Astrocytic stress response is induced by exposure to astrocyte-binding antibodies expressed by plasmablasts from pediatric patients with acute transverse myelitis.

BACKGROUND: Pediatric acute transverse myelitis (ATM) accounts for 20-30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease. METHODS: We compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response. RESULTS: We found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis. CONCLUSIONS: Discordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.

Somatic mutation effects diffused over microRNA dysregulation.

MOTIVATION: As an important player in transcriptome regulation, microRNAs may effectively diffuse somatic mutation impacts to broad cellular processes and ultimately manifest disease and dictate prognosis. Previous studies that tried to correlate mutation with gene expression dysregulation neglected to adjust for the disparate multitudes of false positives associated with unequal sample sizes and uneven class balancing scenarios. RESULTS: To properly address this issue, we developed a statistical framework to rigorously assess the extent of mutation impact on microRNAs in relation to a permutation-based null distribution of a matching sample structure. Carrying out the framework in a pan-cancer study, we ascertained 9008 protein-coding genes with statistically significant mutation impacts on miRNAs. Of these, the collective miRNA expression for 83 genes showed significant prognostic power in nine cancer types. For example, in lower-grade glioma, 10 genes' mutations broadly impacted miRNAs, all of which showed prognostic value with the corresponding miRNA expression. Our framework was further validated with functional analysis and augmented with rich features including the ability to analyze miRNA isoforms; aggregative prognostic analysis; advanced annotations such as mutation type, regulator alteration, somatic motif, and disease association; and instructive visualization such as mutation OncoPrint, Ideogram, and interactive mRNA-miRNA network. AVAILABILITY AND IMPLEMENTATION: The data underlying this article are available in MutMix, at http://innovebioinfo.com/Database/TmiEx/MutMix.php.

Dissecting Intra-tumor Heterogeneity in the Glioblastoma Microenvironment Using Fluorescence-Guided Multiple Sampling.

The treatment of the most aggressive primary brain tumor in adults, glioblastoma (GBM), is challenging due to its heterogeneous nature, invasive potential, and poor response to chemo- and radiotherapy. As a result, GBM inevitably recurs and only a few patients survive 5 years post-diagnosis. GBM is characterized by extensive phenotypic and genetic heterogeneity, creating a diversified genetic landscape and a network of biological interactions between subclones, ultimately promoting tumor growth and therapeutic resistance. This includes spatial and temporal changes in the tumor microenvironment, which influence cellular and molecular programs in GBM and therapeutic responses. However, dissecting phenotypic and genetic heterogeneity at spatial and temporal levels is extremely challenging, and the dynamics of the GBM microenvironment cannot be captured by analysis of a single tumor sample. In this review, we discuss the current research on GBM heterogeneity, in particular, the utility and potential applications of fluorescence-guided multiple sampling to dissect phenotypic and genetic intra-tumor heterogeneity in the GBM microenvironment, identify tumor and non-tumor cell interactions and novel therapeutic targets in areas that are key for tumor growth and recurrence, and improve the molecular classification of GBM.

Dissecting intra-tumor heterogeneity in the glioblastoma microenvironment using fluorescence-guided multiple sampling.

The treatment of the most aggressive primary brain tumor in adults, glioblastoma (GBM), is challenging due to its heterogeneous nature, invasive potential, and poor response to chemo- and radiotherapy. As a result, GBM inevitably recurs and only a few patients survive 5 years post-diagnosis. GBM is characterized by extensive phenotypic and genetic heterogeneity, creating a diversified genetic landscape and a network of biological interactions between subclones, ultimately promoting tumor growth and therapeutic resistance. This includes spatial and temporal changes in the tumor microenvironment, which influence cellular and molecular programs in GBM and therapeutic responses. However, dissecting phenotypic and genetic heterogeneity at spatial and temporal levels is extremely challenging, and the dynamics of the GBM microenvironment cannot be captured by analysis of a single tumor sample. In this review, we discuss the current research on GBM heterogeneity, in particular, the utility and potential applications of fluorescence-guided multiple sampling to dissect phenotypic and genetic intra-tumor heterogeneity in the GBM microenvironment, identify tumor and non-tumor cell interactions and novel therapeutic targets in areas that are key for tumor growth and the recurrence, and improve the molecular classification of GBM.

