Carsten Hagemann

Organotypic brain slice cultures are well-established and valuable three-dimensional (3D) models in neuroscience, particularly for cancer research. However, many existing 3D models rely on adult animals or are restricted to specific brain regions, such as the hippocampus. To address these limitations, we established a whole, large-format brain slice model that preserves the complex cellular architecture of the brain and its diverse cell populations. In addition, the use of 6-9-day-old postnatal mice allows a reduction in animal numbers by sectioning each brain into multiple contiguous slices of 300 µm thickness. The slices were maintained in vitro for 7–14 days under stable conditions and served as carrier tissue for brain tumor cell spheroids, thereby creating an ex vivo glioblastoma (GBM) model. To simulate tumor growth and invasion, spheroids of rat 9L/lacZ GBM cells were seeded onto the murine slices. Two different fluorescence-based methods were used to visualize and quantify the viability of the model. The AlamarBlue assay was used to identify the optimal culture medium and to longitudinally monitor whole-slice viability over a 10-day period. In parallel, the structural integrity of the brain slices and tumor invasion patterns were visualized using live/dead staining for up to 14 days post-slicing. Both methods demonstrated that the slices remained viable and stable for one week before a gradual decrease in viability. When co-cultured with GBM spheroids, the slices’ viability slightly decreased, likely due to the tumor’s invasive nature, which compromised the complex brain structures and cell networks. In summary, our ex vivo whole-brain slice GBM model preserves the complex organotypic brain and tumor structures essential for a robust in vitro GBM 3D model. The model stability for a minimum of 7 days, with the potential for extended culture duration, makes it a valuable tool in brain cancer research.

Background: While chimeric antigen receptor (CAR) T cells have achieved encouraging remission rates in hematological malignancies, they demonstrated limited success in treating Glioblastoma (GBM), particularly due to high intra- and intertumoral heterogeneity. In this study, we identified a relevant and preserved target antigen, Podoplanin (PDPN), and evaluated the potential of a PDPN- and GD2-CAR T cell blend to overcome GBM heterogeneity. Methods: Target antigen screening included clinical samples, healthy tissues and cell lines, as well as publicly available RNA sequencing datasets. The anti-tumor function of CAR T cells were examined in co-culture experiments with GBM cell lines and patient-derived organoids (PDOs), and in vivo after locoregional delivery in orthotopic xenograft models. Results: The generated CAR T cells demonstrated strong anti-tumor activity against several cell lines and PDOs from multiple patients. PDPN and GD2 expression was detectable in all PDOs at varying densities and regardless of the antigenic profile, the CAR T cell blend induced significantly higher levels of apoptosis in organoids than single antigen targeting counterparts. In vivo , we observed efficient tumor regression after locoregional administration of monospecific CAR T cells. While heterogeneous orthotopic tumors eventually relapsed in these groups, blended therapy resulted in a significantly increased overall survival and even achieved cure in the majority of mice. Conclusion: This novel PDPN-/GD2-CAR T cell blend demonstrated strong efficacy in advanced preclinical models of glioblastoma. The results suggest that this approach can overcome GBM heterogeneity in clinical application and address previous limitations of single antigen CAR T cell therapies. KEYPOINTS: PDPN is a relevant and consistent CAR T cell target antigen in primary and recurrent GBM PDPN- and GD2-CAR T cells display synergistic anti-tumor activity in patient-derived organoids Local delivery of our CAR T cell blend confers long-term survival and cure in GBM-bearing mice IMPORTANCE OF THE STUDY: The highly variable landscape of tumor antigens in GBM represents a serious obstacle to single antigen CAR T cell therapies. In this study, we identified Podoplanin (PDPN) and GD2 as the most preserved target antigens in a screening campaign, with an even increased density in recurrent GBM compared to primary GBM. We combined PDPN- and GD2-CAR T cells in a blended treatment approach to counter antigen heterogeneity, and achieved a substantial gain in efficacy against patient-derived organoids (PDOs). In an orthotopic xenograft model, locoregional administration of the PDPN- and GD2-CAR T cell blend conferred long-term complete remission and increased survival compared to single antigen targeting. This study provides a hierarchy of CAR target antigens for treating GBM and illustrates the therapeutic potential of combinatorial antigen targeting using a PDPN/GD2-CAR T cell blend. GRAPHICAL

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.

The use of TTFields, which are alternating electric fields of low intensity (1-3 V/cm) and intermediate frequency (100-500 kHz), has shown its clinical efficacy in the treatment of newly diagnosed glioblastoma (GBM) patients at 200 kHz. Nevertheless, more knowledge is required for the prediction of individual tumors’ responsiveness to treatment. Recently, we developed patient-derived three-dimensional GBM tissue culture models to screen patient tumors for TTFields treatment susceptibility. Here, we extended these models to brain tumors beyond GBM, namely anaplastic ependymoma (AE) and oligodendroglioma. We cultured tumor-organoids as microtumors on murine organotypic hippocampal slice cultures (OHSCs) and subjected them to TTFields at 200 kHz. We prepared OHSCs by slicing the brains of mice 5-8 days postpartum to a thickness of 350 µm using a vibratome and culturing them for 14 days. We obtained organoids from freshly resected tumor tissue from three GBM, one AE, and one oligodendroglioma patient, each. The organoids were cultured for 14 days as well and then stained with carboxyfluorescein succinimidyl ester. Next, we placed the organoids onto the OHSCs and administered TTFields at 200 kHz and 1.5 V/cm for 72 h using the inovitro™ laboratory research system. We quantified microtumor growth by fluorescence imaging. The three tumor entities, in particular organoids from GBM, AE and oligodendroglioma, grew as microtumors after being placed onto OHSCs. TTFields treatment at 200 kHz led to significantly decreased microtumor size of the three analysed GBM samples as well as the AE sample. However, the oligodendroglioma microtumor size was not significantly reduced. Based on clinical experience, we expected that TTFields lead to a reduction in microtumor size. However, to our knowledge, we were the first to apply TTFields to patient-derived AE and oligodendroglioma samples. The lack of TTFields-effects on the latter might be due to different inter-patient sensitivity or suboptimal TTFields frequency. Nevertheless, our work is proof of principle that an organoid-based patient-derived 3D tissue culture model could be used to evaluate effects of TTFields on different brain tumor entities.

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.

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.