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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.

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.

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., What's new? Human cell lines obtained from cancer stem‐like cells represent an invaluable model for studying tumor properties. Cell line authentication by short tandem repeat (STR) profiling is an important tool to identify the potential mislabeling or cross‐contamination of cell lines. Here, the authors characterized 18 colorectal cancer stem‐like cell lines from 17 patients and 103 glioblastoma cancer stem‐like cell lines from 95 patients by STR profiling to create a reference dataset that allows the authentication of these cell lines and their identification through a unique research resource identifier. The results will help further ensure the reliability and reproducibility of research experiments.

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain tumors. In gliomas, Sox2 is essential to maintain CSC. In mouse high-grade glioma pHGG, Sox2 deletion causes cell proliferation arrest and inability to reform tumors in vivo; 134 genes are significantly derepressed. To identify genes mediating the effects of Sox2 deletion, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Cdkn2b, Ebf1, Zfp423 and Hey2, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis, or pharmacological inactivation, of each of these genes, individually, showed that their activity is essential for the proliferation arrest caused by Sox2 deletion. These Sox2-inhibited antioncogenes also inhibited clonogenicity in primary human glioblastoma-derived cancer stem-like cell lines. These experiments identify critical anti-oncogenic factors whose inhibition by Sox2 is involved in CSC maintenance, defining new potential therapeutic targets for gliomas.Table of Contents ImageMain PointsSox2 maintains glioma tumorigenicity by repressing the antioncogenic activity of a regulatory network involving the Ebf1, Hey2, Cdkn2b and Zfp423 genes.Mutation of these genes prevents the cell proliferation arrest of Sox2-deleted glioma cells.

Bevacizumab, a VEGF‐targeting monoclonal antibody, may trigger an infiltrative growth pattern in glioblastoma. We investigated this pattern using both a human specimen and rat models. In the human specimen, a substantial fraction of infiltrating tumor cells were located along perivascular spaces in close relationship with endothelial cells. Brain xenografts of U87MG cells treated with bevacizumab were smaller than controls (p = 0.0055; Student t‐test), however, bands of tumor cells spread through the brain farther than controls (p < 0.001; Student t‐test). Infiltrating tumor Cells exhibited tropism for vascular structures and propensity to form tubules and niches with endothelial cells. Molecularly, bevacizumab triggered an epithelial to mesenchymal transition with over‐expression of the receptor Plexin Domain Containing 1 (PLXDC1). These results were validated using brain xenografts of patient‐derived glioma stem‐like cells. Enforced expression of PLXDC1 in U87MG cells promoted brain infiltration along perivascular spaces. Importantly, PLXDC1 inhibition prevented perivascular infiltration and significantly increased the survival of bevacizumab‐treated rats. Our study indicates that bevacizumab‐induced brain infiltration is driven by vascular endothelium and depends on PLXDC1 activation of tumor cells., What's new? Bevacizumab, a VEGF‐targeting monoclonal antibody, has been observed to trigger an infiltrative growth pattern in glioblastoma as an escape mechanism. The mechanisms underlying this gliomatosis‐like growth pattern, however, remain unclear. Here, the authors found that the infiltrative growth pattern occurs mostly along perivascular spaces and relies on the over‐expression of PLXDC1 by tumor cells and on the restoration of the endothelial component of blood brain barrier. Altogether, the data show that the brain infiltration induced by bevacizumab is mainly driven by the vascular endothelium. Importantly, inhibition of PLXDC1 prevents bevacizumab‐induced infiltrative growth, resulting in significant increase of survival.

