinical data. We used paracellular permeability measurements, immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro.
Results
The BLECs cultured together with brain pericytes in transwells developed a tight monolayer with a correct localization of claudin-5 at the tight junctions (TJ). Several BBB marker proteins such as the TJ proteins claudin-5 and occludin, the glucose transporter GLUT-1 or the efflux pumps PG-P and BCRP were upregulated in these cultures. This was accompanied by a reduced paracellular permeability for fluorescein (400 Da). We then used this model for the treatment with the patient sera. Only the sera of breast cancer patients with cerebral metastases had significantly increased levels of the cytokines fractalkine (CX3CL1) and BCA-1 (CXCL13). The increased levels of fractalkine were associated with the estrogen/progesterone receptor status of the tumour. The treatment of BLECs with these sera selectively increased the expression of CXCL13 and TJ protein occludin. In addition, the permeability of fluorescein was increased after serum treatment.
Conclusion
We demonstrate that the CD34\(^+\) cell-derived human in vitro BBB model can be used as a tool to study the molecular mechanisms underlying cerebrovascular pathologies. We showed that serum from patients with cerebral metastases may affect the integrity of the BBB in vitro, associated with elevated concentrations of specific cytokines such as CX3CL1 and CXCL13.
Objective
Recently, we described a disintegrin and metalloproteinase 9 (ADAM9) overexpression by Schwann cells of vestibular schwannoma (VS) and suggested that it might be a marker for VS tumor growth and invasiveness. This research note provides additional data utilizing a small cohort of VS primary cultures and tissue samples. We examined whether reconstitution of Merlin expression in VS cells regulates ADAM9 protein expression and performed lentiviral ADAM9 knock down to investigate possible effects on VS cells numbers. Moreover, the co-localization of ADAM9 and Integrins α6 and α2β1, respectively, was examined by immunofluorescence double staining.
Results
ADAM9 expression was not regulated by Merlin in VS. However, ADAM9 knock down led to 58% reduction in cell numbers in VS primary cell cultures (p < 0.0001). While ADAM9 and Integrin α2β1 were co-localized in only 22% (2 of 9) of VS, ADAM9 and Integrin α6 were co-localized in 91% (10 of 11) of VS. Therefore, we provide first observations on possible regulatory functions of ADAM9 expression in VS.
Inhibition of the protein kinase MPS1, a mitotic spindle-checkpoint regulator, reinforces the effects of multiple therapies against glioblastoma multiforme (GBM) in experimental settings. We analyzed MPS1 mRNA-expression in gliomas WHO grade II, III and in clinical subgroups of GBM. Data were obtained by qPCR analysis of tumor and healthy brain specimens and correlated with the patients’ clinical data. MPS1 was overexpressed in all gliomas on an mRNA level (ANOVA, p < 0.01) and correlated with tumor aggressiveness. We explain previously published conflicting results on survival: high MPS1 was associated with poorer long term survival when all gliomas were analyzed combined in one group (Cox regression: t < 24 months, p = 0.009, Hazard ratio: 8.0, 95% CI: 1.7–38.4), with poorer survival solely in low-grade gliomas (LogRank: p = 0.02, Cox regression: p = 0.06, Hazard-Ratio: 8.0, 95% CI: 0.9–66.7), but not in GBM (LogRank: p > 0.05). This might be due to their lower tumor volume at the therapy start. GBM patients with high MPS1 mRNA-expression developed clinical symptoms at an earlier stage. This, however, did not benefit their overall survival, most likely due to the more aggressive tumor growth. Since MPS1 mRNA-expression in gliomas was enhanced with increasing tumor aggressiveness, patients with the worst outcome might benefit best from a treatment directed against MPS1.
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.
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 O\(^6\)-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.
Ten thiosemicarbazone ligands obtained by condensation of pyridine-2-carbaldehyde, quinoline-2-carbaldehyde, 2-acetylpyridine, 2-acetylquinoline, or corresponding 2-pyridyl ketones with thiosemicarbazides RNHC(S)NHNH\(_{2}\) and R=CH\(_{3}\), C\(_{6}\)H\(_{5}\) were prepared in good yield. The reaction of [PdCl\(_{2}\)(cod)] with cod=1,5-cyclooctadiene or K\(_{2}\)[PtCl\(_{4}\)] resulted in a total of 17 Pd(II) and Pt(II) complexes isolated in excellent purity, as demonstrated by \(^{1}\)H, \(^{13}\)C, and, where applicable, \(^{195\)Pt NMR spectroscopy combined with CHNS analysis. The cytotoxicity of the title compounds was studied on four human glioblastoma cell lines (GaMG, U87, U138, and U343). The most active compound, with a Pd(II) metal centre, a 2-quinolinyl ring, and methyl groups on both the proximal C and distal N atoms exhibited an EC\(_{50}\) value of 2.1 μM on the GaMG cell lines, thus being slightly more active than cisplatin (EC\(_{50}\) 3.4 μM) and significantly more potent than temozolomide (EC\(_{50}\) 67.1 μM). Surprisingly, the EC\(_{50}\) values were inversely correlated with the lipophilicity, as determined with the “shake-flask method”, and decreased with the length of the alkyl substituents (C\(_{1}\)>C\(_{8}\)>C\(_{10}\)). Correlation with the different structural motifs showed that for the most promising anticancer activity, a maximum of two aromatic rings (either quinolinyl or pyridyl plus phenyl) combined with one methyl group are favoured and the Pd(II) complexes are slightly more potent than their Pt(II) analogues.
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.
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.
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.
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.
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.
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.
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.
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.