Integrated electrophysiological and genomic profiles of single cells reveal spiking tumor cells in human glioma.

Prior studies have described the complex interplay that exists between glioma cells and neurons; however, the electrophysiological properties endogenous to glioma cells remain obscure. To address this, we employed Patch-sequencing (Patch-seq) on human glioma specimens and found that one-third of patched cells in IDH mutant (IDHmut) tumors demonstrate properties of both neurons and glia. To define these hybrid cells (HCs), which fire single, short action potentials, and discern if they are of tumoral origin, we developed the single cell rule association mining (SCRAM) computational tool to annotate each cell individually. SCRAM revealed that HCs possess select features of GABAergic neurons and oligodendrocyte precursor cells, and include both tumor and non-tumor cells. These studies characterize the combined electrophysiological and molecular properties of human glioma cells and describe a cell type in human glioma with unique electrophysiological and transcriptomic properties that may also exist in the non-tumor brain.

Pediatric glioma immune profiling identifies TIM3 as a therapeutic target in BRAF fusion pilocytic astrocytoma.

Despite being the leading cause of cancer-related childhood mortality, pediatric gliomas have been relatively understudied, and the repurposing of immunotherapies has not been successful. Whole-transcriptome sequencing, single-cell sequencing, and sequential multiplex immunofluorescence were used to identify an immunotherapeutic strategy that could be applied to multiple preclinical glioma models. MAPK-driven pediatric gliomas have a higher IFN signature relative to other molecular subgroups. Single-cell sequencing identified an activated and cytotoxic microglia (MG) population designated MG-Act in BRAF-fused, MAPK-activated pilocytic astrocytoma (PA), but not in high-grade gliomas or normal brain. T cell immunoglobulin and mucin domain 3 (TIM3) was expressed on MG-Act and on the myeloid cells lining the tumor vasculature but not normal brain vasculature. TIM3 expression became upregulated on immune cells in the PA microenvironment, and anti-TIM3 reprogrammed ex vivo immune cells from human PAs to a proinflammatory cytotoxic phenotype. In a genetically engineered murine model of MAPK-driven, low-grade gliomas, anti-TIM3 treatment increased median survival over IgG- and anti-PD-1-treated mice. Single-cell RNA-Seq data during the therapeutic window of anti-TIM3 revealed enrichment of the MG-Act population. The therapeutic activity of anti-TIM3 was abrogated in mice on the CX3CR1 MG-KO background. These data support the use of anti-TIM3 in clinical trials of pediatric low-grade, MAPK-driven gliomas.

Histone serotonylation regulates ependymoma tumorigenesis.

Bidirectional communication between tumours and neurons has emerged as a key facet of the tumour microenvironment that drives malignancy1,2. Another hallmark feature of cancer is epigenomic dysregulation, in which alterations in gene expression influence cell states and interactions with the tumour microenvironment3. Ependymoma (EPN) is a paediatric brain tumour that relies on epigenomic remodelling to engender malignancy4,5; however, how these epigenetic mechanisms intersect with extrinsic neuronal signalling during EPN tumour progression is unknown. Here we show that the activity of serotonergic neurons regulates EPN tumorigenesis, and that serotonin itself also serves as an activating modification on histones. We found that inhibiting histone serotonylation blocks EPN tumorigenesis and regulates the expression of a core set of developmental transcription factors. High-throughput, in vivo screening of these transcription factors revealed that ETV5 promotes EPN tumorigenesis and functions by enhancing repressive chromatin states. Neuropeptide Y (NPY) is one of the genes repressed by ETV5, and its overexpression suppresses EPN tumour progression and tumour-associated network hyperactivity through synaptic remodelling. Collectively, this study identifies histone serotonylation as a key driver of EPN tumorigenesis, and also reveals how neuronal signalling, neuro-epigenomics and developmental programs are intertwined to drive malignancy in brain cancer.