Advances from glioma stemlike cell (GSC) research, though increasing our knowledge of glioblastoma (GBM) biology, do not influence clinical decisions yet. We explored the translational power of GSC-enriched cultures from patient-derived tumorspheres (TS) in predicting treatment response.The relationship between TS growth and clinical outcome was investigated in 52 GBMs treated with surgical resection followed by radiotherapy and temozolomide (TMZ). The effect on TS of radiation (6 to 60 Gy) and of TMZ (3.9 μM to 1 mM) was related with patients' survival.Generation of TS was an independent factor for poor overall survival (OS) and poor progression-free survival (PFS) (P.0001 and P = .0010, respectively). Growth rate and clonogenicity of TS predicted poor OS. In general, TS were highly resistant to both radiation and TMZ. Resistance to TMZ was stronger in TS with high clonogenicity and fast growth (P.02). Shorter PFS was associated with radiation LD50 (lethal dose required to kill 50% of TS cells)12 Gy of matched TS (P = .0484). A direct relationship was found between sensitivity of TS to TMZ and patients' survival (P = .0167 and P = .0436 for OS and PFS, respectively). Importantly, values for TMZ half-maximal inhibitory concentration50 μM, which are in the range of plasma levels achieved in vivo, identified cases with longer OS and PFS (P = .0020 and P = .0016, respectively).Analysis of TS holds translational relevance by predicting the response of parent tumors to radiation and, particularly, to TMZ. Dissecting the clonogenic population from proliferating progeny in TS can guide therapeutic strategies to a more effective drug selection and treatment duration.

Glioblastoma (GBM) is the most aggressive and prevalent form of a human brain tumor in adults. Several data have demonstrated the implication of microRNAs (miRNAs) in tumorigenicity of GBM stem-like cells (GSCs). The regulatory functions of miRNAs in GSCs have emerged as potential therapeutic candidates for glioma treatment. The current study aimed at investigating the function of miR-370-3p in glioma progression, as aberrant expression of miR-370-3p, is involved in various human cancers, including glioma. Analyzing our collection of GBM samples and patient-derived GSC lines, we found the expression of miR-370-3p significantly downregulated compared to normal brain tissues and normal neural stem cells. Restoration of miR-370-3p expression in GSCs significantly decreased proliferation, migration, and clonogenic abilities of GSCs, in vitro, and tumor growth in vivo. Gene expression analysis performed on miR-370-3p transduced GSCs, identified several transcripts involved in Epithelial to Mesenchymal Transition (EMT), and Hypoxia signaling pathways. Among the genes downregulated by the restored expression of miR-370-3p, we found the EMT-inducer high-mobility group AT-hook 2 (HMGA2), the master transcriptional regulator of the adaptive response to hypoxia, Hypoxia-inducible factor (HIF)1A, and the long non-coding RNAs (lncRNAs) Nuclear Enriched Abundant Transcript (NEAT)1. NEAT1 acts as an oncogene in a series of human cancers including gliomas, where it is regulated by the Epidermal Growth Factor Receptor (EGFR) pathways, and contributes to tumor growth and invasion. Noteworthy, the expression levels of miR-370-3p and NEAT1 were inversely related in both GBM tumor specimens and GSCs, and a dual-luciferase reporter assay proved the direct binding between miR-370-3p and the lncRNAs NEAT1. Our results identify a critical role of miR-370-3p in the regulation of GBM development, indicating that miR-370-3p acts as a tumor-suppressor factor inhibiting glioma cell growth, migration and invasion by targeting the lncRNAs NEAT1, HMGA2, and HIF1A, thus, providing a potential candidate for GBM patient treatment.

// Michele Signore 1, * , Mariachiara Buccarelli 1, * , Emanuela Pilozzi 2 , Gabriele De Luca 1 , Marianna Cappellari 1 , Maurizio Fanciulli 3 , Frauke Goeman 3 , Elisa Melucci 3 , Mauro Biffoni 1, § , Lucia Ricci-Vitiani 1, § 1 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanita, Rome, Italy 2 Department of Clinical and Molecular Medicine, Sant'Andrea Hospital, University La Sapienza, Rome, Italy 3 Scientific Direction, Regina Elena National Cancer Institute, Rome, Italy * These authors have contributed equally to this work § These two authors shared senior authorship Correspondence to: Michele Signore, email: michele.signore@iss.it Lucia Ricci-Vitiani, email: lriccivitiani@yahoo.it Keywords: colorectal cancer stem-like cells, kinase inhibitors, Chk1, DNA damage, phospho-proteomics Received: October 13, 2015 Accepted: May 13, 2016 Published: June 6, 2016 ABSTRACT Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide. Despite recent progress, the prognosis of advanced stage CRC remains poor, mainly because of cancer recurrence and metastasis. The high morbidity and mortality of CRC has been recently ascribed to a small population of tumor cells that hold the potential of tumor initiation, i.e. cancer stem cells (CSCs), which play a pivotal role in cancer recurrence and metastasis and are not eradicated by current therapy. We screened CRC-SCs in vitro with a library of protein kinase inhibitors and showed that CRC-SCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. Nonetheless, broad-spectrum inhibition by the staurosporin derivative UCN-01 blocks CRC-SC growth and potentiates the activity of irinotecan in vitro and in vivo CRC-SC-derived models. Reverse-Phase Protein Microarrays (RPPA) revealed that, albeit CRC-SCs display individual phospho-proteomic profiles, sensitivity of CRC-SCs to UCN-01 relies on the interference with the DNA damage response mediated by Chk1. Combination of LY2603618, a specific Chk1/2 inhibitor, with irinotecan resulted in a significant reduction of CRC-SC growth in vivo , confirming that irinotecan treatment coupled to inhibition of Chk1 represents a potentially effective therapeutic approach for CRC treatment.

