Patient-derived organoids recapitulate glioma-intrinsic immune program and progenitor populations of glioblastoma.

Glioblastoma multiforme (GBM) is a highly lethal human cancer thought to originate from a self-renewing and therapeutically-resistant population of glioblastoma stem cells (GSCs). The intrinsic mechanisms enacted by GSCs during 3D tumor formation, however, remain unclear, especially in the stages prior to angiogenic/immunological infiltration. In this study, we performed a deep characterization of the genetic, immune, and metabolic profiles of GBM organoids from several patient-derived GSCs (GBMO). Despite being devoid of immune cells, transcriptomic analysis across GBMO revealed a surprising immune-like molecular program, enriched in cytokine, antigen presentation and processing, T-cell receptor inhibitors, and interferon genes. We find two important cell populations thought to drive GBM progression, Special AT-rich sequence-binding protein 2 (SATB2+) and homeodomain-only protein homeobox (HOPX+) progenitors, contribute to this immune landscape in GBMO and GBM in vivo. These progenitors, but not other cell types in GBMO, are resistant to conventional GBM therapies, temozolomide and irradiation. Our work defines a novel intrinsic immune-like landscape in GBMO driven, in part, by SATB2+ and HOPX+ progenitors and deepens our understanding of the intrinsic mechanisms utilized by GSCs in early GBM formation.

Stat1 is an inducible transcriptional repressor of neural stem cells self-renewal program during neuroinflammation.

A central issue in regenerative medicine is understanding the mechanisms that regulate the self-renewal of endogenous stem cells in response to injury and disease. Interferons increase hematopoietic stem cells during infection by activating STAT1, but the mechanisms by which STAT1 regulates intrinsic programs in neural stem cells (NSCs) during neuroinflammation is less known. Here we explored the role of STAT1 on NSC self-renewal. We show that overexpressing Stat1 in NSCs derived from the subventricular zone (SVZ) decreases NSC self-renewal capacity while Stat1 deletion increases NSC self-renewal, neurogenesis, and oligodendrogenesis in isolated NSCs. Importantly, we find upregulation of STAT1 in NSCs in a mouse model of multiple sclerosis (MS) and an increase in pathological T cells expressing IFN-γ rather than interleukin 17 (IL-17) in the cerebrospinal fluid of affected mice. We find IFN-γ is superior to IL-17 in reducing proliferation and precipitating an abnormal NSC phenotype featuring increased STAT1 phosphorylation and Stat1 and p16ink4a gene expression. Notably, Stat1-/- NSCs were resistant to the effect of IFN-γ. Lastly, we identified a Stat1-dependent gene expression profile associated with an increase in the Sox9 transcription factor, a regulator of self-renewal. Stat1 binds and transcriptionally represses Sox9 in a transcriptional luciferase assay. We conclude that Stat1 serves as an inducible checkpoint for NSC self-renewal that is upregulated during chronic brain inflammation leading to decreased self-renewal. As such, Stat1 may be a potential target to modulate for next generation therapies to prevent progression and loss of repair function in NSCs/neural progenitors in MS.

SARS-CoV2 entry factors are expressed in primary human glioblastoma and recapitulated in cerebral organoid models.

PURPOSE: Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults with a median overall survival of only 14.6 months despite aggressive treatment. While immunotherapy has been successful in other cancers, its benefit has been proven elusive in GBM, mainly due to a markedly immunosuppressive tumor microenvironment. SARS-CoV-2 has been associated with the development of a pronounced central nervous system (CNS) inflammatory response when infecting different cells including astrocytes, endothelial cells, and microglia. While SARS-CoV2 entry factors have been described in different tissues, their presence and implication on GBM aggressiveness or microenvironment has not been studied on appropriate preclinical models. METHODS: We evaluated the presence of crucial SARS-CoV-2 entry factors: ACE2, TMPRSS2, and NRP1 in matched surgically-derived GBM tissue, cells lines, and organoids; as well as in human brain derived specimens using immunohistochemistry, confocal pixel line intensity quantification, and transcriptome analysis. RESULTS: We show that patient derived-GBM tissue and cell cultures express SARS-CoV2 entry factors, being NRP1 the most crucial facilitator of SARS-CoV-2 infection in GBM. Moreover, we demonstrate that, receptor expression remains present in our GBM organoids, making them an adequate model to study the effect of this virus in GBM for the potential development of viral therapies in the future. CONCLUSION: Our findings suggest that the SARS-CoV-2 virus entry factors are expressed in primary tissues and organoid models and could be potentially utilized to study the susceptibility of GBM to this virus to target or modulate the tumor microenviroment.