Response of treatment-naive brain metastases to stereotactic radiosurgery.

With improvements in survival for patients with metastatic cancer, long-term local control of brain metastases has become an increasingly important clinical priority. While consensus guidelines recommend surgery followed by stereotactic radiosurgery (SRS) for lesions >3 cm, smaller lesions (≤3 cm) treated with SRS alone elicit variable responses. To determine factors influencing this variable response to SRS, we analyzed outcomes of brain metastases ≤3 cm diameter in patients with no prior systemic therapy treated with frame-based single-fraction SRS. Following SRS, 259 out of 1733 (15%) treated lesions demonstrated MRI findings concerning for local treatment failure (LTF), of which 202 /1733 (12%) demonstrated LTF and 54/1733 (3%) had an adverse radiation effect. Multivariate analysis demonstrated tumor size (>1.5 cm) and melanoma histology were associated with higher LTF rates. Our results demonstrate that brain metastases ≤3 cm are not uniformly responsive to SRS and suggest that prospective studies to evaluate the effect of SRS alone or in combination with surgery on brain metastases ≤3 cm matched by tumor size and histology are warranted. These studies will help establish multi-disciplinary treatment guidelines that improve local control while minimizing radiation necrosis during treatment of brain metastasis ≤3 cm.

Integrated electrophysiological and genomic profiles of single cells reveal spiking tumor cells in human glioma.

Prior studies have described the complex interplay that exists between glioma cells and neurons, however, the electrophysiological properties endogenous to tumor cells remain obscure. To address this, we employed Patch-sequencing on human glioma specimens and found that one third of patched cells in IDH mutant (IDH mut ) tumors demonstrate properties of both neurons and glia by firing single, short action potentials. To define these hybrid cells (HCs) and discern if they are tumor in origin, we developed a computational tool, Single Cell Rule Association Mining (SCRAM), to annotate each cell individually. SCRAM revealed that HCs represent tumor and non-tumor cells that feature GABAergic neuron and oligodendrocyte precursor cell signatures. These studies are the first to characterize the combined electrophysiological and molecular properties of human glioma cells and describe a new cell type in human glioma with unique electrophysiological and transcriptomic properties that are likely also present in the non-tumor mammalian brain.

Association of graph-based spatial features with overall survival status of glioblastoma patients.

Glioblastoma is the most common malignant brain tumor with less than 15 months median survival. To aid prognosis, there is a need for decision tools that leverage diagnostic modalities such as MRI to inform survival. In this study, we examine higher-order spatial proximity characteristics from habitats and propose two graph-based methods (minimum spanning tree and graph run-length matrix) to characterize spatial heterogeneity over tumor MRI-derived intensity habitats and assess their relationships with overall survival as well as the immune signature status of patients with glioblastoma. A data set of 74 patients was studied based on the availability of post-contrast T1-weighted and T2-weighted fluid attenuated inversion recovery (FLAIR) image data in The Cancer Image Archive (TCIA). We assessed the predictive value of MST- and GRLM-derived features from 2D images for prediction of 12-month survival status and immune signature status of patients with glioblastoma via a receiver operating characteristic curve analysis. For 12-month survival prediction using MST-based method, sensitivity and specificity were 0.82 and 0.79 respectively. For GRLM-based method, sensitivity and specificity were 0.73 and 0.77 respectively. For immune status, sensitivity and specificity were 0.91 and 0.69, respectively, for the GRLM-based method with an immune effector. Our results show that the proposed MST- and GRLM-derived features are predictive of 12-month survival status as well as the immune signature status of patients with glioblastoma. To our knowledge, this is the first application of MST- and GRLM-based proximity analyses for the study of radiologically-defined tumor habitats in glioblastoma.

Prevalence of pathogenic germline variants in adult-type diffuse glioma.