Epigenetic alterations, including dysregulated DNA methylation and histone modifications, govern the progression of colorectal cancer (CRC). Cancer cells exploit epigenetic regulation to control cellular pathways, including apoptotic and metastatic signals. Since aberrations in epigenome can be pharmacologically reversed by DNA methyltransferase and histone deacetylase inhibitors, epigenetics in combination with standard agents are currently envisaged as a new therapeutic frontier in cancer, expected to overcome drug resistance associated with current treatments. In this study, we challenged this idea and demonstrated that the combination of azacitidine and romidepsin with IFN-α owns a high therapeutic potential, targeting the most aggressive cellular components of CRC, such as metastatic cells and cancer stem cells (CSCs), via tight control of key survival and death pathways. Moreover, the antitumor efficacy of this novel pharmacological approach is associated with induction of signals of immunogenic cell death. Of note, a previously undisclosed key role of IFN-α in inducing both antiproliferative and pro-apoptotic effects on CSCs of CRC was also found. Overall, these findings open a new frontier on the suitability of IFN-α in association with epigenetics as a novel and promising therapeutic approach for CRC management.

Glioblastoma (GBM) possesses glioma stem cells (GSCs) that exhibit aggressive invasion behavior in the brain. Current preclinical GBM invasion assays using mouse brain xenografts are time consuming and less efficient. Here, we demonstrate an array of methods that allow rapid and efficient assaying of GSCs invasion in human brain organoids. The assays are versatile to characterize various aspects of GSCs, such as invasion, integration, and interaction with mature neurons of brain organoids. Tissue clearing and quantitative 3D imaging of GSCs in host organoids reveal that invasiveness is inversely correlated with the organoids' age. Importantly, the described invasion assays can distinguish the invasive behaviors of primary and recurrent GSCs. The assays are also amenable to test pharmacological agents. As an example, we show that GI254023X, an inhibitor of ADAM10, could prevent the integration of GSCs into the organoids.

The role of autophagy in cancer onset and progression appears still controversial. On one hand, autophagy allows cancer cell to survive in unfavorable environmental conditions, on the other hand, once internal energy resources are exhausted, it leads to cell death. In addition, autophagy interpheres with cell cycle progression, de facto exerting a cytostatic activity. Hence, it represents an important target for anticancer therapy. For example, temozolomide (TMZ), of use for glioblastoma (GBM) treatment, appears as capable of inducing autophagy partially inhibiting cancer cell proliferation. However, GBM, a very aggressive brain tumor with poor prognosis even after surgery and radio-chemotherapy, invariably recurs and leads to patient death. Since cancer stem cells have been hypothesized to play a role in refractory/relapsing cancers, in the present work we investigated if autophagy could represent a constitutive cytoprotection mechanism for glioblastoma stem-like cells (GSCs) and if the modulation of autophagic process could affect GBM growth and survival. Thus, in the present study we first evaluated the relevance of autophagy in GBM tumor specimens, then its occurrence in GSCs and, finally, if modulation of autophagy could influence GSC response to TMZ. Our results suggested that, in vitro, the impairing autophagic process with quinacrine, a compound able to cross the blood-brain barrier, increased GSC susceptibility to TMZ. Death of GSCs was apparently due to the iron dependent form of programmed cell death characterized by the accumulation of lipid peroxides called ferroptosis. These results underscore the relevance of the modulation of autophagy in the GSC survival and death and suggest that triggering of ferroptosis in GSCs could represent a novel and important target for the management of glioblastoma.