Resveratrol: A New Potential Therapeutic Agent for Melanoma?

Melanoma is the most life-threatening and aggressive class of skin malignancies. The incidence of melanoma has steadily increased. Metastatic melanoma is greatly resistant to standard antimelanoma treatments such as chemotherapy, and the 5-year survival rate of cases with melanoma who have a metastatic form of the disease is less than 10%. The contributing role of apoptosis, angiogenesis and autophagy in the pathophysiology of melanoma has been previously demonstrated. Thus, it is extremely urgent to search for complementary therapeutic approaches that could enhance the quality of life of subjects and reduce treatment resistance and adverse effects. Resveratrol, known as a polyphenol component present in grapes and some plants, has anti-cancer properties due to its function as an apoptosis inducer in tumor cells, and anti-angiogenic agent to prevent metastasis. However, more clinical trials should be conducted to prove resveratrol efficacy. Herein, for the first time, we summarize the current knowledge of anti-cancerous activities of resveratrol in melanoma.

Gliomas Interact with Non-glioma Brain Cells via Extracellular Vesicles.

Emerging evidence suggests that crosstalk between glioma cells and the brain microenvironment may influence brain tumor growth. To date, known reciprocal interactions among these cells have been limited to the release of paracrine factors. Combining a genetic strategy with longitudinal live imaging, we find that individual gliomas communicate with distinct sets of non-glioma cells, including glial cells, neurons, and vascular cells. Transfer of genetic material is achieved mainly through extracellular vesicles (EVs), although cell fusion also plays a minor role. We further demonstrate that EV-mediated communication leads to the increase of synaptic activity in neurons. Blocking EV release causes a reduction of glioma growth in vivo. Our findings indicate that EV-mediated interaction between glioma cells and non-glioma brain cells alters the tumor microenvironment and contributes to glioma development.

In vivo MRS measurement of 2-hydroxyglutarate in patient-derived IDH-mutant xenograft mouse models versus glioma patients.

PURPOSE: To generate a preclinical model of isocitrate dehydrogenase (IDH) mutant gliomas from glioma patients and design a MRS method to test the compatibility of 2-hydroxyglutarate (2HG) production between the preclinical model and patients. METHODS: Five patient-derived xenograft (PDX) mice were generated from two glioma patients with IDH1 R132H mutation. A PRESS sequence was tailored at 9.4 T, with computer simulation and phantom analyses, for improving 2HG detection in mice. 2HG and other metabolites in the PDX mice were measured using the optimized MRS at 9.4 T and compared with 3 T MRS measurements of the metabolites in the parental-tumor patients. Spectral fitting was performed with LCModel using in-house basis spectra. Metabolite levels were quantified with reference to water. RESULTS: The PRESS TE was optimized to be 96 ms, at which the 2HG 2.25 ppm signal was narrow and inverted, thereby leading to unequivocal separation of the 2HG resonance from adjacent signals from other metabolites. The optimized MRS provided precise detection of 2HG in mice compared to short-TE MRS at 9.4 T. The 2HG estimates in PDX mice were in excellent agreement with the 2HG measurements in the patients. CONCLUSION: The similarity of 2HG production between PDX models and parental-tumor patients indicates that PDX tumors retain the parental IDH metabolic fingerprint and can serve as a preclinical model for improving our understanding of the IDH-mutation associated metabolic reprogramming.

Distinct Expression Patterns of Carbonic Anhydrase IX in Clear Cell, Microcystic, and Angiomatous Meningiomas.