Background: No consensus germline testing guidelines currently exist for glioma patients, so the prevalence of germline pathogenic variants remains unknown. This study aims to determine the prevalence and type of pathogenic germline variants in adult glioma. Methods: A retrospective review at a single institution with paired tumor/normal sequencing from August 2018-April 2022 was performed and corresponding clinical data were collected. Results: We identified 152 glioma patients of which 15 (9.8%) had pathogenic germline variants. Pathogenic germline variants were seen in 11/84 (13.1%) of Glioblastoma, IDH wild type; 3/42 (7.1%) of Astrocytoma, IDH mutant; and 1/26 (3.8%) of Oligodendroglioma, IDH mutant, and 1p/19q co-deleted patients. Pathogenic variants in BRCA2, MUTYH, and CHEK2 were most common (3/15, 20% each). BRCA1 variants occurred in 2/15 (13%) patients, with variants in NF1, ATM, MSH2, and MSH3 occurring in one patient (7%) each. Prior cancer diagnosis was found in 5/15 patients (33%). Second-hit somatic variants were seen in 3/15 patients (20%) in NF1, MUTYH, and MSH2. Referral to genetics was performed in 6/15 (40%) patients with pathogenic germline variants. 14/15 (93%) of patients discovered their pathogenic variant as a result of their paired glioma sequencing. Conclusions: These findings suggest a possible overlooked opportunity for determination of hereditary cancer syndromes with impact on surveillance as well as potential broader treatment options. Further studies that can determine the role of variants in gliomagenesis and confirm the occurrence and types of pathogenic germline variants in patients with IDH wild type compared to IDH mutant tumors are necessary.

Remote neuronal activity drives glioma progression through SEMA4F.

The tumour microenvironment plays an essential role in malignancy, and neurons have emerged as a key component of the tumour microenvironment that promotes tumourigenesis across a host of cancers1,2. Recent studies on glioblastoma (GBM) highlight bidirectional signalling between tumours and neurons that propagates a vicious cycle of proliferation, synaptic integration and brain hyperactivity3-8; however, the identity of neuronal subtypes and tumour subpopulations driving this phenomenon is incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumours promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity-dependent infiltrating population present at the leading edge of mouse and human tumours that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified SEMA4F as a key regulator of tumourigenesis and activity-dependent progression. Furthermore, SEMA4F promotes the activity-dependent infiltrating population and propagates bidirectional signalling with neurons by remodelling tumour-adjacent synapses towards brain network hyperactivity. Collectively our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, and also show new mechanisms of glioma progression that are regulated by neuronal activity.

Aspirin and immunotherapy: a Faustian bargain?

Fibrinogen-like protein 1 (FGL1) has been associated with improved survival in hepatocellular carcinoma (HCC). However, recent evidence suggests that FGL1 may bind to surface receptors on lymphocytes and induce immune senescence. In this issue of the JCI, Lin and co-authors show that FGL1 may be acetylated by aspirin and targeted for degradation, which is associated with increased antitumor immunity and improved survival. Similar findings were obtained with inhibitors of sirtuin 2 (SIRT2), a histone deacetylase. These findings expand our current understanding of the role of FGL1 in cancer and provide an impetus for the evaluation of alternative immunotherapy combinations in HCC.

Concurrent IDH1 and IDH2 mutations in glioblastoma: A case report.

Isocitrate dehydrogenase (IDH) mutations are cornerstone diagnostic features in glioma classification. IDH mutations are typically characterized by mutually exclusive amino acid substitutions in the genes encoding for the IDH1 and the IDH2 enzyme isoforms. We report our institutional case of a diffuse astrocytoma with progression to secondary glioblastoma and concurrent IDH1/IDH2 mutations. A 49-year-old male underwent a subtotal resection of a lobular lesion within the right insula in 2013, revealing a WHO grade 3 anaplastic oligoastrocytoma, IDH1 mutated, 1p19q intact. Symptomatic tumor progression was suspected in 2018, leading to a surgical tumor biopsy that demonstrated WHO grade 4 IDH1 and IDH2 mutant diffuse astrocytoma. The patient subsequently underwent surgical resection followed by medical management and finally died in 2021. Although concurrent IDH1/IDH2 mutations have been rarely reported in the current literature, further study is required to better define their impact on patients' prognoses and their response to targeted therapies.

