Brain Tumour Research

Tumor-associated macrophages (TAMs) are key contributors to the brain tumor microenvironment. However, their role in medulloblastoma (MB) progression and chemoresistance remains elusive. We utilized CD11b-diphtheria toxin receptor (DTR)/Ptch1-deficient MB model to genetically delete TAMs. NeuroD2-SmoA1 MB mice were treated with CSF1R inhibitor PLX3397, PI3K inhibitor buparlisib, and a combination of PLX3397 with chemotherapy to examine the functional significance of TAMs in MB. Tumor tissues and cell co-culture system were analyzed using RNA sequencing (RNA-seq), Western blotting, flow cytometry, immunohistochemistry, quantitative PCR and 3D bioprinted MB mono-culture to delineate mechanistic interactions. Myeloid-derived TAMs are abundant in MB tissues and primarily exhibit M2-like polarization. We demonstrate that TAMs depletion, achieved either genetically via diphtheria toxin (DT) or pharmacologically via PLX3397-mediated preferential targeting of M2-like TAMs, markedly downregulates Zic1 expression and impedes MB growth. Mechanistically, M2-like TAMs secreted high levels of IGF1 to activate the PI3K/mTOR/Zic1 axis in MB cells; whereas the PI3K inhibitor buparlisib effectively reduced the population of Zic1-expresssing cells and restricted MB growth. Notably, the PI3K/mTOR/Zic1 axis was demonstrated to confer chemoresistance. While chemotherapy exacerbates M2-like TAMs accumulation within MB, combining PLX3397 with chemotherapy abrogates this infiltration and attenuates IGF1/PI3K/Zic1 signaling axis. This combination strategy synergistically inhibits tumor growthand extends survival inmice. Our findings demonstrate that inhibiting the TAMs-induced IGF1/PI3K/Zic1 signaling axis results in MB regression and chemosensitization. These results underscore the therapeutic potential of combining clinical CSF1R inhibitor with standard chemotherapy as an effective treatment strategy for SHH MB.

The utility of chemotherapy for childhood ependymoma is uncertain without a prior randomized trial. ACNS0831, a multicenter phase 3 randomized study conducted through the Children's Oncology Group (COG), included patients 1-21 years with newly diagnosed intracranial ependymoma. Patients with complete/near-total resections (GTR/NTR) or complete response (CR) to induction therapy were randomized to radiation (RT) alone or RT followed by chemotherapy (RT-CHEMO). Primary outcomes were event-free survival (EFS) and overall survival (OS). Due to anticipated noncompliance with chemotherapy, an "as treated" analysis was planned. Patients with subtotal resection (STR) were non-randomly assigned to RT-CHEMO. Grade 2 supratentorial tumors with GTR or CR to induction therapy were observed. Of 449 eligible patients, 325 with GTR/NTR or CR were randomized. Five-year EFS was 63.7% (95% CI: 55.1%-71.1%) for RT only (n = 161) versus 69.2% (60.8%-76.3%) for RT-CHEMO (n = 164) (1-sided log-rank P = .299, HR = 0.866). Five-year OS was 86.9% (79.8%-91.6%) for RT only versus 88.3% (81.8%-92.6%) for RT-CHEMO (1-sided log-rank P-value = .172, HR = 0.757). The "as treated" and "as randomized" analysis results were similar. Sixty-three subjects with STR were assigned to RT-CHEMO; 5-year EFS was 33.6% (22.1%-45.5%) and OS 74.0% (60.5%-83.5%). Supratentorial grade 2 tumors with GTR or CR were observed with a 5-year EFS of 66.9% (49.0%-79.7%) and OS of 100%. Molecular classification was provided for 94% (n = 422) of all subjects. Primary analysis showed no benefit for maintenance chemotherapy. Further follow-up is important to assess its effect on late relapses. This is the largest cohort of molecularly classified ependymomas treated on a Phase 3 randomized trial.