Clear cell, microcytic, and angiomatous meningiomas are 3 vasculature-rich variants with overlapping morphological features but different prognostic and treatment implications. Distinction between them is not always straightforward. We compared the expression patterns of the hypoxia marker carbonic anhydrase IX (CA-IX) in meningiomas with predominant clear cell (n = 15), microcystic (n = 9), or angiomatous (n = 11) morphologies, as well as 117 cases of other World Health Organization recognized histological meningioma variants. Immunostaining for SMARCE1 protein, whose loss-of-function has been associated with clear cell meningiomas, was performed on all clear cell meningiomas, and selected variants of meningiomas as controls. All clear cell meningiomas showed absence of CA-IX expression and loss of nuclear SMARCE1 expression. All microcystic and angiomatous meningiomas showed diffuse CA-IX immunoreactivity and retained nuclear SMARCE1 expression. In other meningioma variants, CA-IX was expressed in a hypoxia-restricted pattern and was highly associated with atypical features such as necrosis, small cell change, and focal clear cell change. In conclusion, CA-IX may serve as a useful diagnostic marker in differentiating clear cell, microcystic, and angiomatous meningiomas.

Human osteogenic differentiation in Space: proteomic and epigenetic clues to better understand osteoporosis.

In the frame of the VITA mission of the Italian Space Agency (ASI), we addressed the problem of Space osteoporosis by using human blood-derived stem cells (BDSCs) as a suitable osteogenic differentiation model. In particular, we investigated proteomic and epigenetic changes in BDSCs during osteoblastic differentiation induced by rapamycin under microgravity conditions. A decrease in the expression of 4 embryonic markers (Sox2, Oct3/4, Nanog and E-cadherin) was found to occur to a larger extent on board the ISS than on Earth, along with an earlier activation of the differentiation process towards the osteogenic lineage. The changes in the expression of 4 transcription factors (Otx2, Snail, GATA4 and Sox17) engaged in osteogenesis supported these findings. We then ascertained whether osteogenic differentiation of BDSCs could depend on epigenetic regulation, and interrogated changes of histone H3 that is crucial in this type of gene control. Indeed, we found that H3K4me3, H3K27me2/3, H3K79me2/3 and H3K9me2/3 residues are engaged in cellular reprogramming that drives gene expression. Overall, we suggest that rapamycin induces transcriptional activation of BDSCs towards osteogenic differentiation, through increased GATA4 and Sox17 that modulate downstream transcription factors (like Runx2), critical for bone formation. Additional studies are warranted to ascertain the possible exploitation of these data to identify new biomarkers and therapeutic targets to treat osteoporosis, not only in Space but also on Earth.

A non-hierarchical organization of tumorigenic NG2 cells in glioblastoma promoted by EGFR.

BACKGROUND: Expression of neuron-glial antigen 2 (NG2) identifies an aggressive malignant phenotype in glioblastoma (GBM). Mouse models have implicated NG2 in the genesis, evolution, and maintenance of glial cancers and have highlighted potential interactions between NG2 and epidermal growth factor receptor (EGFR). However, it is unknown whether the lineage relationship of NG2+ and NG2- cells follows a hierarchical or stochastic mode of growth. Furthermore, the interaction between NG2 and EGFR signaling in human GBM is also unclear. METHODS: Single GBM NG2+ and NG2- cells were studied longitudinally to assess lineage relationships. Short hairpin RNA knockdown of NG2 was used to assess the mechanistic role of NG2 in human GBM cells. NG2+ and NG2- cells and NG2 knockdown (NG2-KD) and wild type (NG2-WT) cells were analyzed for differential effects on EGFR signaling. RESULTS: Expression of NG2 endows an aggressive phenotype both at single cell and population levels. Progeny derived from single GBM NG2- or GBM NG2+ cells consistently establish phenotypic equilibrium, indicating the absence of a cellular hierarchy. NG2 knockdown reduces proliferation, and mice grafted with NG2-KD survive longer than controls. Finally, NG2 promotes EGFR signaling and is associated with EGFR expression. CONCLUSIONS: These data support a dynamic evolution in which a bidirectional relationship exists between GBM NG2+ and GBM NG2- cells. Such findings have implications for understanding phenotypic heterogeneity, the emergence of resistant disease, and developing novel therapeutics.