Development of Experimental Three-Dimensional Tumor Models to Study Glioblastoma Cancer Stem Cells and Tumor Microenvironment.

Glioblastoma (GBM) is the most common and dismal primary brain tumor. Unfortunately, despite multidisciplinary treatment, most patients will perish approximately 15 months after diagnosis. For this reason, there is an urgent need to improve our understanding of GBM tumor biology and develop novel therapies that can achieve better clinical outcomes. In this setting, three-dimensional tumor models have risen as more appropriate preclinical tools when compared to traditional cell cultures, given that two-dimensional (2D) cultures have failed to accurately recapitulate tumor biology and translate preclinical findings into patient benefits. Three-dimensional cultures using neurospheres, organoids, and organotypic better resemble original tumor genetic and epigenetic profiles, maintaining tumor microenvironment characteristics and mimicking cell-cell and cell-matrix interactions. This chapter summarizes our methods to generate well-characterized glioblastoma neurospheres, organoids, and organotypics.

Monthly triple-dose gadolinium administration: potential associations with leukopenia, hypophosphatemia, and bone marrow T1 hyperintensity.

OBJECTIVE: Monthly scanning with triple-dose gadopentetate dimeglumine has been shown to be associated with progressive increases in bone T1 hyperintensity, hypophosphatemia, and leukopenia. This study was performed to retrospectively investigate the potential associations among these phenomena. METHODS: This retrospective analysis involved patients who had received monthly triple-dose gadopentetate dimeglumine for up to 2 years as part of treatment for multiple sclerosis. Monthly magnetic resonance imaging scans of the brain (n = 67) were segmented to evaluate the signal intensity in the cranial marrow. Potential associations among the marrow T1 hyperintensity, serum phosphate concentration, and white blood cell count were examined. RESULTS: Patients in the no leukopenia group showed a statistically significant mean monthly increase in the bone marrow signal-to-noise ratio of 0.0430/month. Patients in the leukopenia group showed a mean monthly increase in the bone marrow signal-to-noise ratio of 0.0398/month, but this was not statistically significant. Patients in the hypophosphatemia group were significantly less likely to develop leukopenia than patients who had never developed hypophosphatemia. CONCLUSIONS: Although monthly administration of triple-dose gadopentetate dimeglumine over 13 months has been associated with progressive increases in leukopenia, hypophosphatemia, and T1 signal intensity of bone, this study showed an inverse relationship between leukopenia and hypophosphatemia.

Real-world experience of ocrelizumab initiation in a diverse multiple sclerosis population.

BACKGROUND: Ocrelizumab (OCR) is a humanized monoclonal antibody directed against CD20 positive B-lymphocytes. It was approved for use in 2017 by the U.S. Food and Drug Administration (FDA) for both the relapsing-remitting and primary progressive forms of multiple sclerosis (MS). OBJECTIVE: To provide real-world data for patients with MS treated with OCR in our center and evaluate both the safety and efficacy across different ethnic groups not studied in previous clinical trials. METHODS: We performed a retrospective observational analysis of MS patients who were treated with OCR from March 31, 2017 to April 30, 2020. We collected data on patients who had received at least a one dose infusion of OCR at our MS center. Patient characteristics, including demographics, clinical disease course, and documented side effects, were collected and analyzed. RESULTS: A total of 82 patients were eligible for this study, of which 72% had relapsing-remitting MS (RRMS), 14% had primary progressive MS (PPMS), and 11% active/relapsing secondary progressive MS (SPMS). 22% of our patients were of African American descent, 61% Caucasian, and 17% of Hispanic descent. The mean age of starting OCR was 41 ± 11 years. 47% were treatment naïve when started on OCR, 24% were previously treated with one disease-modifying therapy (DMT), 14% were treated with two DMTs, and 15% were treated with more than two DMTs prior to OCR. 50% of patients had at least one adverse event while on OCR; 4.8% had adverse events requiring to OCR discontinuation, 36% had infusion-related reactions, and 7.3% had viral infections. We found two cases of severe babesiosis along with index cases of re-activation of lichen planus, agranulocytosis, severe lymphopenia, and ectopic pregnancy. There were no cases of malignancy, progressive multifocal leukoencephalopathy, or death within our cohort. The mean time after OCR initiation was 17.3 months in the RRMS group, 22.2 months in the PPMS group, and 28.2 months in SPMS group. The annualized relapse rate reduced from 1.33 to 0.15 in the RRMS group. The mean extended disability status scale (EDSS) scores did not worsen across MS phenotypes and ethnic groups while being treated with OCR. CONCLUSIONS: In a diverse patient population, OCR was well-tolerated without significant adverse events. There were novel cases of severe babesiosis, re-activation of lichen planus, lymphopenia, agranulocytosis, and ectopic pregnancy. It is vital to consider geographic risk factors that may expose patients to Babesia microti (B. microti) when either considering or initiating OCR therapy. There were an additional six cases of severe B. microti cases associated with OCR that were reported to the FDA adverse event reporting system (FAERS) along with multiple babesiosis cases associated with other DMTs, including rituximab. OCR was found in our cohort to be effective by decreasing relapse rates and maintaining EDSS scores. Our study extends the generalizability of OCR from clinical trials to a real-world setting consisting of a diverse population.