Medulloblastoma (MB) is one of the most prevalent malignant brain tumors in children, with tremendous cognitive and neuroendocrine disability among survivors. Group 3 MB (G3MB) has poor overall survival at <50%, high frequencies of metastases, and no targeted therapies. Amplification of MYC and activation of TGFβ signaling occur frequently in G3MB. Many tumors have no reported mutations, suggesting epigenetic drivers. We here describe novel humanized models for G3MB from human induced pluripotent stem cells (hiPSC). By transducing hiPSC-derived neuroepithelial stem cells (NESC), we determined that: 1) both MYC and TGFβ effectors drove tumors in vivo; 2) MYC/TGFβR1 in combination led to more aggressive tumors and resistance to clinical inhibitors of TGFβ, and 3) NESC-derived tumors clustered with human G3MB. To decipher mechanisms, we integrated RNA-sequencing with CUT&RUN (for MYC genomic localization and post-translational modification of histones). MYC-bound neural developmental genes were repressed in MYC/TGFβR1 co-driven lines. Gene signatures associated with the Polycomb Repressive Complex (PRC) demarcated with H3K27me3; the histone mark directly regulated by PRC. We identified JARID1B, a MYC binding partner and H3K4me3 demethylase, as a regulator of repressed neural genes. Primary G3MB also showed increased levels of H3K27me3 concurrent with higher expression of JARID1B. Knockdown of JARID1B in human G3MB cell lines reduced growth, supporting potential as a therapeutic target. We conclude that a MYC-TGFβ-JARID1B axis represses target genes to drive G3MB and present new humanized models for G3MB to understand epigenetic dysregulation in G3MB.

Abstract Ependymoma (EPN) is the second most malignant paediatric brain tumour. The PF-A subgroup, associated with a hypoxic microenvironment and overexpression of the epigenetic regulator EZHIP, has a dismal survival rate of 50%, with clinical trials failing to demonstrate a significant survival advantage from chemotherapy to date. We present a multi-omic integration investigating whether spatially-distinct tumour microenvironments represent targetable metabolic niches in PF-A EPN. Surgical sampling of spatially-distinct regions from eight PF-A patients was performed, with metabolites and RNA simultaneously extracted and analysed using LC-MS and RNA-seq. Integration of metabolites and RNA was performed using Metscape3 with functional assays evaluating proliferation and invasion in 2D and 3D patient-derived in-vitro cell models. LC-MS and qPCR were used to analyse post-treatment changes of our lead drug combination under hypoxia and normoxia. Clonogenic assays investigated radiosensitisation (±2 Gy) and in-vivo tolerability was evaluated in non-tumour-bearing mice (Disulfiram (Dsf)/Cu2 + 200/4 mg/kg, i.p.). Multi-omic integration identified 124 dysregulated metabolic pathways, demonstrating heterogeneity within and across PF-A tumours. Based on the identified metabolically relevant genes, Dsf and Cu2+ were highlighted as potential therapeutic agents showing impaired metabolic viability and invasion in 2D and 3D models of PF-A EPN, with radiosensitising effects in 2D clonogenic assays and limited chemosensitivity observed in human cerebellar astrocytes. LC-MS revealed Dsf/Cu2+ disrupts mitochondrial fatty acid oxidation (normoxia) and TCA cycle intermediates (hypoxia), whereas qPCR analysis identified changes in EPOP, EZH2 and PSMB7, implicating EZHIP-mediated regulation and proteasomal alterations. In vivo, DSF/Cu2+ showed no weight loss or overt toxicity in non-tumour-bearing mice. This combination also demonstrated efficacy in additional paediatric in-vitro models, suggesting pan-cancer potential. This is the first instance where multi-omic data integration and intra-tumor heterogeneity have been investigated for paediatric EPN, revealing novel therapeutic targets in the context of gene-metabolite correlations. In-vivo efficacy studies using the MAF-928 model are currently ongoing.

The connective tissue support of female pelvic viscera-endopelvic fascia-has been studied in fetal and immunohistochemical models to demonstrate its relationship with the autonomic nerves of the female pelvis. Due to a paucity of literature examining the gross anatomical relationships between endopelvic fascia and autonomic nerves in adult female pelvises, it remains unknown whether defects in endopelvic fascia predisposing pelvic organ prolapse and/or manipulation of endopelvic fascia during prolapse repair may be the cause of prolapse-related pelvic pain and sexual dysfunction. Through the dissection of formalin-fixed hemipelvises (n = 10) the present study aimed to map the loci of the visceral branches of the inferior hypogastric plexus and associate them with endopelvic fascia of the female pelvis. In all specimens, two gross bundles of autonomic nerves were identified within pubocervical fascia traveling caudally to innervate the clitoral crura, vestibular bulbs, and anterior vaginal wall. Moreover, a distinct bundle was found traveling within the rectovaginal septum innervating the posterior vaginal wall. Immunohistochemical staining for S100 protein confirmed the presence of nerves and ganglia within pubocervical fascia that were localized near the vaginal arteries. Additionally, our dissections revealed that the visceral branches of the inferior hypogastric plexus can be stratified to correspond to different named components of endopelvic fascia. Clinically, our results provide a robust anatomical characterization that will inform the development of surgical approaches to reduce the risk of pelvic neuropathy during vaginal prolapse surgery.