Modulation of gene expression in rat muscle cells following treatment with nanoceria in different gravity regimes.

AIM: Oxidative stress (OS) is strictly associated with senescence/pathogenesis of biological systems. As putative countermeasure to environmental OS, cerium oxide nanoparticles (nanoceria [NC]) were administered to muscle cells on ground and aboard the International Space Station. MATERIALS & METHODS: Transcriptional analyses were conducted through microarray technology and hierarchical clustering. Venn diagram and gene ontology analyses were also performed on selected gene lists. RESULTS: Adaptive responses to both NC administration and to permanence in real microgravity conditions occurred. Enrichment in the biological processes related to aging, body fat development and mesodermal tissue proliferation for NC-treated samples were found. CONCLUSION: Nanotechnology antioxidants promise applications to pathological conditions governed by OS on Earth and in life-hostile environments (low Earth orbit and deep space).

Oncogenes Activate an Autonomous Transcriptional Regulatory Circuit That Drives Glioblastoma.

Efforts to identify and target glioblastoma (GBM) drivers have primarily focused on receptor tyrosine kinases (RTKs). Clinical benefits, however, have been elusive. Here, we identify an SRY-related box 2 (SOX2) transcriptional regulatory network that is independent of upstream RTKs and capable of driving glioma-initiating cells. We identified oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc-finger E-box binding homeobox 1 (ZEB1), which are frequently co-expressed irrespective of driver mutations, as potential SOX2 targets. In murine glioma models, we show that different combinations of tumor suppressor and oncogene mutations can activate Sox2, Olig2, and Zeb1 expression. We demonstrate that ectopic co-expression of the three transcription factors can transform tumor-suppressor-deficient astrocytes into glioma-initiating cells in the absence of an upstream RTK oncogene. Finally, we demonstrate that the transcriptional inhibitor mithramycin downregulates SOX2 and its target genes, resulting in markedly reduced proliferation of GBM cells in vivo.

EGFRvIII mutations can emerge as late and heterogenous events in glioblastoma development and promote angiogenesis through Src activation.

BACKGROUND: Amplification of the epidermal growth factor receptor (EGFR) and its mutant EGFRvIII are among the most common genetic alterations in glioblastoma (GBM), the most frequent and most aggressive primary brain tumor. METHODS: In the present work, we analyzed the clonal evolution of these major EGFR aberrations in a small cohort of GBM patients using a unique surgical multisampling technique. Furthermore, we overexpressed both receptors separately and together in 2 patient-derived GBM stem cell lines (GSCs) to analyze their functions in vivo in orthotopic xenograft models. RESULTS: In human GBM biopsies, we identified EGFR amplification as an early event because EGFRvIII mutations emerge from intratumoral heterogeneity later in tumor development. To investigate the biological relevance of this distinct developmental pattern, we established experimental model systems. In these models, EGFR+ tumor cells showed activation of classical downstream signaling pathways upon EGF stimulation and displayed enhanced invasive growth without evidence of angiogenesis in vivo. In contrast, EGFRvIII+ tumors were driven by activation of the prototypical Src family kinase c-Src that promoted VEGF secretion leading to angiogenic tumor growth. CONCLUSIONS: The presented work shows that sequential EGFR amplification and EGFRvIII mutations might represent concerted evolutionary events that drive the aggressive nature of GBM by promoting invasion and angiogenesis via distinct signaling pathways. In particular, c-SRC may be an attractive therapeutic target for tumors harboring EGFRvIII as we identified this protein specifically mediating angiogenic tumor growth downstream of EGFRvIII.