APOSTEL 2.0 Recommendations for Reporting Quantitative Optical Coherence Tomography Studies.

OBJECTIVE: To update the consensus recommendations for reporting of quantitative optical coherence tomography (OCT) study results, thus revising the previously published Advised Protocol for OCT Study Terminology and Elements (APOSTEL) recommendations. METHODS: To identify studies reporting quantitative OCT results, we performed a PubMed search for the terms "quantitative" and "optical coherence tomography" from 2015 to 2017. Corresponding authors of the identified publications were invited to provide feedback on the initial APOSTEL recommendations via online surveys following the principle of a modified Delphi method. The results were evaluated and discussed by a panel of experts and changes to the initial recommendations were proposed. A final survey was recirculated among the corresponding authors to obtain a majority vote on the proposed changes. RESULTS: A total of 116 authors participated in the surveys, resulting in 15 suggestions, of which 12 were finally accepted and incorporated into an updated 9-point checklist. We harmonized the nomenclature of the outer retinal layers, added the exact area of measurement to the description of volume scans, and suggested reporting device-specific features. We advised to address potential bias in manual segmentation or manual correction of segmentation errors. References to specific reporting guidelines and room light conditions were removed. The participants' consensus with the recommendations increased from 80% for the previous APOSTEL version to greater than 90%. CONCLUSIONS: The modified Delphi method resulted in an expert-led guideline (evidence Class III; Grading of Recommendations, Assessment, Development and Evaluations [GRADE] criteria) concerning study protocol, acquisition device, acquisition settings, scanning protocol, funduscopic imaging, postacquisition data selection, postacquisition analysis, nomenclature and abbreviations, and statistical approach. It will be essential to update these recommendations to new research and practices regularly.

Babesia microti infection in a patient with multiple sclerosis treated with ocrelizumab.

Ocrelizumab is a humanized monoclonal anti-CD20 antibody approved for treatment of relapsing-remitting and primary progressive multiple sclerosis (MS). Rare parasitic infections have been reported in patients with lymphoproliferative disorders using rituximab, a chimeric anti-CD20 antibody used off-label for the treatment of MS. Here, we report a patient with MS on ocrelizumab with B-cell depletion who developed severe Babesia microti (B. microti) infection with neutropenia, hemolytic anemia, and thrombocytopenia. He recovered after prompt diagnosis and treatment. This case represents the first published occurrence of babesiosis in a patient with MS on ocrelizumab. It also adds to the accumulating evidence from databases of emergent severe or relapsing B. microti infection in patients receiving anti-CD20 antibodies. This presentation stresses the diagnostic vigilance required by MS neurologists in endemic areas to identify cases of babesiosis in patients on anti-CD20 therapy and to better counsel these individuals on their risks of B. microti infection.

Inflammatory Mediators in Glioma Microenvironment Play a Dual Role in Gliomagenesis and Mesenchymal Stem Cell Homing: Implication for Cellular Therapy.