Clustering of patient-derived glioma stem-like cells (GSCs) through unsupervised analysis of metabolites detected by magnetic resonance spectroscopy (MRS) evidenced three subgroups, namely clusters 1a and 1b, with high intergroup similarity and neural fingerprints, and cluster 2, with a metabolism typical of commercial tumor lines. In addition, subclones generated by the same GSC line showed different metabolic phenotypes. Aerobic glycolysis prevailed in cluster 2 cells as demonstrated by higher lactate production compared to cluster 1 cells. Oligomycin, a mitochondrial ATPase inhibitor, induced high lactate extrusion only in cluster 1 cells, where it produced neutral lipid accumulation detected as mobile lipid signals by MRS and lipid droplets by confocal microscopy. These results indicate a relevant role of mitochondrial fatty acid oxidation for energy production in GSCs. On the other hand, further metabolic differences, likely accounting for different therapy responsiveness observed after etomoxir treatment, suggest that caution must be used in considering patient treatment with mitochondria FAO blockers. Metabolomics and metabolic profiling may contribute to discover new diagnostic or prognostic biomarkers to be used for personalized therapies.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor in adults, characterized by aggressive growth, limited response to therapy, and inexorable recurrence. Because of the extremely unfavorable prognosis of GBM, it is important to develop more effective diagnostic and therapeutic strategies based on biologically and clinically relevant patient stratification systems. Analyzing a collection of patient-derived GBM stem-like cells (GSCs) by gene expression profiling, nuclear magnetic resonance (NMR) spectroscopy and signal transduction pathway activation, we identified two GSC clusters characterized by different clinical features. Due to the widely documented role played by microRNAs (miRNAs) in the tumorigenesis process, in this study we explored whether these two GBM patient subtypes could also be discriminated by different miRNA signatures. Global miRNA expression pattern was analyzed by oblique principal component (OPC) analysis and principal component analysis (PCA). By a combined inferential strategy on PCA results, we identified a reduced set of three miRNAs – miR-23a, miR-27a and miR-9* (miR-9-3p) – able to discriminate the proneural- and mesenchymal-like GSC phenotypes as well as mesenchymal and proneural subtypes of primary GBM included in The Cancer Genome Atlas (TCGA) dataset. Kaplan-Meier analysis showed a significant correlation between the selected miRNAs and overall survival in 429 GBM specimens from TCGA-identifying patients who had an unfavorable outcome. The survival prognostic capability of the three miRNA signatures could have important implications for the understanding of the biology of GBM subtypes and could be useful in patient stratification to facilitate interpretation of results from clinical trials.

Glioblastoma multiforme (GBM) is the most common and fatal malignant adult primary brain tumor. Currently, the overall prognosis for GBM patients remains poor despite advances in neurosurgery and adjuvant treatments. MicroRNAs (miRNAs) contribute to the pathogenesis of various types of tumor, including GBM. In this study we analyzed the expression of a panel of miRNAs, which are known to be differentially expressed by the brain and GBM tumor, in a collection of patient-derived GBM stem-like cells (GSCs). Notably, the average expression level of miR-135b, was the most downregulated compared to its normal counterpart, suggesting a potential role as anti-oncogene. Restoration of miR-135b in GSCs significantly decreased proliferation, migration and clonogenic abilities. More importantly, miR-135b restoration was able to significantly reduce brain infiltration in mouse models of GBM obtained by intracerebral injection of GSC lines. We identified ADAM12 and confirmed SMAD5 and GSK3β as miR-135b targets and potential mediators of its effects. The whole transcriptome analysis ascertained that the expression of miR-135b downmodulated additional genes driving key pathways in GBM survival and infiltration capabilities. Our results identify a critical role of miR-135b in the regulation of GBM development, suggesting that miR-135b might act as a tumor-suppressor factor and thus providing a potential candidate for the treatment of GBM patients.