Characterization of recurrence patterns and outcomes of medulloblastoma in adults: The University of Texas MD Anderson Cancer Center experience.

Background: Medulloblastoma in adults is rare and treatment decisions are largely driven from pediatric literature. We sought to characterize recurrent medulloblastoma in adults. Methods: From a single-institution dataset of 200 adult patients diagnosed with medulloblastoma during 1978-2017, those with recurrence were analyzed for clinical features, treatment, and outcome. Results: Of the 200 patients, 82 (41%) with median age of 29 years (18-59) had recurrence after a median follow-up time of 8.4 years (95% CI = 7.1, 10.3). Of these, 30 (37%) were standard-risk, 31 (38%) were high-risk, and 21 (26%) had unknown-risk diseases at the time of initial diagnosis. Forty-eight (58%) presented with recurrence outside the posterior fossa, of whom 35 (43%) had distant recurrence only. Median Progression-free survival (PFS) and OS from initial surgery were 33.5 and 62.4 months, respectively. Neither PFS nor OS from initial diagnosis differed between the standard-risk and high-risk groups in those who experience recurrence (P = .505 and .463, respectively). Median OS from first recurrence was 20.3 months, also with no difference between the standard-risk and high-risk groups (P = .518). Recurrences were treated with combinations of re-resection (20 patients; 25%), systemic chemotherapy (61 patients; 76%), radiation (29 patients; 36%), stem cell transplant (6 patients; 8%), and intrathecal chemotherapy (4 patients; 5%). Patients who received radiation at recurrence had better OS (32.9 months) than those who did not (19.2 months) (P = .034). Conclusions: Recurrent medulloblastoma in adults has a poor prognosis irrespective of initial risk stratification. Recurrence commonly arises outside the posterior fossa years after initial diagnosis.

Single-cell RNA sequencing reveals immunosuppressive myeloid cell diversity during malignant progression in a murine model of glioma.

Recent studies have shown the importance of the dynamic tumor microenvironment (TME) in high-grade gliomas (HGGs). In particular, myeloid cells are known to mediate immunosuppression in glioma; however, it is still unclear if myeloid cells play a role in low-grade glioma (LGG) malignant progression. Here, we investigate the cellular heterogeneity of the TME using single-cell RNA sequencing in a murine glioma model that recapitulates the malignant progression of LGG to HGG. LGGs show increased infiltrating CD4+ and CD8+ T cells and natural killer (NK) cells in the TME, whereas HGGs abrogate this infiltration. Our study identifies distinct macrophage clusters in the TME that show an immune-activated phenotype in LGG but then evolve to an immunosuppressive state in HGG. We identify CD74 and macrophage migration inhibition factor (MIF) as potential targets for these distinct macrophage populations. Targeting these intra-tumoral macrophages in the LGG stage may attenuate their immunosuppressive properties and impair malignant progression.

Remote neuronal activity drives glioma infiltration via Sema4f.

The tumor microenvironment (TME) plays an essential role in malignancy and neurons have emerged as a key component of the TME that promotes tumorigenesis across a host of cancers. Recent studies on glioblastoma (GBM) highlight bi-directional signaling between tumors and neurons that propagates a vicious cycle of proliferation, synaptic integration, and brain hyperactivity; however, the identity of neuronal subtypes and tumor subpopulations driving this phenomenon are incompletely understood. Here we show that callosal projection neurons located in the hemisphere contralateral to primary GBM tumors promote progression and widespread infiltration. Using this platform to examine GBM infiltration, we identified an activity dependent infiltrating population present at the leading edge of mouse and human tumors that is enriched for axon guidance genes. High-throughput, in vivo screening of these genes identified Sema4F as a key regulator of tumorigenesis and activity-dependent infiltration. Furthermore, Sema4F promotes the activity-dependent infiltrating population and propagates bi-directional signaling with neurons by remodeling tumor adjacent synapses towards brain network hyperactivity. Collectively, our studies demonstrate that subsets of neurons in locations remote to primary GBM promote malignant progression, while revealing new mechanisms of tumor infiltration that are regulated by neuronal activity.

