Phase I clinical trial of intracerebroventricular transplantation of allogeneic neural stem cells in people with progressive multiple sclerosis.

We report the analysis of 1 year of data from the first cohort of 15 patients enrolled in an open-label, first-in-human, dose-escalation phase I study (ClinicalTrials.gov: NCT03282760, EudraCT2015-004855-37) to determine the feasibility, safety, and tolerability of the transplantation of allogeneic human neural stem/progenitor cells (hNSCs) for the treatment of secondary progressive multiple sclerosis. Participants were treated with hNSCs delivered via intracerebroventricular injection in combination with an immunosuppressive regimen. No treatment-related deaths nor serious adverse events (AEs) were observed. All participants displayed stability of clinical and laboratory outcomes, as well as lesion load and brain activity (MRI), compared with the study entry. Longitudinal metabolomics and lipidomics of biological fluids identified time- and dose-dependent responses with increased levels of acyl-carnitines and fatty acids in the cerebrospinal fluid (CSF). The absence of AEs and the stability of functional and structural outcomes are reassuring and represent a milestone for the safe translation of stem cells into regenerative medicines.

Different states of stemness of glioblastoma stem cells sustain glioblastoma subtypes indicating novel clinical biomarkers and high-efficacy customized therapies.

BACKGROUND: Glioblastoma (GBM) is the most malignant among gliomas with an inevitable lethal outcome. The elucidation of the physiology and regulation of this tumor is mandatory to unravel novel target and effective therapeutics. Emerging concepts show that the minor subset of glioblastoma stem cells (GSCs) accounts for tumorigenicity, representing the true target for innovative therapies in GBM. METHODS: Here, we isolated and established functionally stable and steadily expanding GSCs lines from a large cohort of GBM patients. The molecular, functional and antigenic landscape of GBM tissues and their derivative GSCs was highlited in a side-by-side comprehensive genomic and transcriptomic characterization by ANOVA and Fisher's exact tests. GSCs' physio-pathological hallmarks were delineated by comparing over time in vitro and in vivo their expansion, self-renewal and tumorigenic ability with hierarchical linear models for repeated measurements and Kaplan-Meier method. Candidate biomarkers performance in discriminating GBM patients' classification emerged by classification tree and patients' survival analysis. RESULTS: Here, distinct biomarker signatures together with aberrant functional programs were shown to stratify GBM patients as well as their sibling GSCs population into TCGA clusters. Of importance, GSCs cells were demonstrated to fully resemble over time the molecular features of their patient of origin. Furthermore, we pointed out the existence of distinct GSCs subsets within GBM classification, inherently endowed with different self-renewal and tumorigenic potential. Particularly, classical GSCs were identified by more undifferentiated biological hallmarks, enhanced expansion and clonal capacity as compared to the more mature, relatively slow-propagating mesenchymal and proneural cells, likely endowed with a higher potential for infiltration either ex vivo or in vivo. Importantly, the combination of DCX and EGFR markers, selectively enriched among GSCs pools, almost exactly predicted GBM patients' clusters together with their survival and drug response. CONCLUSIONS: In this study we report that an inherent enrichment of distinct GSCs pools underpin the functional inter-cluster variances displayed by GBM patients. We uncover two selectively represented novel functional biomarkers capable of discriminating GBM patients' stratification, survival and drug response, setting the stage for the determination of patient-tailored diagnostic and prognostic strategies and, mostly, for the design of appropriate, patient-selective treatment protocols.

AQP4-dependent glioma cell features affect the phenotype of surrounding cells via extracellular vesicles.

