Shivani Baisiwala

Glioblastoma (GBM) is a devastating primary brain tumor with remarkable inter- and intra-tumoral heterogeneity. GBM cells assume a spectrum of neurodevelopmental-like phenotypes and co-opt normal neurophysiological processes, which include synaptic integration with their neuronal microenvironment. This is mediated by neuron-tumor synapses (NTS) that predominantly involve glutamatergic receptors, which drive calcium elevations that promote tumor proliferation and invasion. The exact relationship between synaptic signaling and tumor cell fate specification, however, remains largely unexplored. Here, we develop and leverage a synapse-optimized human organoid tumor transplantation (so-HOTT) model of GBM to decipher how glutamatergic signaling impacts GBM lineage trajectories. so-HOTT preserves patient tumor heterogeneity, features excitatory NTS, and enables clonal lineage tracing of tumor cells after NTS perturbations. Genetic and pharmacological inhibition of AMPA and kainate receptors in so-HOTT shifts tumor cell composition from neuronal fates toward progenitor-proximal astrocytic/mesenchymal states. This occurs through the attenuation of calcium signaling and reduced plasticity of malignant radial glia (RG)-like progenitors, a previously unrecognized target of NTS. Through the integration of inputs from the neuronal microenvironment into glutamatergic signaling, progenitor populations modulate their transcriptional programs and cell fate, ultimately shaping GBM tumor heterogeneity. Targeting synaptic input may thus constrain the heterogeneity that fuels GBM adaptation and therapeutic escape.

BACKGROUND AND OBJECTIVES: Glioblastoma often extends along the subventricular zone and resection frequently results in ventricular entry, which is associated with increased risk of postoperative hydrocephalus. Choroid plexus cauterization (CPC) is a treatment for pediatric hydrocephalus, but its utility in preventing postoperative hydrocephalus following glioblastoma resection with ventricular entry is unknown. We sought to characterize CPC safety and efficacy in preventing postoperative hydrocephalus in this setting. METHODS: We evaluated a historical cohort of 260 patients who underwent craniotomy for glioblastoma with ventricular entry, assessing postoperative hydrocephalus, survival, and functional outcomes. Furthermore, we prospectively performed CPC in a matched cohort with ventricular entry. We quantitated the volume of choroid plexus that was successfully cauterized using magnetic resonance segmentation and evaluated the safety and efficacy of CPC. RESULTS: 25.8% of patients with glioblastoma resection with ventricular entry developed postoperative hydrocephalus. Hydrocephalus was associated with more readmissions (2.3 vs 0.6; P < .0001), longer hospital stays (10.0 vs 6.5 days; P = .0047), and lower 3-month Karnofsky Performance Status (64.4 vs 78.6; P < .0001), although overall survival was unaffected. Thirty patients underwent prospective CPC with no procedure-related postoperative complications. Choroid plexus volume in the involved ventricle decreased by 50% on postoperative MRI segmentations ( P = .0020). In a matched analysis, there was a reduction in postoperative hydrocephalus compared with the retrospective cohort (3.3% vs 25.8%; P = .0060) associated with fewer hospital readmissions ( P = .0330). CONCLUSION: As ventricular entry becomes increasingly common during supramaximal glioblastoma resection, our data demonstrate that patients who develop postoperative hydrocephalus have more readmissions, longer hospital stays, and worse functional status. Thus, strategies to reduce postoperative hydrocephalus are critical. Although it does not alter the natural course of glioblastoma itself, we find that CPC represents a safe and effective adjunct that prevents postoperative hydrocephalus, reduces hospital readmissions, and downstream interventions, which is extremely important to a patient cohort with limited expected survival.

Extensive molecular profiling has revealed profound heterogeneity in glioblastoma (GBM), yet how cellular lineages organize over time to govern tumor propagation and therapeutic response remains poorly understood. Existing single-cell approaches define transcriptional states but provide limited insight into how clonal dynamics shape functional tumor behavior. Here, we integrate high-complexity combinatorial DNA barcoding with single-cell transcriptomics in direct-from-patient IDH1-wild-type GBM, enabling lineage-resolved mapping of progenitor organization in a human microenvironmental context. Across 235,155 malignant cells from nine tumors, clonal relationships form reproducible lineage tracks in which distinct progenitor populations give rise to specific differentiated cell types, revealing that tumor growth is sustained by multiple non-redundant progenitors rather than a single dominant population. These progenitors exhibit distinct propensities for self-renewal, fate restriction, and cross-compartment interactions, collectively accounting for the full spectrum of tumor states. Using this lineage-resolved framework, we identify complementary drug targets in distinct progenitor compartments and demonstrate that hierarchy-informed combination therapies disrupt progenitor-progenitor interactions and reshape lineage output. These findings move beyond descriptive heterogeneity to define functional logic underlying GBM propagation and establish a generalizable framework for rational, cell type-specific combinatorial therapies.

A major obstacle to identifying effective therapies for the aggressive brain tumor glioblastoma is the lack of human-specific, immunocompetent models that reflect the human tumor microenvironment. To address this, we developed the immune-Human Organoid Tumor Transplantation (iHOTT) model. This is an autologous co-culture platform that integrates patient-derived tumor cells and matched peripheral blood mononuclear cells (PBMCs) within human cortical organoids, enabling the study of the patient-specific immune response to the tumor and tumor-immune interactions. This platform preserves tumor and immune populations, immune signaling, and cell-cell interactions observed in patient tumors. Treatment of iHOTT with pembrolizumab, a checkpoint inhibitor, mirrored cell type shifts and cell interactions observed in patients. TCR sequencing further revealed pembrolizumab-driven expansion of stem-like CD4-T-cell clonotypes exhibiting patient-specific repertoires. These findings establish iHOTT as a physiologically relevant platform for exploring autologous tumor–immune interactions and underscore the critical need for antigen-targeted strategies to enhance immunotherapy in glioblastoma.

