Pervasive structural heterogeneity rewires glioblastoma chromosomes to sustain patient-specific transcriptional programs.

Glioblastoma multiforme (GBM) encompasses brain malignancies marked by phenotypic and transcriptional heterogeneity thought to render these tumors aggressive, resistant to therapy, and inevitably recurrent. However, little is known about how the spatial organization of GBM genomes underlies this heterogeneity and its effects. Here, we compile a cohort of 28 patient-derived glioblastoma stem cell-like lines (GSCs) known to reflect the properties of their tumor-of-origin; six of these were primary-relapse tumor pairs from the same patient. We generate and analyze 5 kbp-resolution chromosome conformation capture (Hi-C) data from all GSCs to systematically map thousands of standalone and complex structural variants (SVs) and the multitude of neoloops arising as a result. By combining Hi-C, histone modification, and gene expression data with chromatin folding simulations, we explain how the pervasive, uneven, and idiosyncratic occurrence of neoloops sustains tumor-specific transcriptional programs via the formation of new enhancer-promoter contacts. We also show how even moderately recurrent neoloops can relate to patient-specific vulnerabilities. Together, our data provide a resource for dissecting GBM biology and heterogeneity, as well as for informing therapeutic approaches.

Biological Implications and Functional Significance of Transglutaminase Type 2 in Nervous System Tumors.

Transglutaminase type 2 (TG2) is the most ubiquitously expressed member of the transglutaminase family. TG2 catalyzes the transamidation reaction leading to several protein post-translational modifications and it is also implicated in signal transduction thanks to its GTP binding/hydrolyzing activity. In the nervous system, TG2 regulates multiple physiological processes, such as development, neuronal cell death and differentiation, and synaptic plasticity. Given its different enzymatic activities, aberrant expression or activity of TG2 can contribute to tumorigenesis, including in peripheral and central nervous system tumors. Indeed, TG2 dysregulation has been reported in meningiomas, medulloblastomas, neuroblastomas, glioblastomas, and other adult-type diffuse gliomas. The aim of this review is to provide an overview of the biological and functional relevance of TG2 in the pathogenesis of nervous system tumors, highlighting its involvement in survival, tumor inflammation, differentiation, and in the resistance to standard therapies.

Extracellular vesicles produced by irradiated endothelial or Glioblastoma stem cells promote tumor growth and vascularization modulating tumor microenvironment.

BACKGROUND: Glioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment. METHODS: In this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays. RESULTS: We confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration. CONCLUSION: In this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

CD8+CD103+PD1+TIM3+ T cells in glioblastoma microenvironment correlate with prognosis.

Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.

BRAF Mutations in Melanoma: Biological Aspects, Therapeutic Implications, and Circulating Biomarkers.

Melanoma is an aggressive form of skin cancer resulting from the malignant transformation of melanocytes. Recent therapeutic approaches, including targeted therapy and immunotherapy, have improved the prognosis and outcome of melanoma patients. BRAF is one of the most frequently mutated oncogenes recognised in melanoma. The most frequent oncogenic BRAF mutations consist of a single point mutation at codon 600 (mostly V600E) that leads to constitutive activation of the BRAF/MEK/ERK (MAPK) signalling pathway. Therefore, mutated BRAF has become a useful target for molecular therapy and the use of BRAF kinase inhibitors has shown promising results. However, several resistance mechanisms invariably develop leading to therapeutic failure. The aim of this manuscript is to review the role of BRAF mutational status in the pathogenesis of melanoma and its impact on differentiation and inflammation. Moreover, this review focuses on the mechanisms responsible for resistance to targeted therapies in BRAF-mutated melanoma and provides an overview of circulating biomarkers including circulating tumour cells, circulating tumour DNA, and non-coding RNAs.

Effectiveness of Flattening-Filter-Free versus Flattened Beams in V79 and Glioblastoma Patient-Derived Stem-like Cells.