Glioblastoma is the most aggressive malignant primary brain tumor, with a dismal prognosis and a devastating overall survival. Despite aggressive surgical resection and adjuvant treatment, average survival remains approximately 14.6 months. The brain tumor microenvironment is heterogeneous, comprising multiple populations of tumor, stromal, and immune cells. Tumor cells evade the immune system by suppressing several immune functions to enable survival. Gliomas release immunosuppressive and tumor-supportive soluble factors into the microenvironment, leading to accelerated cancer proliferation, invasion, and immune escape. Mesenchymal stem cells (MSCs) isolated from bone marrow, adipose tissue, or umbilical cord are a promising tool for cell-based therapies. One crucial mechanism mediating the therapeutic outcomes often seen in MSC application is their tropism to sites of injury. Furthermore, MSCs interact with host immune cells to regulate the inflammatory response, and data points to the possibility of using MSCs to achieve immunomodulation in solid tumors. Interleukin 1ß, interleukin 6, tumor necrosis factor α, transforming growth factor ß, and stromal cell-derived factor 1 are notably up-regulated in glioblastoma and dually promote immune and MSC trafficking. Mesenchymal stem cells have widely been regarded as hypoimmunogenic, enabling this cell-based administration across major histocompatibility barriers. In this review, we will highlight (1) the bidirectional communication of glioma cells and tumor-associated immune cells, (2) the inflammatory mediators enabling leukocytes and transplantable MSC migration, and (3) review preclinical and human clinical trials using MSCs as delivery vehicles. Mesenchymal stem cells possess innate abilities to migrate great distances, cross the blood-brain barrier, and communicate with surrounding cells, all of which make them desirable "Trojan horses" for brain cancer therapy.

Complications from "Stem Cell Tourism" in Neurology.

"Stem cell tourism," the practice of offering unproven cellular preparations to patients as approved therapy, is rising in neurology. Currently, the experiences of patients and reported complications from these procedures are unknown in the United States. We evaluate academic neurologists' experiences with stem cell tourism and assess perceived competency on discussing this topic with patients. We found a lack of neurologist preparedness to discuss stem cell therapies with patients and an alarming list of unreported complications from these unregulated procedures. We also identified an urgent need for neurologist education and the creation of a national registry for reporting patient complications resulting from experimental stem cell interventions. ANN NEUROL 2020;88:661-668.

A possible link between intracranial lipomas and localization-related relapses in multiple sclerosis.

Intracranial lipomas are congenital malformations representing less than 0.5% of intracranial tumors. They are found incidentally and are asymptomatic in the majority of patients. Here we present three patients with Multiple sclerosis (MS) and intracranial lipomas (IL). The patients showed increased flares and burden of disabling and worsening MS symptoms with cognitive, neurovestibular dysfunction, and gait alterations associated with the localization of the Lipoma. The parenchyma near the Lipomas showed areas of demyelination and atrophy. We postulate that the location and content of the Lipomas may participate in the pathophysiology of MS symptoms in these patients. We conclude that in concurrent IL and MS, the lipomas localization may provoke incapacitating relapses.

Convergence of human cellular models and genetics to study neural stem cell signaling to enhance central nervous system regeneration and repair.

Human central nervous system (CNS) regeneration is considered the holy grail of neuroscience research, and is one of the most pressing and difficult questions in biology and science. Despite more than 20 years of work in the field of neural stem cells (NSCs), the area remains in its infancy as our understanding of the fundamental mechanisms that can be leveraged to improve CNS regeneration in neurological diseases is still growing. Here, we focus on the recent lessons from lower organism CNS regeneration genetics and how such findings are starting to illuminate our understanding of NSC signaling pathways in humans. These findings will allow us to improve upon our knowledge of endogenous NSC function, the utility of exogenous NSCs, and the limitations of NSCs as therapeutic vehicles for providing relief from devastating human neurological diseases. We also discuss the limitations of activating NSC signaling for CNS repair in humans, especially the potential for tumor formation. Finally, we will review the recent advances in new culture techniques, including patient-derived cells and cerebral organoids to model the genetic regulation of signaling pathways controlling the function of NSCs during injury and disease states.

Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation.