BACKGROUND: Extracellular vesicles (EVs) are membrane-enclosed particles released systemically by all cells, including tumours. Tumour EVs have been shown to manipulate their local environments as well as distal targets to sustain the tumour in a variety of tumours, including glioblastoma (GBM). We have previously demonstrated the dual role of the glial water channel aquaporin-4 (AQP4) protein in glioma progression or suppression depending on its aggregation state. However, its possible role in communication mechanisms in the microenvironment of malignant gliomas remains to be unveiled. RESULTS: Here we show that in GBM cells AQP4 is released via EVs that are able to affect the GBM microenvironment. To explore this role, EVs derived from invasive GBM cells expressing AQP4-tetramers or apoptotic GBM cells expressing orthogonal arrays of particles (AQP4-OAPs) were isolated, using a differential ultracentrifugation method, and were added to pre-seeded GBM cells. Confocal microscopy analysis was used to visualize the interaction and uptake of AQP4-containing EVs by recipient cells. Chemoinvasion and Caspase3/7 activation assay, performed on recipient cells after EVs uptake, revealed that EVs produced by AQP4-tetramers expressing cells were able to drive surrounding tumour cells toward the migratory phenotype, whereas EVs produced by AQP4-OAPs expressing cells drive them toward the apoptosis pathway. CONCLUSION: This study demonstrates that the different GBM cell phenotypes can be transferred by AQP4-containing EVs able to influence tumour cell fate toward invasiveness or apoptosis. This study opens a new perspective on the role of AQP4 in the brain tumour microenvironment associated with the EV-dependent communication mechanism.

Growth factor independence underpins a paroxysmal, aggressive Wnt5aHigh/EphA2Low phenotype in glioblastoma stem cells, conducive to experimental combinatorial therapy.

BACKGROUND: Glioblastoma multiforme (GBM) is an incurable tumor, with a median survival rate of only 14-15 months. Along with heterogeneity and unregulated growth, a central matter in dealing with GBMs is cell invasiveness. Thus, improving prognosis requires finding new agents to inhibit key multiple pathways, even simultaneously. A subset of GBM stem-like cells (GSCs) may account for tumorigenicity, representing, through their pathways, the proper cellular target in the therapeutics of glioblastomas. GSCs cells are routinely enriched and expanded due to continuous exposure to specific growth factors, which might alter some of their intrinsic characteristic and hide therapeutically relevant traits. METHODS: By removing exogenous growth factors stimulation, here we isolated and characterized a subset of GSCs with a "mitogen-independent" phenotype (I-GSCs) from patient's tumor specimens. Differential side-by-side comparative functional and molecular analyses were performed either in vitro or in vivo on these cells versus their classical growth factor (GF)-dependent counterpart (D-GSCs) as well as their tissue of origin. This was performed to pinpoint the inherent GSCs' critical regulators, with particular emphasis on those involved in spreading and tumorigenic potential. Transcriptomic fingerprints were pointed out by ANOVA with Benjamini-Hochberg False Discovery Rate (FDR) and association of copy number alterations or somatic mutations was determined by comparing each subgroup with a two-tailed Fisher's exact test. The combined effects of interacting in vitro and in vivo with two emerging GSCs' key regulators, such as Wnt5a and EphA2, were then predicted under in vivo experimental settings that are conducive to clinical applications. In vivo comparisons were carried out in mouse-human xenografts GBM model by a hierarchical linear model for repeated measurements and Dunnett's multiple comparison test with the distribution of survival compared by Kaplan-Meier method. RESULTS: Here, we assessed that a subset of GSCs from high-grade gliomas is self-sufficient in the activation of regulatory growth signaling. Furthermore, while constitutively present within the same GBM tissue, these GF-independent GSCs cells were endowed with a distinctive functional and molecular repertoire, defined by highly aggressive Wnt5aHigh/EphA2Low profile, as opposed to Wnt5aLow/EphA2High expression in sibling D-GSCs. Regardless of their GBM subtype of origin, I-GSCs, are endowed with a raised in vivo tumorigenic potential than matched D-GSCs, which were fast-growing ex-vivo but less lethal and invasive in vivo. Also, the malignant I-GSCs' transcriptomic fingerprint faithfully mirrored the original tumor, bringing into evidence key regulators of invasiveness, angiogenesis and immuno-modulators, which became candidates for glioma diagnostic/prognostic markers and therapeutic targets. Particularly, simultaneously counteracting the activity of the tissue invasive mediator Wnt5a and EphA2 tyrosine kinase receptor addictively hindered GSCs' tumorigenic and invasive ability, thus increasing survival. CONCLUSION: We show how the preservation of a mitogen-independent phenotype in GSCs plays a central role in determining the exacerbated tumorigenic and high mobility features distinctive of GBM. The exploitation of the I-GSCs' peculiar features shown here offers new ways to identify novel, GSCs-specific effectors, whose modulation can be used in order to identify novel, potential molecular therapeutic targets. Furthermore, we show how the combined use of PepA, the anti-Wnt5a drug, and of ephrinA1-Fc to can hinder GSCs' lethality in a clinically relevant xenogeneic in vivo model thus being conducive to perspective, novel combinatorial clinical application.