Abstract AIMS Developing a translational programme for brain tumours involves navigating numerous challenges, including robustness of preclinical studies, safety, clinical trial design, understanding and addressing regulatory require- ments, and funding. Recognising the difficulty of this journey, the Brain Tumour Research - Novel Therapeutics Accelerator (BTR-NTA) was launched in 2023. The BTR-NTA is an accelerator programme providing indepen- dent expert guidance to de-risk drug or device development through systematic evaluation. The international, multidisciplinary BTR-NTA Committee spans expertise across the entire therapeutic development pipeline. Sup- ported by Brain Tumour Research, the BTR-NTA programme is free for academic applicants. METHODS Researchers at any stage of therapeutic development may apply to BTR-NTA. Accepted applicants receive up to 240 hours of expert input, with feedback on strengths, identification of potential risks, and guidance on next steps. As part of the BTR-NTA review, the applicants and Committee meet in-person to constructively evaluate and discuss the proposed therapeutic strategy. RESULTS Since the launch of BTR-NTA in 2023, 24 international groups have applied to the programme, eleven therapeu- tics have been advanced to the in-person review by the BTR-NTA Committee, and 34 multidisciplinary experts have been involved. All applicants who provided feedback on the programme intended to refine their future work based on the Committee’s input, 86% said the process provided new insights or ideas they had not previ- ously considered, and the average rating of the programme’s usefulness was 9.7 out of 10. A number of com- mon challenges to developing therapies in the brain tumour field have been identified and these lessons can be shared with the wider brain tumour community to help accelerate the translation of future therapies. CONCLUSION The ultimate goal of the BTR-NTA programme is to help researchers with a potential therapy for brain tumours navigate the development journey and reach the clinic in an efficient way.

Isocitrate dehydrogenase wild-type glioblastoma is the most aggressive primary brain tumor classified as grade 4 of malignancy. Standard treatment, combining surgical resection, radiotherapy, and chemotherapy, often leads to severe side effects, with the emergence of tumor recurrence in all cases. Nucleic acid-based therapy has emerged as a promising strategy for cancer treatment. Non-viral nanosystems have become the vehicles of choice for gene delivery, due to their efficient nucleic acid encapsulation, protection, and intracellular transport. This work explores the potential of a formulation of low molecular weight protamine (LMWP) and dextran sulfate for gene delivery. The nanoparticles (NPs) were evaluated in terms of particle size, surface charge, morphology, and capacity to condense different nucleic acids. NPs formed by ionic complexation resulted in a homogeneous population of spherical particles with a low polydispersity index (PDI), small size, and positive surface charge. Competitive displacement assay demonstrated that the NPs could condense nucleic acids without alterations in their morphology and physicochemical characteristics, even after long-term storage. The efficacy of this formulation as a gene delivery system was evaluated in vitro in different glioblastoma cell lines and three-dimensional (3D) spheroids and in vivo using zebrafish models, showing negligible toxicity, efficient internalization, and consistent expression of fluorescent/luminescent proteins. Overall, these cationic polymeric NPs show promising features for their use as non-viral gene delivery vehicles for glioblastoma treatments.

Central nervous system germ cell tumors (CNS-GCT) are malignant neoplasms that arise predominantly during adolescence and young adulthood. These tumors are typically sensitive to treatment, but resulting long-term health deficits are common. Additional clinical challenges include surgical risks associated with tumor biopsy, and need to determine treatment response for adapting radiotherapy protocols. The aim of this study was to establish the detectability of circulating-tumor DNA (ctDNA) from cerebrospinal fluid (CSF) of children with CNS-GCT as a potential biomarker. We obtained CSF from patients with CNS-GCT by lumbar puncture or intra-operatively. Cell-free DNA (cfDNA) was extracted and subjected to low-pass whole genome sequencing (LP-WGS). Copy-number alterations (CNAs) were inferred and served as a marker of measurable residual disease (MRD). Comparisons with imaging findings and tumor marker levels were made. A total of 29 CSF samples from 21 patients (16 with germinoma, 5 with non-germinomatous GCT) were sequenced. Twenty samples from 19 patients were collected at diagnosis, and 9 samples from 7 patients were collected during or after therapy. Among the diagnostic samples, CNAs were detected in samples from 17/19 patients (89%), which included 8 with marker-negative tumors. Specific clinical scenarios suggested that serial cfDNA analysis may carry utility in tracking treatment responses as well as clarifying indeterminate imaging findings. Our results provide evidence for the high-sensitivity in detecting ctDNA from CSF of CNS-GCT patients using LP-WGS, with potential utility for non-invasive diagnosis and disease monitoring in upcoming CNS-GCT studies.