In demyelinating diseases including multiple sclerosis (MS), neural stem cells (NSCs) can replace damaged oligodendrocytes if the local microenvironment supports the required differentiation process. Although chitinase-like proteins (CLPs) form part of this microenvironment, their function in this differentiation process is unknown. Here, we demonstrate that murine Chitinase 3-like-3 (Chi3l3/Ym1), human Chi3L1 and Chit1 induce oligodendrogenesis. In mice, Chi3l3 is highly expressed in the subventricular zone, a stem cell niche of the adult brain, and in inflammatory brain lesions during experimental autoimmune encephalomyelitis (EAE). We find that silencing Chi3l3 increases severity of EAE. We present evidence that in NSCs Chi3l3 activates the epidermal growth factor receptor (EGFR), thereby inducing Pyk2-and Erk1/2- dependent expression of a pro-oligodendrogenic transcription factor signature. Our results implicate CLP-EGFR-Pyk2-MEK-ERK as a key intrinsic pathway controlling oligodendrogenesis.

The "Growing" Reality of the Neurological Complications of Global "Stem Cell Tourism".

"Stem cell tourism" is defined as the unethical practice of offering unproven cellular preparations to patients suffering from various medical conditions. This phenomenon is rising in the field of neurology as patients are requesting information and opportunities for treatment with stem cells for incurable conditions such as multiple sclerosis and amyotrophic lateral sclerosis, despite their clinical research and experimental designation. Here, we review the recent trends in "stem cell tourism" in both the United States and abroad, and discuss the recent reports of neurological complications from these activities. Finally, we frame critical questions for the field of neurology regarding training in the ethical, legal, and societal issues of the global "stem cell tourism," as well as suggest strategies to alleviate this problem. Although there are ongoing legitimate clinical trials with stem cells for neurological diseases, procedures offered by "stem cell clinics" cannot be defined as clinical research. They lack the experimental and state-of-the-art framework defined by peers and the FDA that focus on human research that safeguard the protection of human subjects against economical exploitation, unwanted side effects, and futility of unproven procedures. "Stem cell tourism" ultimately exploits therapeutic hope of patients and families with incurable neurological diseases and can put in danger the legitimacy of stem cell research as a whole. We posit that an improvement in education, regulation, legislation, and involvement of authorities in global health in neurology and neurosurgery is required.

PRMT5 as a druggable target for glioblastoma therapy.

Background: In spite of standard multimodal therapy consisting of surgical resection followed by radiation and concurrent chemotherapy, prognosis for glioblastoma (GBM) patients remains poor. The identification of both differentiated and undifferentiated "stem cell like" populations in the tumor highlights the significance of finding novel targets that affect the heterogeneous tumor cell population. Protein arginine methyltransferase 5 (PRMT5) is one such candidate gene whose nuclear expression correlates with poor survival and has been reported to be required for survival of differentiated GBM cells and self-renewal of undifferentiated GBM cells. In the current study we screened the specificity and efficacy of 4 novel PRMT5 inhibitors in the treatment of GBM. Methods: Efficacies of these inhibitors were screened using an in vitro GBM neurosphere model and an in vivo intracranial zebrafish model of glioma. Standard molecular biology methods were employed to investigate changes in cell cycle, growth, and senescence. Results: In vitro and in vivo studies revealed that among the 4 PRMT5 inhibitors, treatment of GBM cells with compound 5 (CMP5) mirrored the effects of PRMT5 knockdown wherein it led to apoptosis of differentiated GBM cells and drove undifferentiated primary patient derived GBM cells into a nonreplicative senescent state. Conclusion: In vivo antitumor efficacy combined with the specificity of CMP5 underscores the importance of developing it for translation.

Progressive multifocal leukoencephalopathy in a patient without apparent immunosuppression.

Progressive multifocal leukoencephalopathy (PML) is a viral demyelinating disease due to the reactivation of the JC virus (JCV), which usually occurs in the context of immunosuppression in HIV infection, malignancy, or in patients on disease modifying therapy for autoimmune diseases, such as multiple sclerosis (MS) and Crohn's disease. Notably, there is growing recognition that PML can occur in patients with transient immune dysfunction. Here, we present a case of a 55-year-old man without history of immunosuppression or evidence of ICL who was diagnosed with PML on brain biopsy. We will discuss the potential etiologies of mild and transient immunosuppression that can lead to PML with non-apparent immunosuppression.

Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity.

Glioblastoma (GBM) is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP) or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2(+) cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a platform for drug screening.