BRAFV600E mutation impinges on gut microbial markers defining novel biomarkers for serrated colorectal cancer effective therapies.

BACKGROUND: Colorectal cancer (CRC) harboring BRAFV600E mutation exhibits low response to conventional therapy and poorest prognosis. Due to the emerging correlation between gut microbiota and CRC carcinogenesis, we investigated in serrated BRAFV600E cases the existence of a peculiar fecal microbial fingerprint and specific bacterial markers, which might represent a tool for the development of more effective clinical strategies. METHODS: By injecting human CRC stem-like cells isolated from BRAFV600E patients in immunocompromised mice, we described a new xenogeneic model of this subtype of CRC. By performing bacterial 16S rRNA sequencing, the fecal microbiota profile was then investigated either in CRC-carrying mice or in a cohort of human CRC subjects. The microbial communities' functional profile was also predicted. Data were compared with Mann-Whitney U, Welch's t-test for unequal variances and Kruskal-Wallis test with Benjamini-Hochberg false discovery rate (FDR) correction, extracted as potential BRAF class biomarkers and selected as model features. The obtained mean test prediction scores were subjected to Receiver Operating characteristic (ROC) analysis. To discriminate the BRAF status, a Random Forest classifier (RF) was employed. RESULTS: A specific microbial signature distinctive for BRAF status emerged, being the BRAF-mutated cases closer to healthy controls than BRAF wild-type counterpart. In agreement, a considerable score of correlation was also pointed out between bacteria abundance from BRAF-mutated cases and the level of markers distinctive of BRAFV600E pathway, including those involved in inflammation, innate immune response and epithelial-mesenchymal transition. We provide evidence that two candidate bacterial markers, Prevotella enoeca and Ruthenibacterium lactatiformans, more abundant in BRAFV600E and BRAF wild-type subjects respectively, emerged as single factors with the best performance in distinguishing BRAF status (AUROC = 0.72 and 0.74, respectively, 95% confidence interval). Furthermore, the combination of the 10 differentially represented microorganisms between the two groups improved performance in discriminating serrated CRC driven by BRAF mutation from BRAF wild-type CRC cases (AUROC = 0.85, 95% confidence interval, 0.69-1.01). CONCLUSION: Overall, our results suggest that BRAFV600E mutation itself drives a distinctive gut microbiota signature and provide predictive CRC-associated bacterial biomarkers able to discriminate BRAF status in CRC patients and, thus, useful to devise non-invasive patient-selective diagnostic strategies and patient-tailored optimized therapies.

Results from Phase I Clinical Trial with Intraspinal Injection of Neural Stem Cells in Amyotrophic Lateral Sclerosis: A Long-Term Outcome.

The main objective of this phase I trial was to assess the feasibility and safety of microtransplanting human neural stem cell (hNSC) lines into the spinal cord of patients with amyotrophic lateral sclerosis (ALS). Eighteen patients with a definite diagnosis of ALS received microinjections of hNSCs into the gray matter tracts of the lumbar or cervical spinal cord. Patients were monitored before and after transplantation by clinical, psychological, neuroradiological, and neurophysiological assessment. For up to 60 months after surgery, none of the patients manifested severe adverse effects or increased disease progression because of the treatment. Eleven patients died, and two underwent tracheotomy as a result of the natural history of the disease. We detected a transitory decrease in progression of ALS Functional Rating Scale Revised, starting within the first month after surgery and up to 4 months after transplantation. Our results show that transplantation of hNSC is a safe procedure that causes no major deleterious effects over the short or long term. This study is the first example of medical transplantation of a highly standardized cell drug product, which can be reproducibly and stably expanded ex vivo, comprising hNSC that are not immortalized, and are derived from the forebrain of the same two donors throughout this entire study as well as across future trials. Our experimental design provides benefits in terms of enhancing both intra- and interstudy reproducibility and homogeneity. Given the potential therapeutic effects of the hNSCs, our observations support undertaking future phase II clinical studies in which increased cell dosages are studied in larger cohorts of patients. Stem Cells Translational Medicine 2019;8:887&897.