Abstract Developing a comprehensive translational programme for brain tumours involves navigating numerous challenges, including robustness of preclinical studies, safety, clinical trial design, understanding and addressing regulatory requirements, and funding. Recognising the difficulty of this journey, the Brain Tumour Research Novel Therapeutics Accelerator (BTR-NTA) was launched in 2023. The BTR-NTA is an accelerator programme providing independent expert guidance to de-risk drug or device development through systematic evaluation. The international, multidisciplinary BTR-NTA Committee spans expertise across the entire therapeutic development pipeline. Supported by Brain Tumour Research, the BTR-NTA programme is free for academic applicants. Researchers at any stage of therapeutic development may apply to BTR-NTA. Accepted applicants proceed as follows: 1. Applicants outline their current and prospective data and findings. The proposal and all supporting materials are covered under a confidentiality agreement that protects applicants’ intellectual property; 2. Proposals are reviewed by the BTR-NTA Committee; 3. Applicants and BTR-NTA Committee meet in-person to constructively evaluate the work; and 4. Applicants receive a comprehensive report with feedback on strengths, identification of potential risks, and guidance on next steps. The BTR-NTA Committee dedicates time for in-person meetings with each applicant, and extensive time pre- and post-meeting. Since the launch of BTR-NTA in 2023,14 international groups have applied to the programme and seven therapies have been advanced to the in-person review by the BTR-NTA Committee. Of the applicants who provided feedback on the BTR-NTA programme, all intended to adjust or refine their future work based on the Committee’s input and the average rating of the programme’s helpfulness was 9.5 out of 10. The ultimate goal of BTR-NTA is to help researchers with a potential therapy for brain tumours to navigate the development journey and reach the clinic in an efficient way. This programme will support the development of novel therapies to improve outcomes for patients with brain tumours.

Abstract AIMS Relapse rates of 30% and 50% in medulloblastoma and ependymoma lead to poor outcomes for paediatric brain tumours. Occurring usually within two years, relapses imply minimal residual disease (MRD) is present below the imaging detection threshold. Patient outcomes might be improved by diagnosing MRD using a sensitive, minimally invasive cerebrospinal fluid (CSF) liquid biopsy, as CSF is proximal to tumour. Metabolites arising from aberrant cancer metabolism may be biomarkers in CSF. METHOD With only 25µl CSF, extracted with 75µl methanol, liquid chromatography coupled with tandem high-resolution mass spectrometry (LC-MS/MS) could differentiate these paediatric brain tumours (n=47; day 14 post-surgery) from controls (n=49). RESULTS Fourteen metabolites were increased in abundance in tumours and four decreased compared to controls. These included amino acids and derivatives (n=8), metabolites in one carbon metabolism (n=1), carnitine cycle (n=2), aerobic glycolysis (n=2), purine catabolism (n=2), ascorbate degradation (n=1) and creatinine synthesis (n=2). From those, betaine/creatinine ratio gave an area under the curve (AUC) of 0.96 (excellent) in receiver operating curve analysis. Using a cut-off ratio of 0.1775 for discriminating brain tumours from controls, had sensitivity and specificity of 92% and 89%. Specifically identifying tumours, a ratio of betaine/threonate with cut-off 2.1514 discriminated ependymomas from controls (sensitivity/specificity 92%/91%). A ratio of malic acid/hypoxanthine with cut-off 0.04429 discriminated medulloblastomas from controls (sensitivity/specificity 100%/98%). Medulloblastomas (n=14) were largely similar to ependymomas (n=33); nevertheless, three metabolites met the criteria of corrected p&amp;lt;0.05. A ratio of glutarate semialdehyde/malic acid with cut-off 0.04243 discriminated ependymomas from medulloblastomas (sensitivity/specificity 79%/82%) in a good model (AUC 0.85). CONCLUSION This is the first report of CSF metabolites in ependymoma. Ratios between metabolites levels could be useful as biomarkers to detect brain tumours and distinguish brain tumour type specifically and sensitively. Further work to develop a liquid biopsy test for the detection of MRD is ongoing.