Assessing and monitoring intratumor heterogeneity in glioblastoma: how far has multimodal imaging come?

Glioblastoma demonstrates imaging features of intratumor heterogeneity that result from underlying heterogeneous biological properties. This stems from variations in cellular behavior that result from genetic mutations that either drive, or are driven by, heterogeneous microenvironment conditions. Among all imaging methods available, only T1-weighted contrast-enhancing and T2-weighted fluid-attenuated inversion recovery are used in standard clinical glioblastoma assessment and monitoring. Advanced imaging modalities are still considered emerging techniques as appropriate end points and robust methodologies are missing from clinical trials. Discovering how these images specifically relate to the underlying tumor biology may aid in improving quality of clinical trials and understanding the factors involved in regional responses to treatment, including variable drug uptake and effect of radiotherapy. Upon validation and standardization of emerging MR techniques, providing information based on the underlying tumor biology, these images may allow for clinical decision-making that is tailored to an individual's response to treatment.

Clonal diversity in glioblastoma: is it clinically relevant?

"Serial tumor sampling, single-cell genomics and quantitative imaging are all available technologies, but their integration into current pathways of care will require a paradigm shift in the clinical management of patients with glioblastoma."

CD15 Expression Does Not Identify a Phenotypically or Genetically Distinct Glioblastoma Population.

UNLABELLED: : Recent research has focused on the hypothesis that the growth and regeneration of glioblastoma (GB) is sustained by a subpopulation of self-renewing stem-like cells. This has led to the prediction that molecular markers for cancer stem cells in GB may provide a treatment target. One candidate marker is CD15: we wanted to determine if CD15 represented a credible stem cell marker in GB. We first demonstrated that CD15-positive (CD15+) cells were less proliferative than their CD15-negative (CD15-) counterparts in 10 patient GB tumors. Next we compared the proliferative activity of CD15+ and CD15- cells in vitro using tumor-initiating primary GB cell lines (TICs) and found no difference in proliferative behavior. Furthermore, TICs sorted for CD15+ and CD15- were not significantly different cytogenetically or in terms of gene expression profile. Sorted single CD15+ and CD15- cells were equally capable of reconstituting a heterogeneous population containing both CD15+ and CD15- cells over time, and both CD15+ and CD15- cells were able to generate tumors in vivo. No difference was found in the phenotypic or genomic behavior of CD15+ cells compared with CD15- cells from the same patient. Moreover, we found that in vitro, cells were able to interconvert between the CD15+ and CD15- states. Our data challenge the utility of CD15 as a cancer stem cell marker. SIGNIFICANCE: The data from this study contribute to the ongoing debate about the role of cancer stem cells in gliomagenesis. Results showed that CD15, a marker previously thought to be a cancer stem-like marker in glioblastoma, could not isolate a phenotypically or genetically distinct population. Moreover, isolated CD15-positive and -negative cells were able to generate mixed populations of glioblastoma cells in vitro.

Genetic and functional diversity of propagating cells in glioblastoma.

Glioblastoma (GBM) is a lethal malignancy whose clinical intransigence has been linked to extensive intraclonal genetic and phenotypic diversity and the common emergence of therapeutic resistance. This interpretation embodies the implicit assumption that cancer stem cells or tumor-propagating cells are themselves genetically and functionally diverse. To test this, we screened primary GBM tumors by SNP array to identify copy number alterations (a minimum of three) that could be visualized in single cells by multicolor fluorescence in situ hybridization. Interrogation of neurosphere-derived cells (from four patients) and cells derived from secondary transplants of these same cells in NOD-SCID mice allowed us to infer the clonal and phylogenetic architectures. Whole-exome sequencing and single-cell genetic analysis in one case revealed a more complex clonal structure. This proof-of-principle experiment revealed that subclones in each GBM had variable regenerative or stem cell activity, and highlighted genetic alterations associated with more competitive propagating activity in vivo.