Stemness underpinning all steps of human colorectal cancer defines the core of effective therapeutic strategies.

BACKGROUND: Despite their lethality and ensuing clinical and therapeutic relevance, circulating tumor cells (CTCs) from colorectal carcinoma (CRC) remain elusive, poorly characterized biological entities. METHODS AND FINDINGS: We perfected a cell system of stable, primary lines from human CRC showing that they possess the full complement of ex- and in-vivo, in xenogeneic models, characteristics of CRC stem cells (CCSCs). Here we show how tumor-initiating, CCSCs cells can establish faithful orthotopic phenocopies of the original disease, which contain cells that spread into the circulatory system. While in the vascular bed, these cells retain stemness, thus qualifying as circulating CCSCs (cCCSCs). This is followed by the establishment of lesions in distant organs, which also contain resident metastatic CCSCs (mCCSCs). INTERPRETATION: Our results support the concept that throughout all the stages of CRC, stemness is retained as a continuous property by some of their tumor cells. Importantly, we describe a useful standardized model that can enable isolation and stable perpetuation of human CRC's CCSCs, cCCSCs and mCCSCs, providing a useful platform for studies of CRC initiation and progression that is suitable for the discovery of reliable stage-specific biomarkers and the refinement of new patient-tailored therapies. FUND: This work was financially supported by grants from "Ministero della Salute Italiano"(GR-2011-02351534, RC1703IC36 and RC1803IC35) to Elena Binda and from "Associazione Italiana Cancro" (IG-14368) Angelo L. Vescovi. None of the above funders have any role in study design, data collection, data analysis, interpretation, writing the project.

AQP4 Aggregation State Is a Determinant for Glioma Cell Fate.

The glial water channel protein aquaporin-4 (AQP4) forms heterotetramers in the plasma membrane made of the M23-AQP4 and M1-AQP4 isoforms. The isoform ratio controls AQP4 aggregation into supramolecular structures called orthogonal arrays of particles (AQP4-OAP). The role of AQP4 aggregation into OAP in malignant gliomas is still unclear. In this study, we demonstrate that AQP4 aggregation/disaggregation into OAP influences the biology of glioma cells. Selective expression of the OAP-forming isoform M23-AQP4 (AQP4-OAP) triggered cell shape changes in glioma cells associated with alterations to the F-actin cytoskeleton that affected apoptosis. By contrast, expression of M1-AQP4 (AQP4-tetramers), which is unable to aggregate into OAP, ameliorated glioma cell invasiveness, improved cell migration, and increased methalloproteinase-9 activity. Two prolines (254 and 296) at the C-terminus tail were shown to be important in mediating the relationship between the actin cytoskeleton and AQP4-OAP and AQP4-tetramers. In conclusion, this study demonstrates that AQP4 aggregation state might be an important determinant in orienting glioma cells to persist or perish. AQP4 disaggregation may potentiate invasiveness potential, whereas AQP4 aggregation may activate the apoptotic path. This study shows a new perspective on the role of AQP4 in brain tumors not necessarily associated with edema formation but with AQP4 aggregation/disaggregation dynamics and their link with the actin cytoskeleton. SIGNIFICANCE: This study demonstrates how AQP4 aggregation influences plasma membrane dynamics to alter cell proliferation, invasiveness, migration, and apoptotic potential in glioma cells.

Targeting APLN/APLNR Improves Antiangiogenic Efficiency and Blunts Proinvasive Side Effects of VEGFA/VEGFR2 Blockade in Glioblastoma.