Literature data on the administration of conventional high-dose beams with (FF) or without flattening filters (FFF) show conflicting results on biological effects at the cellular level. To contribute to this field, we irradiated V79 Chinese hamster lung fibroblasts and two patient-derived glioblastoma stem-like cell lines (GSCs-named #1 and #83) using a clinical 10 MV accelerator with FF (at 4 Gy/min) and FFF (at two dose rates 4 and 24 Gy/min). Cell killing and DNA damage induction, determined using the γ-H2AX assay, and gene expression were studied. No significant differences in the early survival of V79 cells were observed as a function of dose rates and FF or FFF beams, while a trend of reduction in late survival was observed at the highest dose rate with the FFF beam. GSCs showed similar survival levels as a function of dose rates, both delivered in the FFF regimen. The amount of DNA damage measured for both dose rates after 2 h was much higher in line #1 than in line #83, with statistically significant differences between the two dose rates only in line #83. The gene expression analysis of the two GSC lines indicates gene signatures mimicking the prognosis of glioblastoma (GBM) patients derived from a public database. Overall, the results support the current use of FFF and highlight the possibility of identifying patients with candidate gene signatures that could benefit from irradiation with FFF beams at a high dose rate.

Characterization of Glioblastoma Cells Response to Regorafenib.

Glioblastoma (GBM), the most malignant primary brain tumor in adults. Although not frequent, it has a relevant social impact because the peak incidence coincides with the age of professional maturity. A number of novel treatments have been proposed, yet clinical trials have been disappointing. Recently, a phase II clinical trial (REGOMA) demonstrated that the multikinase inhibitor regorafenib significantly increased the median overall survival (OS) of GBM patients when compared to lomustine-treated patients. On this basis, the National Comprehensive Cancer Network (NCCN) 2020 Guidelines included regorafenib as a preferred regimen in relapsed GBM treatment. Despite the use in GBM patients' therapy, little is known about the molecular mechanisms governing regorafenib effectiveness on the GBM tumor. Here we report an in vitro characterization of GBM tumor cells' response to regorafenib, performed both on cell lines and on patient-derived glioma stem cells (GSCs). Overall, regorafenib significantly reduced cell growth of 2D tumor cell cultures and of 3D tumor spheroids. Strikingly, this effect was accompanied by transcriptional regulation of epithelial to mesenchymal transition (EMT) genes and by an increased ability of surviving tumor cells to invade the surrounding matrix. Taken together, our data suggest that regorafenib limits cell growth, however, it might induce an invasive phenotype.

Glioblastoma-Specific Strategies of Vascularization: Implications in Anti-Angiogenic Therapy Resistance.

Angiogenesis has long been implicated as a crucial process in GBM growth and progression. GBM can adopt several strategies to build up its abundant and aberrant vasculature. Targeting GBM angiogenesis has gained more and more attention in anti-cancer therapy, and many strategies have been developed to interfere with this hallmark. However, recent findings reveal that the effects of anti-angiogenic treatments are temporally limited and that tumors become refractory to therapy and more aggressive. In this review, we summarize the GBM-associated neovascularization processes and their implication in drug resistance mechanisms underlying the transient efficacy of current anti-angiogenic therapies. Moreover, we describe potential strategies and perspectives to overcome the mechanisms adopted by GBM to develop resistance to anti-angiogenic therapy as new potential therapeutic approaches.

Development and Validation of a HPLC-MS/MS Method to Measure Nifuroxazide and Its Application in Healthy and Glioblastoma-Bearing Mice.

Nifuroxazide (NAZ), a nitrofuran derivative used to treat diarrhea, has been recently shown to possess anticancer activity. However, its pharmacokinetic profile is poorly known. The pharmacokinetic profile of NAZ was thus investigated in mice using a newly developed method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). We determined the concentrations of NAZ in the plasma and brain tissue of mice treated with the drug. The method proved to be specific, reproducible, precise, and accurate. It also demonstrated high sensitivity, reaching an LOQ in the order of ppb for both matrices, using samples of 100 µL or 0.2 g. The new HPLC-MS/MS assay was successfully applied to study the pharmacokinetics of NAZ after chronic intraperitoneal administration in mice at a dose of 30 mg/kg. One hour after treatment, plasma concentrations of NAZ were in the range of 336-2640 ng/mL. Moreover, unlike the brains of healthy mice or those with healed mechanical injuries, we found that NAZ was able to cross the injured blood-brain barrier of tumor-infiltrated brains. Thus, following i.p. administration, NAZ reaches systemic levels suitable for testing its efficacy in preclinical models of glioblastoma. Overall, these pharmacokinetic data provide robust evidence supporting the repositioning of NAZ as an antitumor drug.