Glioma epileptiform activity and progression are driven by IGSF3-mediated potassium dysregulation.

Seizures are a frequent pathophysiological feature of malignant glioma. Recent studies implicate peritumoral synaptic dysregulation as a driver of brain hyperactivity and tumor progression; however, the molecular mechanisms that govern these phenomena remain elusive. Using scRNA-seq and intraoperative patient ECoG recordings, we show that tumors from seizure patients are enriched for gene signatures regulating synapse formation. Employing a human-to-mouse in vivo functionalization pipeline to screen these genes, we identify IGSF3 as a mediator of glioma progression and dysregulated neural circuitry that manifests as spreading depolarization (SD). Mechanistically, we discover that IGSF3 interacts with Kir4.1 to suppress potassium buffering and found that seizure patients exhibit reduced expression of potassium handlers in proliferating tumor cells. In vivo imaging reveals that dysregulated synaptic activity emanates from the tumor-neuron interface, which we confirm in patients. Our studies reveal that tumor progression and seizures are enabled by ion dyshomeostasis and identify SD as a driver of disease.

The Role of Hyperexcitability in Gliomagenesis.

Glioblastoma is the most common malignant primary brain tumor. Recent studies have demonstrated that excitatory or activity-dependent signaling-both synaptic and non-synaptic-contribute to the progression of glioblastoma. Glutamatergic receptors may be stimulated via neuron-tumor synapses or release of glutamate by the tumor itself. Ion currents generated by these receptors directly alter the structure of membrane adhesion molecules and cytoskeletal proteins to promote migratory behavior. Additionally, the hyperexcitable milieu surrounding glioma increases the rate at which tumor cells proliferate and drive recurrent disease. Inhibition of excitatory signaling has shown to effectively reduce its pro-migratory and -proliferative effects.

CXCR4 expression is associated with proneural-to-mesenchymal transition in glioblastoma.

Glioblastoma (GBM) is the most common primary intracranial malignant tumor and consists of three molecular subtypes: proneural (PN), mesenchymal (MES) and classical (CL). Transition between PN to MES subtypes (PMT) is the glioma analog of the epithelial-mesenchymal transition (EMT) in carcinomas and is associated with resistance to therapy. CXCR4 signaling increases the expression of MES genes in glioma cell lines and promotes EMT in other cancers. RNA sequencing (RNAseq) data of PN GBMs in The Cancer Genome Atlas (TCGA) and secondary high-grade gliomas (HGGs) from an internal cohort were examined for correlation between CXCR4 expression and survival as well as expression of MES markers. Publicly available single-cell RNA sequencing (scRNAseq) data was analyzed for cell type specific CXCR4 expression. These results were validated in a genetic mouse model of PN GBM. Higher CXCR4 expression was associated with significantly reduced survival and increased expression of MES markers in TCGA and internal cohorts. CXCR4 was expressed in immune and tumor cells based on scRNAseq analysis. Higher CXCR4 expression within tumor cells on scRNAseq was associated with increased MES phenotype, suggesting a cell-autonomous effect. In a genetically engineered mouse model, tumors induced with CXCR4 exhibited a mesenchymal phenotype and shortened survival. These results suggest that CXCR4 signaling promotes PMT and shortens survival in GBM and highlights its inhibition as a potential therapeutic strategy.

Fibrinogen-like protein 2: Its biological function across cell types and the potential to serve as an immunotherapy target for brain tumors.