Antiangiogenic therapy of glioblastoma (GBM) with bevacizumab, a VEGFA-blocking antibody, may accelerate tumor cell invasion and induce alternative angiogenic pathways. Here we investigate the roles of the proangiogenic apelin receptor APLNR and its cognate ligand apelin in VEGFA/VEGFR2 antiangiogenic therapy against distinct subtypes of GBM. In proneural GBM, apelin levels were downregulated by VEGFA or VEGFR2 blockade. A central role for apelin/APLNR in controlling GBM vascularization was corroborated in a serial implantation model of the angiogenic switch that occurs in human GBM. Apelin and APLNR are broadly expressed in human GBM, and knockdown or knockout of APLN in orthotopic models of proneural or classical GBM subtypes significantly reduced GBM vascularization compared with controls. However, reduction in apelin expression led to accelerated GBM cell invasion. Analysis of stereotactic GBM biopsies from patients as well as from in vitro and in vivo experiments revealed increased dissemination of APLNR-positive tumor cells when apelin levels were reduced. Application of apelin-F13A, a mutant APLNR ligand, blocked tumor angiogenesis and GBM cell invasion. Furthermore, cotargeting VEGFR2 and APLNR synergistically improved survival of mice bearing proneural GBM. In summary, we show that apelin/APLNR signaling controls GBM angiogenesis and invasion and that both pathologic features are blunted by apelin-F13A. We suggest that apelin-F13A can improve the efficiency and reduce the side effects of established antiangiogenic treatments for distinct GBM subtypes. SIGNIFICANCE: Pharmacologic targeting of the APLNR acts synergistically with established antiangiogenic treatments in glioblastoma and blunts therapy resistance to current strategies for antiangiogenesis.See related commentary by Amoozgar et al., p. 2104.

Establishment of stable iPS-derived human neural stem cell lines suitable for cell therapies.

Establishing specific cell lineages from human induced pluripotent stem cells (hiPSCs) is vital for cell therapy approaches in regenerative medicine, particularly for neurodegenerative disorders. While neural precursors have been induced from hiPSCs, the establishment of hiPSC-derived human neural stem cells (hiNSCs), with characteristics that match foetal hNSCs and abide by cGMP standards, thus allowing clinical applications, has not been described. We generated hiNSCs by a virus-free technique, whose properties recapitulate those of the clinical-grade hNSCs successfully used in an Amyotrophic Lateral Sclerosis (ALS) phase I clinical trial. Ex vivo, hiNSCs critically depend on exogenous mitogens for stable self-renewal and amplification and spontaneously differentiate into astrocytes, oligodendrocytes and neurons upon their removal. In the brain of immunodeficient mice, hiNSCs engraft and differentiate into neurons and glia, without tumour formation. These findings now warrant the establishment of clinical-grade, autologous and continuous hiNSC lines for clinical trials in neurological diseases such as Huntington's, Parkinson's and Alzheimer's, among others.

Advances in stem cell therapy for amyotrophic lateral sclerosis.

INTRODUCTION: Amyotrophic Lateral Sclerosis (ALS) is a progressive, incurable neurodegenerative disease that targets motoneurons. Cell-based therapies have generated widespread interest as a potential therapeutic approach but no conclusive results have yet been reported either from pre-clinical or clinical studies. AREAS COVERED: This is an integrated review of pre-clinical and clinical studies focused on the development of cell-based therapies for ALS. We analyze the biology of stem cell treatments and results obtained from pre-clinical models of ALS and examine the methods and the results obtained to date from clinical trials. We discuss scientific, clinical, and ethical issues and propose some directions for future studies. EXPERT OPINION: While data from individual studies are encouraging, stem-cell-based therapies do not yet represent a satisfactory, reliable clinical option. The field will critically benefit from the introduction of well-designed, randomized and reproducible, powered clinical trials. Comparative studies addressing key issues such as the nature, properties, and number of donor cells, the delivery mode and the selection of proper patient populations that may benefit the most from cell-based therapies are now of the essence. Multidisciplinary networks of experts should be established to empower effective translation of research into the clinic.

Cancer stem cells from peritumoral tissue of glioblastoma multiforme: the possible missing link between tumor development and progression.