Glioblastoma Stem-Like Cells (GSCs) with Mesenchymal Signature: Lipid Profiles of Mobile Lipids Obtained with MRS before and after Radio/Chemical Treatments.

Glioblastoma is the most common and lethal primary malignant brain tumor in adults. Glioblastoma stem cells (GSCs) promote and are responsible for glioblastoma intratumoral heterogeneity and therapy resistance, due to their two main features: self-renewal and differentiation. Lipids have important biological and physiological functions that are critical for understanding the regulation and control of stem cell fate; lipid metabolism and related unsaturation levels play a possible role as the target of therapeutics to overcome glioblastoma radioresistance. This paper aimed at an in-depth analysis of 13 GSC mesenchymal (MES) lines, two subclones, and a stabilized glioblastoma line (T98G) by magnetic resonance spectroscopy (MRS). Particularly, 2D MRS was used to investigate lipid unsaturation behavior during growth in culture and after treatment with etomoxir and photon beams. MES lines, although belonging to the same genetic and metabolic cluster, showed metabolic heterogeneity when observed by MRS, focusing on lipid signals. Nonetheless, the observed unsaturation level stability for two representative lines after stressful treatments suggests unusual robustness of the unsaturation levels for each line, as a peculiar and intrinsic characteristic of GSCs.

FMRP modulates the Wnt signalling pathway in glioblastoma.

Converging evidence indicates that the Fragile X Messenger Ribonucleoprotein (FMRP), which absent or mutated in Fragile X Syndrome (FXS), plays a role in many types of cancers. However, while FMRP roles in brain development and function have been extensively studied, its involvement in the biology of brain tumors remains largely unexplored. Here we show, in human glioblastoma (GBM) biopsies, that increased expression of FMRP directly correlates with a worse patient outcome. In contrast, reductions in FMRP correlate with a diminished tumor growth and proliferation of human GBM stem-like cells (GSCs) in vitro in a cell culture model and in vivo in mouse brain GSC xenografts. Consistently, increased FMRP levels promote GSC proliferation. To characterize the mechanism(s) by which FMRP regulates GSC proliferation, we performed GSC transcriptome analyses in GSCs expressing high levels of FMRP, and in these GSCs after knockdown of FMRP. We show that the WNT signalling is the most significantly enriched among the published FMRP target genes and genes involved in ASD. Consistently, we find that reductions in FMRP downregulate both the canonical WNT/ß-Catenin and the non-canonical WNT-ERK1/2 signalling pathways, reducing the stability of several key transcription factors (i.e. ß-Catenin, CREB and ETS1) previously implicated in the modulation of malignant features of glioma cells. Our findings support a key role for FMRP in GBM cancer progression, acting via regulation of WNT signalling.

MiR-378a-3p Acts as a Tumor Suppressor in Colorectal Cancer Stem-Like Cells and Affects the Expression of MALAT1 and NEAT1 lncRNAs.

MiR-378a-3p plays a critical role in carcinogenesis acting as a tumor suppressor, promoting apoptosis and cell cycle arrest and reducing invasion and drug resistance in several human cancers, including colorectal cancer (CRC), where its expression is significantly associated with histological classification and prognosis. In this study, we investigated the biological and cellular processes affected by miR-378a-3p in the context of CRC carcinogenesis. In agreement with the literature, miR-378a-3p is downregulated in our cohort of CRC patients as well as, in 15 patient-derived colorectal cancer stem-like cell (CRC-SC) lines and 8 CRC cell lines, compared to normal mucosae. Restoration of miR-378a-3p restrains tumorigenic properties of CRC and CRC-SC lines, as well as, significantly reduces tumor growth in two CRC-SC xenograft mouse models. We reported that miR-378a-3p modulates the expression of the lncRNAs MALAT1 and NEAT1. Their expression is inversely correlated with that of miR-378a-3p in patient-derived CRC-SC lines. Silencing of miR-378a-3p targets, MALAT1 and NEAT1, significantly impairs tumorigenic properties of CRC-SCs, supporting the critical role of miR-378a-3p in CRC carcinogenesis as a tumor-suppressor factor by establishing a finely tuned crosstalk with lncRNAs MALAT1 and NEAT1.

