Leptomeningeal dissemination in pediatric brain tumors.

Leptomeningeal disease (LMD) in pediatric brain tumors (PBTs) is a poorly understood and categorized phenomenon. LMD incidence rates, as well as diagnosis, treatment, and screening practices, vary greatly depending on the primary tumor pathology. While LMD is encountered most frequently in medulloblastoma, reports of LMD have been described across a wide variety of PBT pathologies. LMD may be diagnosed simultaneously with the primary tumor, at time of recurrence, or as primary LMD without a primary intraparenchymal lesion. Dissemination and seeding of the cerebrospinal fluid (CSF) involves a modified invasion-metastasis cascade and is often the result of direct deposition of tumor cells into the CSF. Cells develop select environmental advantages to survive the harsh, nutrient poor and turbulent environment of the CSF and leptomeninges. Improved understanding of the molecular mechanisms that underlie LMD, along with improved diagnostic and treatment approaches, will help the prognosis of children affected by primary brain tumors.

Pediatric Postmortem Tissue Donation in the Confines of a Pandemic: A Model of Collaboration.

BACKGROUND: Obtaining postmortem tissue from pediatric oncology patients is critical to research and may help grieving families heal. Since 2019, the national Gift from a Child program has made significant progress in collecting postmortem tissue from pediatric patients with central nervous system tumors to advance research. This progress was at risk during the onset of the severe acute respiratory syndrome coronavirus 2 pandemic, when some autopsy programs came to a halt. METHODS: We retrospectively reviewed autopsies of four patients treated at Memorial Sloan Kettering Cancer Center who underwent postmortem examination at Weill Cornell Medicine from June 2020 to March 2021. We collected patient demographics, Do not resuscitate status, time of death and procedure, restrictions due to the coronavirus disease 2019 (COVID-19) pandemic, and results of the tissue analysis. RESULTS: Three of four specimens were processed within 12 hours of the time of death. Two families required interpreter services to obtain consent. In all cases, tumor aliquots were flash frozen for further study. Cell line generation was successful in one case. All families expressed gratitude both for the opportunity to participate and for the handling of the procedures. CONCLUSIONS: Despite the sensitive nature of these cases and the challenges presented by COVID-19 restrictions, clinicians should offer the option of a rapid autopsy to caregivers of pediatric patients based on the scientific need and the positive effect it has on grieving families. This article outlines the logistic efforts required for these donations to take place and provides a framework for providers to offer rapid autopsy as an option for families through this program.

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.

Immune landscapes associated with different glioblastoma molecular subtypes.

Recent work has highlighted the tumor microenvironment as a central player in cancer. In particular, interactions between tumor and immune cells may help drive the development of brain tumors such as glioblastoma multiforme (GBM). Despite significant research into the molecular classification of glioblastoma, few studies have characterized in a comprehensive manner the immune infiltrate in situ and within different GBM subtypes.In this study, we use an unbiased, automated immunohistochemistry-based approach to determine the immune phenotype of the four GBM subtypes (classical, mesenchymal, neural and proneural) in a cohort of 98 patients. Tissue Micro Arrays (TMA) were stained for CD20 (B lymphocytes), CD5, CD3, CD4, CD8 (T lymphocytes), CD68 (microglia), and CD163 (bone marrow derived macrophages) antibodies. Using automated image analysis, the percentage of each immune population was calculated with respect to the total tumor cells. Mesenchymal GBMs displayed the highest percentage of microglia, macrophage, and lymphocyte infiltration. CD68+ and CD163+ cells were the most abundant cell populations in all four GBM subtypes, and a higher percentage of CD163+ cells was associated with a worse prognosis. We also compared our results to the relative composition of immune cell type infiltration (using RNA-seq data) across TCGA GBM tumors and validated our results obtained with immunohistochemistry with an external cohort and a different method. The results of this study offer a comprehensive analysis of the distribution and the infiltration of the immune components across the four commonly described GBM subgroups, setting the basis for a more detailed patient classification and new insights that may be used to better apply or design immunotherapies for GBM.

Ex vivo imaging of active caspase 3 by a FRET-based molecular probe demonstrates the cellular dynamics and localization of the protease in cerebellar granule cells and its regulation by the apoptosis-inhibiting protein survivin.