In glioblastoma multiforme (GBM), cancer stem cells (CSCs) are thought to be responsible for gliomagenesis, resistance to treatment and recurrence. Unfortunately, the prognosis for GBM remains poor and recurrence frequently occurs in the peritumoral tissue within 2 cm from the tumor edge. In this area, a population of CSCs has been demonstrated which may recapitulate the tumor after surgical resection. In the present study, we aimed to characterize CSCs derived from both peritumoral tissue (PCSCs) and GBM (GCSCs) in order to deepen their significance in GBM development and progression. The stemness of PCSC/GCSC pairs obtained from four human GBM surgical specimens was investigated by comparing the expression of specific stem cell markers such as Nestin, Musashi-1 and SOX2. In addition, the growth rate, the ultrastructural features and the expression of other molecules such as c-Met, pMet and MAP kinases, involved in cell migration/invasion, maintenance of tumor stemness and/or resistance to treatments were evaluated. Since it has been recently demonstrated the involvement of the long non-coding RNAs (lncRNAs) in the progression of gliomas, the expression of H19 lncRNA, as well as of one of its two mature products miR-675-5p was evaluated in neurospheres. Our results show significant differences between GCSCs and PCSCs in terms of proliferation, ultrastructural peculiarities and, at a lower extent, stemness profile. These differences might be important in view of their potential role as a therapeutic target.

Wnt5a Drives an Invasive Phenotype in Human Glioblastoma Stem-like Cells.

Brain invasion by glioblastoma determines prognosis, recurrence, and lethality in patients, but no master factor coordinating the invasive properties of glioblastoma has been identified. Here we report evidence favoring such a role for the noncanonical WNT family member Wnt5a. We found the most invasive gliomas to be characterized by Wnt5a overexpression, which correlated with poor prognosis and also discriminated infiltrating mesenchymal glioblastoma from poorly motile proneural and classical glioblastoma. Indeed, Wnt5a overexpression associated with tumor-promoting stem-like characteristics (TPC) in defining the character of highly infiltrating mesenchymal glioblastoma cells (Wnt5aHigh). Inhibiting Wnt5a in mesenchymal glioblastoma TPC suppressed their infiltrating capability. Conversely, enforcing high levels of Wnt5a activated an infiltrative, mesenchymal-like program in classical glioblastoma TPC and Wnt5aLow mesenchymal TPC. In intracranial mouse xenograft models of glioblastoma, inhibiting Wnt5a activity blocked brain invasion and increased host survival. Overall, our results highlight Wnt5a as a master regulator of brain invasion, specifically TPC, and they provide a therapeutic rationale to target it in patients with glioblastoma. Cancer Res; 77(4); 996-1007. ©2016 AACR.

Human neural stem cell transplantation in ALS: initial results from a phase I trial.

BACKGROUND: We report the initial results from a phase I clinical trial for ALS. We transplanted GMP-grade, fetal human neural stem cells from natural in utero death (hNSCs) into the anterior horns of the spinal cord to test for the safety of both cells and neurosurgical procedures in these patients. The trial was approved by the Istituto Superiore di Sanità and the competent Ethics Committees and was monitored by an external Safety Board. METHODS: Six non-ambulatory patients were treated. Three of them received 3 unilateral hNSCs microinjections into the lumbar cord tract, while the remaining ones received bilateral (n = 3 + 3) microinjections. None manifested severe adverse events related to the treatment, even though nearly 5 times more cells were injected in the patients receiving bilateral implants and a much milder immune-suppression regimen was used as compared to previous trials. RESULTS: No increase of disease progression due to the treatment was observed for up to18 months after surgery. Rather, two patients showed a transitory improvement of the subscore ambulation on the ALS-FRS-R scale (from 1 to 2). A third patient showed improvement of the MRC score for tibialis anterior, which persisted for as long as 7 months. The latter and two additional patients refused PEG and invasive ventilation and died 8 months after surgery due to the progression of respiratory failure. The autopsies confirmed that this was related to the evolution of the disease. CONCLUSIONS: We describe a safe cell therapy approach that will allow for the treatment of larger pools of patients for later-phase ALS clinical trials, while warranting good reproducibility. These can now be carried out under more standardized conditions, based on a more homogenous repertoire of clinical grade hNSCs. The use of brain tissue from natural miscarriages eliminates the ethical concerns that may arise from the use of fetal material. TRIAL REGISTRATION: EudraCT:2009-014484-39 .

Phenotypic variation in Aicardi-Goutières syndrome explained by cell-specific IFN-stimulated gene response and cytokine release.