Brain tumors are among the 10 leading causes of cancer-related death and present unique treatment challenges due to their critical location, genetic heterogeneity, and the blood-brain barrier. Recent advances in targeted immunotherapy and immune checkpoint blocking therapy provide alternative therapeutic strategies for brain tumors. Fibrinogen-like protein 2 (FGL2), which induces transformation from low-grade glioma to high-grade glioblastoma, is a type II membrane protein that is highly expressed in both host immune cells and tumor cells. Studies have uncovered multiple forms of FGL2 proteins with a broad range of roles in inducing immune tolerance and avoiding immune surveillance in tumor cells. Of note, presence of FGL2 transforms low grade to high grade brain tumors via promoting Treg, macrophages, and perhaps stemness. Absence (knockout) of FGL2 in tumor cells (not in host cells) induces CD103 DC cells, which triggers tumor specific CD8 +T cell activity to reject brain tumor progression. Immunotherapies targeting FGL2 have shown great promise in improving survival time in murine models. In this article, we will summarize the biological function of FGL2 in immune and tumor cells.

The impact of a night float system on operative experience in neurosurgery residency.

OBJECTIVE: Since the Accreditation Council for Graduate Medical Education (ACGME) implemented duty-hour restrictions in 2003, many residency programs have adopted a night float system to comply with time constraints. However, some surgical subspecialities have been concerned that use of a night float system deprives residents of operative experience. In this study, the authors describe their training program's transition to a night float system and its impact on resident operative experience. METHODS: The authors conducted a single-program study of resident surgical case volume before and after implementing the night float system at 3 of their 5 hospitals from 2014 to 2020. The authors obtained surgical case numbers from the ACGME case log database. RESULTS: Junior residents received a concentrated educational experience, whereas senior residents saw a significant decrease from 112 calls/year to 17. Logged cases significantly increased after implementation of the night float system (8846 vs 10,547, p = 0.04), whereas cases at non-night float hospitals remained the same. This increase was concurrent with an increase in hospital cases. This difference was mainly driven by senior resident cases (p = 0.010), as junior and chief residents did not show significant differences in logged cases (p > 0.40). Lead resident cases increased significantly after implementation of the night float system (6852 vs 8860, p = 0.04). When normalized for increased hospital cases, resident case increases were not statistically significant. CONCLUSIONS: Transitioning to a night float call system at the authors' institution increased overall resident operative cases, particularly for lead resident surgeons. Based on the results of this study, the authors recommend the use of a night float call system to consolidate night calls, which increases junior resident-level educational opportunities and senior resident cases.

In vivo functional characterization of EGFR variants identifies novel drivers of glioblastoma.

BACKGROUND: Glioblastoma is the most common and aggressive primary brain tumor. Large-scale sequencing initiatives have cataloged its mutational landscape in hopes of elucidating mechanisms driving this deadly disease. However, a major bottleneck in harnessing this data for new therapies is deciphering "driver" and "passenger" events amongst the vast volume of information. METHODS: We utilized an autochthonous, in vivo screening approach to identify driver, EGFR variants. RNA-Seq identified unique molecular signatures of mouse gliomas across these variants, which only differ by a single amino acid change. In particular, we identified alterations to lipid metabolism, which we further validated through an unbiased lipidomics screen. RESULTS: Our screen identified A289I as the most potent EGFR variant, which has previously not been characterized. One of the mechanisms through which A289I promotes gliomagenesis is to alter cellular triacylglycerides through MTTP. Knockout of Mttp in mouse gliomas, reduces gliomagenesis in multiple models. CONCLUSIONS: EGFR variants that differ by a single amino acid residue differentially promote gliomagenesis. Among the identified mechanism that drives glioma growth include lipid metabolism through MTTP. Understanding triacylglyceride accumulation may present a prospective therapeutic pathway for this deadly disease.