Effects of the Combined Treatment with a G-Quadruplex-Stabilizing Ligand and Photon Beams on Glioblastoma Stem-like Cells: A Magnetic Resonance Study.

Glioblastoma multiforme is a malignant primary brain tumor with a poor prognosis and high rates of chemo-radiotherapy failure, mainly due to a small cell fraction with stem-like properties (GSCs). The mechanisms underlying GSC response to radiation need to be elucidated to enhance sensitivity to treatments and to develop new therapeutic strategies. In a previous study, two GSC lines, named line #1 and line #83, responded differently to carbon ions and photon beams, with the differences likely attributable to their own different metabolic fingerprint rather than to radiation type. Data from the literature showed the capability of RHPS4, a G-quadruplex stabilizing ligand, to sensitize the glioblastoma radioresistant U251MG cells to X-rays. The combined metabolic effect of ligand #190, a new RHPS4-derivative showing reduced cardiotoxicity, and a photon beam has been monitored by magnetic resonance (MR) spectroscopy for the two GSC lines, #1 and #83, to reveal whether a synergistic response occurs. MR spectra from both lines were affected by single and combined treatments, but the variations of the analysed metabolites were statistically significant mainly in line #1, without synergistic effects due to combination. The multivariate analysis of ten metabolites shows a separation between control and treated samples in line #1 regardless of treatment type, while separation was not detected in line #83.

Elesclomol-induced increase of mitochondrial reactive oxygen species impairs glioblastoma stem-like cell survival and tumor growth.

BACKGROUND: Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults, characterized by a poor prognosis mainly due to recurrence and therapeutic resistance. It has been widely demonstrated that glioblastoma stem-like cells (GSCs), a subpopulation of tumor cells endowed with stem-like properties is responsible for tumor maintenance and progression. Moreover, it has been demonstrated that GSCs contribute to GBM-associated neovascularization processes, through different mechanisms including the transdifferentiation into GSC-derived endothelial cells (GdECs). METHODS: In order to identify druggable cancer-related pathways in GBM, we assessed the effect of a selection of 349 compounds on both GSCs and GdECs and we selected elesclomol (STA-4783) as the most effective agent in inducing cell death on both GSC and GdEC lines tested. RESULTS: Elesclomol has been already described to be a potent oxidative stress inducer. In depth investigation of the molecular mechanisms underlying GSC and GdEC response to elesclomol, confirmed that this compound induces a strong increase in mitochondrial reactive oxygen species (ROS) in both GSCs and GdECs ultimately leading to a non-apoptotic copper-dependent cell death. Moreover, combined in vitro treatment with elesclomol and the alkylating agent temozolomide (TMZ) enhanced the cytotoxicity compared to TMZ alone. Finally, we used our experimental model of mouse brain xenografts to test the combination of elesclomol and TMZ and confirmed their efficacy in vivo. CONCLUSIONS: Our results support further evaluation of therapeutics targeting oxidative stress such as elesclomol with the aim of satisfying the high unmet medical need in the management of GBM.

Inhibition of mitochondrial translation suppresses glioblastoma stem cell growth.

Glioblastoma stem cells (GSCs) resist current glioblastoma (GBM) therapies. GSCs rely highly on oxidative phosphorylation (OXPHOS), whose function requires mitochondrial translation. Here we explore the therapeutic potential of targeting mitochondrial translation and report the results of high-content screening with putative blockers of mitochondrial ribosomes. We identify the bacterial antibiotic quinupristin/dalfopristin (Q/D) as an effective suppressor of GSC growth. Q/D also decreases the clonogenicity of GSCs in vitro, consequently dysregulating the cell cycle and inducing apoptosis. Cryoelectron microscopy (cryo-EM) reveals that Q/D binds to the large mitoribosomal subunit, inhibiting mitochondrial protein synthesis and functionally dysregulating OXPHOS complexes. These data suggest that targeting mitochondrial translation could be explored to therapeutically suppress GSC growth in GBM and that Q/D could potentially be repurposed for cancer treatment.