Aicardi-Goutières syndrome (AGS) is a monogenic inflammatory encephalopathy caused by mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, or MDA5. Mutations in those genes affect normal RNA/DNA intracellular metabolism and detection, triggering an autoimmune response with an increase in cerebral IFN-α production by astrocytes. Microangiopathy and vascular disease also contribute to the neuropathology in AGS. In this study, we report that AGS gene silencing of TREX1, SAMHD1, RNASEH2A, and ADAR1 by short hairpin RNAs in human neural stem cell-derived astrocytes, human primary astrocytes, and brain-derived endothelial cells leads to an antiviral status of these cells compared with nontarget short hairpin RNA-treated cells. We observed a distinct activation of the IFN-stimulated gene signature with a substantial increase in the release of proinflammatory cytokines (IL-6) and chemokines (CXCL10 and CCL5). A differential impact of AGS gene silencing was noted; silencing TREX1 gave rise to the most dramatic in both cell types. Our findings fit well with the observation that patients carrying mutations in TREX1 experience an earlier onset and fatal outcome. We provide in the present study, to our knowledge for the first time, insight into how astrocytic and endothelial activation of antiviral status may differentially lead to cerebral pathology, suggesting a rational link between proinflammatory mediators and disease severity in AGS.

Vaccinia virus expressing bone morphogenetic protein-4 in novel glioblastoma orthotopic models facilitates enhanced tumor regression and long-term survival.

BACKGROUND: Glioblastoma multiforme (GBM) is one of the most aggressive forms of cancer with a high rate of recurrence. We propose a novel oncolytic vaccinia virus (VACV)-based therapy using expression of the bone morphogenetic protein (BMP)-4 for treating GBM and preventing recurrence. METHODS: We have utilized clinically relevant, orthotopic xenograft models of GBM based on tumor-biopsy derived, primary cancer stem cell (CSC) lines. One of the cell lines, after being transduced with a cDNA encoding firefly luciferase, could be used for real time tumor imaging. A VACV that expresses BMP-4 was constructed and utilized for infecting several primary glioma cultures besides conventional serum-grown glioma cell lines. This virus was also delivered intracranially upon implantation of the GBM CSCs in mice to determine effects on tumor growth. RESULTS: We found that the VACV that overexpresses BMP-4 demonstrated heightened replication and cytotoxic activity in GBM CSC cultures with a broad spectrum of activity across several different patient-biopsy cultures. Intracranial inoculation of mice with this virus resulted in a tumor size equal to or below that at the time of injection. This resulted in survival of 100% of the treated mice up to 84 days post inoculation, significantly superior to that of a VACV lacking BMP-4 expression. When mice with a higher tumor burden were injected with the VACV lacking BMP-4, 80% of the mice showed tumor recurrence. In contrast, no recurrence was seen when mice were injected with the VACV expressing BMP-4, possibly due to induction of differentiation in the CSC population and subsequently serving as a better host for VACV infection and oncolysis. This lack of recurrence resulted in superior survival in the BMP-4 VACV treated group. CONCLUSIONS: Based on these findings we propose a novel VACV therapy for treating GBM, which would allow tumor specific production of drugs in the future in combination with BMPs which would simultaneously control tumor maintenance and facilitate CSC differentiation, respectively, thereby causing sustained tumor regression without recurrence.

Chronic exposure of astrocytes to interferon-α reveals molecular changes related to Aicardi-Goutieres syndrome.

Aicardi-Goutières syndrome is a genetically determined infantile encephalopathy, manifesting as progressive microcephaly, psychomotor retardation, and in ∼25% of patients, death in early childhood. Aicardi-Goutières syndrome is caused by mutations in any of the genes encoding TREX1, RNASEH2-A, -B, -C and SAMHD1, with protein dysfunction hypothesized to result in the accumulation of nucleic acids within the cell, thus triggering an autoinflammatory response with increased interferon-α production. Astrocytes have been identified as a major source of interferon-α production in the brains of patients with Aicardi-Goutières syndrome. Here, we study the effect of interferon-α treatment on astrocytes derived from immortalized human neural stem cells. Chronic interferon-α treatment promoted astrocyte activation and a reduction in cell proliferation. Moreover, chronic exposure resulted in an alteration of genes and proteins involved in the stability of white matter (ATF4, eIF2Bα, cathepsin D, cystatin F), an increase of antigen-presenting genes (human leukocyte antigen class I) and downregulation of pro-angiogenic factors and other cytokines (vascular endothelial growth factor and IL-1). Interestingly, withdrawal of interferon-α for 7 days barely reversed these cellular alterations, demonstrating that the interferon-α mediated effects persist over time. We confirmed our in vitro findings using brain samples from patients with Aicardi-Goutières syndrome. Our results support the idea of interferon-α as a key factor in the pathogenesis of Aicardi-Goutières syndrome relating to the observed leukodystrophy and microangiopathy. Because of the sustained interferon-α effect, even after withdrawal, therapeutic targets for Aicardi-Goutières syndrome, and other interferon-α-mediated encephalopathies, may include downstream interferon-α signalling cascade effectors rather than interferon-α alone.