Benign-appearing intracranial cysts may become symptomatic due to mass effect and require surgical treatment. Mechanisms underlying cyst formation and growth remain poorly understood. This study identified 16 patients who underwent surgical treatment for benign intracranial cysts. Cyst wall pathologic samples (n = 8) were characterized as arachnoid or glioependymal cysts using H&E and immunofluorescence staining. Five samples (62.5%) were found to be glioependymal while three (37.5%) were arachnoid cysts. Cyst fluid examined in 4 cases resembled cerebrospinal fluid (CSF) and showed no significant differences in composition between pathological subtypes. Single-cell sequencing and RNA expression profile comparisons between glioependymal and arachnoid cysts revealed distinct cellular profiles. Analyses of the innermost cell layer (ependymal cells versus arachnoid cells) suggested differing mechanisms of pathogenesis. Glioependymal cysts harbored upregulated expression of sodium transporters and aquaporin channels, which suggests possible CSF-like fluid production whereas arachnoid cysts may be associated with mechanical fluid accumulation. Thus, intracranial cysts represent multiple unique pathologic entities with differing cell types and RNA expression profiles. Further study into mechanisms of glioependymal cyst formation may allow for targeted interventions to reduce fluid production and avoid surgery.

Tumor invasion and metastasis remain the leading causes of mortality for patients with cancer despite current treatment strategies. In some cancer types, recurrence is considered inevitable due to the lack of effective anti-metastatic therapies. Recent studies across many cancer types demonstrate a close relationship between cancer-initiating cells (CICs) and metastasis, as well as general cancer progression. First, this review describes CICs’ contribution to cancer progression. Then we discuss our recent understanding of mechanisms through which CICs promote tumor invasion and metastasis by examining the role of CICs in each stage. Finally, we examine the current understanding of CICs’ contribution to therapeutic resistance and recent developments in CIC-targeting drugs. We believe this understanding is key to advancing anti-CIC clinical therapeutics. Graphical Abstract

Glioblastoma is a primary brain cancer with a near 100% recurrence rate. Upon recurrence, the tumour is resistant to all conventional therapies, and because of this, 5-year survival is dismal. One of the major drivers of this high recurrence rate is the ability of glioblastoma cells to adapt to complex changes within the tumour microenvironment. To elucidate this adaptation's molecular mechanisms, specifically during temozolomide chemotherapy, we used chromatin immunoprecipitation followed by sequencing and gene expression analysis. We identified a molecular circuit in which the expression of ciliary protein ADP-ribosylation factor-like protein 13B (ARL13B) is epigenetically regulated to promote adaptation to chemotherapy. Immuno-precipitation combined with liquid chromatography-mass spectrometry binding partner analysis revealed that that ARL13B interacts with the purine biosynthetic enzyme inosine-5′-monophosphate dehydrogenase 2 (IMPDH2). Further, radioisotope tracing revealed that this interaction functions as a negative regulator for purine salvaging. Inhibition of the ARL13B-IMPDH2 interaction enhances temozolomide-induced DNA damage by forcing glioblastoma cells to rely on the purine salvage pathway. Targeting the ARLI3B-IMPDH2 circuit can be achieved using the Food and Drug Administration-approved drug, mycophenolate mofetil, which can block IMPDH2 activity and enhance the therapeutic efficacy of temozolomide. Our results suggest and support clinical evaluation of MMF in combination with temozolomide treatment in glioma patients.

Glioblastoma (GBM) is the most common primary brain malignancy in adults, with a 100% recurrence rate and 21-month median survival. Our lab and others have shown that GBM contains a subpopulation of glioma stem cells (GSCs) that expand during chemotherapy and may contribute to therapeutic resistance and recurrence in GBM. To investigate the mechanism behind this expansion, we applied gene set expression analysis (GSEA) to patient-derived xenograft (PDX) cells in response to temozolomide (TMZ), the most commonly used chemotherapy against GBM. Results showed significant enrichment of cancer stem cell and cell cycle pathways (False Discovery Rate (FDR) < 0.25). The ligand of numb protein 1 (LNX1), a known regulator of Notch signaling by targeting negative regulator Numb, is strongly upregulated after TMZ therapy (p < 0.0001) and is negatively correlated with survival of GBM patients. LNX1 is also upregulated after TMZ therapy in multiple PDX lines with concomitant downregulations in Numb and upregulations in intracellular Notch1 (NICD). Overexpression of LNX1 results in Notch1 signaling activation and increased GSC populations. In contrast, knocking down LNX1 reverses these changes, causing a significant downregulation of NICD, reduction in stemness after TMZ therapy, and resulting in more prolonged median survival in a mouse model. Based on this, we propose that during anti-GBM chemotherapy, LNX1-regulated Notch1 signaling promotes stemness and contributes to therapeutic resistance.

This year nearly 20,000 lives will be lost to Glioblastoma (GBM), a treatment-resistant primary brain cancer. In this study, we identified a molecular circuit driven by epigenetic regulation that regulates the expression of ciliary protein ALR13B. We also demonstrated that ARL13B subsequently interacts with purine biosynthetic enzyme IMPDH2. Removal of ARL13B enhanced TMZ-induced DNA damage by reducing de-novo purine biosynthesis and forcing GBM cells to rely on the purine salvage pathway. Furthermore, targeting can be achieved by using an FDA-approved drug, Mycophenolate Moefitil. Our results suggest a clinical evaluation of MMF in combination with TMZ treatment in glioma patients.