BACKGROUND: Apoptosis takes place in naturally occurring neuronal death, but also in aging, neurodegenerative disorders, and traumatic brain injuries. Caspase 3 (Casp3) is the most important effector protease in apoptosis: being inactive inside the cell, it undergoes enzymatic cleavage and - hence - activation once the apoptotic cascade is triggered. Immunological techniques with antibodies against cleaved Casp3 (cCasp3) or assays with colorimetric/fluorogenic substrates are commonly in use, but they do not allow to directly follow the dynamics of activation in alive neurons that may be committed to die. RESULTS: By combined biolistic transfection, confocal microscopy, and fluorescence resonance energy transfer (FRET), we have implemented a methodology to dynamically monitor Casp3 activation in organotypic cerebellar slices from postnatal mice. After transfection with pSCAT3 FRET probes, we measured the ratio of the emissions of the donor/acceptor pair (ECFPem/Venusem) in fixed or alive cultures. In so doing, we i. discriminated the cellular compartment(s) of enzyme activation (nucleus, perikaryon, neurites); ii. demonstrated that Casp3 was constitutively active in the granule cells; iii. followed the fluctuations of ECFPem/Venusem, and its response to 25 mM KCl depolarization, or to increased intracellular Ca(++) after NMDA (1 mM), kainic acid (1 mM), or A23187 (100-200 µM). The specificity of the active pSCAT3-DEVD probe was confirmed with RNA interference and after inhibition of Casp3 with Ac-DEVD-CMK (100 µM), as both sets of experiments brought ECFPem/Venusem to the values recorded with the control probe pSCAT3-DEVG. After double-transfection with pSCAT3-DEVD + pHcRed1-C1-survivin, we also showed a 44-56% reduction of basal Casp3 activity in cells overexpressing survivin, a protein-member of the family of apoptosis inhibitors, with augmented survival (2.82 folds). Survivin-rescued cells were sensitive to 5 mM H2O2 oxidative stress but died without intervention of Casp3. CONCLUSIONS: This ex vivo FRET-based methodology provides quantitative information on the functional and histological dynamics of Casp3 activation in individual neurons at a cell level resolution. Not only it can be combined with experimental manipulation of the apoptotic machinery inside the cell, but offers several advantages over existing protocols for monitoring apoptosis in live mammalian neurons, and has potential to be transferred in vivo. Due to the pivotal role of Casp3 in apoptosis, our approach is relevant for a better comprehension of molecular neurodegeneration in the normal and pathological brain.

Alterations of Cell Proliferation and Apoptosis in the Hypoplastic Reeler Cerebellum.

A mutation of the reln gene gives rise to the Reeler mouse (reln (-∕-)) displaying an ataxic phenotype and cerebellar hypoplasia. We have characterized the neurochemistry of postnatal (P0-P60) reln (-∕-) mouse cerebella with specific attention to the intervention of cell proliferation and apoptosis in the P0-P25 interval. Homozygous reln (-∕-) mice and age-matched controls were analyzed by immunofluorescence using primary antibodies against NeuN, calbindin, GFAP, vimentin, SMI32, and GAD67. Proliferation and apoptosis were detected after a single intraperitoneal BrdU injection and by the TUNEL assay with anti-digoxigenin rhodamine-conjugated antibodies. Quantitative analysis with descriptive and predictive statistics was used to calculate cell densities (number/mm(2)) after fluorescent nuclear stain (TCD, total cell density), labeling with BrdU (PrCD, proliferating cell density), or TUNEL (ApoCD, apoptotic cell density). By this approach we first have shown that the temporal pattern of expression of neuronal/glial markers in postnatal cerebellum is not affected by the Reeler mutation. Then, we have demonstrated that the hypoplasia in the Reeler mouse cerebellum is consequent to reduction of cortical size and cellularity (TCD), and that TCD is, in turn, linked to quantitative differences in the extent of cell proliferation and apoptosis, as well as derangements in their temporal trends during postnatal maturation. Finally, we have calculated that PrCD is the most important predictive factor to determine TCD in the cerebellar cortex of the mutants. These results support the notion that, beside the well-known consequences onto the migration of the cerebellar neurons, the lack of Reelin results in a measurable deficit in neural proliferation.

The number of Purkinje neurons and their topology in the cerebellar vermis of normal and reln haplodeficient mouse.

The Reeler heterozygous mice (reln(+/-)) are haplodeficient in the gene (reln) encoding for the reelin glycoprotein (RELN) and display reductions in brain/peripheral RELN similar to autistic or schizophrenic patients. Cytoarchitectonic alterations of the reln(+/-) brain may be subtle, and are difficult to demonstrate by current histological approaches. We analyzed the number and topological organization of the Purkinje neurons (PNs) in five vermal lobules - central (II-III), culmen (IV-V), tuber (VIIb), uvula (IX), and nodulus (X) - that process different types of afferent functional inputs in reln(+/+) and reln(+/-) adult mice (P60) of both sexes (n=24). Animals were crossed with L7GFP mice so that the GFP-tagged PNs could be directly identified in cryosections. Digital images from these sections were processed with different open source software for quantitative topological and statistical analyses. Diversity indices calculated were: maximum caliper, density, area of soma, dispersion along the XZ axis, and dispersion along the YZ axis. We demonstrate: i. reduction in density of PNs in reln(+/-) males (14.37%) and reln(+/-) females (17.73%) compared to reln(+/+) males; ii. that reln(+/-) males have larger PNs than other genotypes, and females (irrespective of the reln genetic background) have smaller PNs than reln(+/+) males; iii. PNs are more chaotically arranged along the YZ axis in reln(+/-) males than in reln(+/+) males and, except in central lobulus, reln(+/-) females. Therefore, image processing and statistics reveal previously unforeseen gender and genotype-related structural differences in cerebellum that may be clues for the definition of novel biomarkers in human psychiatric disorders.

Real-time visualization of caspase-3 activation by fluorescence resonance energy transfer (FRET).

As apoptosis occurs via a complex signaling cascade that is tightly regulated at multiple cell points, different methods exist to evaluate the activity of the proteins involved in the intracellular apoptotic pathways and the phenotype of apoptotic neurons. Detention of the activity of the enzyme caspase-3, the key executioner caspase in programmed cell death, by laser scanning confocal fluorescence microscopy and the fluorescence resonance energy transfer technology is an alternative approach to classical standard techniques, such as Western blotting, activity assays, or histological techniques, and allows working with both fixed and living cells. This technique combined with the organotypic culture approach ex vivo represents a valid tool for the study of the mechanisms of neuronal survival /death and neuroprotection.