The EphA2 receptor drives self-renewal and tumorigenicity in stem-like tumor-propagating cells from human glioblastomas.

In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2(High) and EphA2(Low) populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications.

CD90 is identified as a candidate marker for cancer stem cells in primary high-grade gliomas using tissue microarrays.

Although CD90 has been identified as a marker for various kinds of stem cells including liver cancer stem cells (CSCs) that are responsible for tumorigenesis, the potential role of CD90 as a marker for CSCs in gliomas has not been characterized. To address the issue, we investigated the expression of CD90 in tissue microarrays containing 15 glioblastoma multiformes (GBMs), 19 WHO grade III astrocytomas, 13 WHO grade II astrocytomas, 3 WHO grade I astrocytomas and 8 normal brain tissues. Immunohistochemical analysis showed that CD90 was expressed at a medium to high level in all tested high-grade gliomas (grade III and GBM) whereas it was barely detectable in low-grade gliomas (grade I and grade II) and normal brains. Double immunofluorescence staining for CD90 and CD133 in GBM tissues revealed that CD133(+) CSCs are a subpopulation of CD90(+) cells in GBMs in vivo. Flow cytometry analysis of the expression of CD90 and CD133 in GBM-derived stem-like neurospheres further confirmed the conclusion in vitro. The expression levels of both CD90 and CD133 were reduced along with the loss of stem cells after differentiation. Furthermore, the limiting dilution assay demonstrated that the sphere formation ability was comparable between the CD90(+)/CD133(+) and the CD90(+)/CD133(-) populations of GBM neurospheres, which is much higher than that of the CD90(-)/CD133(-) population. We also performed double staining for CD90 and a vascular endothelial cell marker CD31 in tissue microarrays which revealed that the CD90(+) cells were clustered around the tumor vasculatures in high-grade glioma tissues. These findings suggest that CD90 is not only a potential prognostic marker for high-grade gliomas but also a marker for CSCs within gliomas, and it resides within endothelial niche and may also play a critical role in the generation of tumor vasculatures via differentiation into endothelial cells.

Endothelial cells create a stem cell niche in glioblastoma by providing NOTCH ligands that nurture self-renewal of cancer stem-like cells.

One important function of endothelial cells in glioblastoma multiforme (GBM) is to create a niche that helps promote self-renewal of cancer stem-like cells (CSLC). However, the underlying molecular mechanism for this endothelial function is not known. Since activation of NOTCH signaling has been found to be required for propagation of GBM CSLCs, we hypothesized that the GBM endothelium may provide the source of NOTCH ligands. Here, we report a corroboration of this concept with a demonstration that NOTCH ligands are expressed in endothelial cells adjacent to NESTIN and NOTCH receptor-positive cancer cells in primary GBMs. Coculturing human brain microvascular endothelial cells (hBMEC) or NOTCH ligand with GBM neurospheres promoted GBM cell growth and increased CSLC self-renewal. Notably, RNAi-mediated knockdown of NOTCH ligands in hBMECs abrogated their ability to induce CSLC self-renewal and GBM tumor growth, both in vitro and in vivo. Thus, our findings establish that NOTCH activation in GBM CSLCs is driven by juxtacrine signaling between tumor cells and their surrounding endothelial cells in the tumor microenvironment, suggesting that targeting both CSLCs and their niche may provide a novel strategy to deplete CSLCs and improve GBM treatment.