Dilation of Brain Veins and Perivascular Infiltration by Glioblastoma Cells in an In Vivo Assay of Early Tumor Angiogenesis.

The cranial window (CW) technique provides a simple and low-cost method to assess tumor angiogenesis in the brain. The CW combined with histology using selective markers for tumor and endothelial cells can allow a sensitive monitoring of novel antiangiogenesis therapies in preclinical models. The CW was established in cyclosporine immunosuppressed rats that were stereotactically grafted with fluorescent U87MG glioblastoma cells. One to 3 weeks after grafting, brain vasculature was visualized in vivo and assessed by immunofluorescence microscopy using antibodies against endothelial and smooth-muscle cells and blood brain barrier. At 1-2 weeks after grafting, the CW reliably detected the hypertrophy of venous-venous anastomoses and cortical veins. These structures increased highly significantly their pregrafting diameter. Arterialized veins and hemorrhages were seen by three weeks after grafting. Immunofluorescence microscopy showed significant branching and dilation of microvessels, particularly those surrounded by tumor cells. Mechanistically, these changes lead to loss of vascular resistance, increased venous outflow, and opening of venous-venous anastomoses on the cortical surface. Data from the present study, namely, the hypertrophy of cortical venous-venous anastomoses, microvessel branching, and dilation of the microvessels surrounded by tumor cells, indicate the power of this in vivo model for the sensitive monitoring of early tumor angiogenesis.

Anticancer Properties of the Antipsychotic Drug Chlorpromazine and Its Synergism With Temozolomide in Restraining Human Glioblastoma Proliferation In Vitro.

The extremely poor prognosis of patients affected by glioblastoma (GBM, grade IV glioma) prompts the search for new and more effective therapies. In this regard, drug repurposing or repositioning can represent a safe, swift, and inexpensive way to bring novel pharmacological approaches from bench to bedside. Chlorpromazine, a medication used since six decades for the therapy of psychiatric disorders, shows in vitro several features that make it eligible for repositioning in cancer therapy. Using six GBM cell lines, three of which growing as patient-derived neurospheres and displaying stem-like properties, we found that chlorpromazine was able to inhibit viability in an apoptosis-independent way, induce hyperdiploidy, reduce cloning efficiency as well as neurosphere formation and downregulate the expression of stemness genes in all these cell lines. Notably, chlorpromazine synergized with temozolomide, the first-line therapeutic in GBM patients, in hindering GBM cell viability, and both drugs strongly cooperated in reducing cloning efficiency and inducing cell death in vitro for all the GBM cell lines assayed. These results prompted us to start a Phase II clinical trial on GBM patients (EudraCT # 2019-001988-75; ClinicalTrials.gov Identifier: NCT04224441) by adding chlorpromazine to temozolomide in the adjuvant phase of the standard first-line therapeutic protocol.

Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423, and Hey2.

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high-grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2-deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma-derived CSC-like lines. These experiments identify a network of critical tumor suppressive Sox2-targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets.

Short tandem repeat profiling for the authentication of cancer stem-like cells.

Colorectal and glioblastoma cancer stem-like cells (CSCs) are essential for translational research. Cell line authentication by short tandem repeat (STR) profiling ensures reproducibility of results in oncology research. This technique enables to identify mislabeling or cross-contamination of cell lines. In our study, we provide a reference dataset for a panel of colorectal and glioblastoma CSCs that allows authentication. Each cell line was entered into the cell Line Integrated Molecular Authentication database 2.1 to be compared to the STR profiles of 4485 tumor cell lines. This article also provides clinical data of patients from whom CSCs arose and data on the parent tumor stage and mutations. STR profiles and information of our CSCs are also available in the Cellosaurus database (ExPASy) as identified by unique research resource identifier codes.