Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma.

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

Galectin functions in cancer-associated inflammation and thrombosis.

Galectins are carbohydrate-binding proteins that regulate many cellular functions including proliferation, adhesion, migration, and phagocytosis. Increasing experimental and clinical evidence indicates that galectins influence many steps of cancer development by inducing the recruitment of immune cells to the inflammatory sites and modulating the effector function of neutrophils, monocytes, and lymphocytes. Recent studies described that different isoforms of galectins can induce platelet adhesion, aggregation, and granule release through the interaction with platelet-specific glycoproteins and integrins. Patients with cancer and/or deep-venous thrombosis have increased levels of galectins in the vasculature, suggesting that these proteins could be important contributors to cancer-associated inflammation and thrombosis. In this review, we summarize the pathological role of galectins in inflammatory and thrombotic events, influencing tumor progression and metastasis. We also discuss the potential of anti-cancer therapies targeting galectins in the pathological context of cancer-associated inflammation and thrombosis.

Articular Cartilage Repair After Implantation of Hyaline Cartilage Beads Engineered From Adult Dedifferentiated Chondrocytes: Cartibeads Preclinical Efficacy Study in a Large Animal Model.

BACKGROUND: Chondrocyte-based cell therapy to repair cartilage has been used for >25 years despite current limitations. This work presents a new treatment option for cartilage lesions. HYPOTHESIS: High-quality hyaline cartilage microtissues called Cartibeads are capable of treating focal chondral lesions once implanted in the defect, by complete fusion of Cartibeads among themselves and their integration with the surrounding native cartilage and subchondral bone. STUDY DESIGN: Controlled laboratory study. METHODS: Cartibeads were first produced from human donors and characterized using histology (safranin O staining of glycosaminoglycan [GAG] and immunohistochemistry of collagen I and II) and GAG dosage. Cartibeads from 6 Göttingen minipigs were engineered and implanted in an autologous condition in the knee (4 or 5 lesions per knee). One group was followed up for 3 months and the other for 6 months. Feasibility and efficacy were measured using histological analysis and macroscopic and microscopic scores. RESULTS: Cartibeads revealed hyaline features with strong staining of GAG and collagen II. High GAG content was obtained: 24.6-µg/mg tissue (wet weight), 15.52-µg/mg tissue (dry weight), and 35 ± 3-µg GAG/bead (mean ± SD). Histological analysis of Göttingen minipigs showed good integration of Cartibeads grafts at 3 and 6 months after implantation. The Bern Score of the histological assay comparing grafted versus empty lesions was significant at 3 months (grafted, n = 10; nongrafted, n = 4; score, 3.3 and 5.3, respectively) and 6 months (grafted, n = 11; nongrafted, n = 3; score, 1.6 and 5.1). CONCLUSION: We developed an innovative 3-step method allowing, for the first time, the use of fully dedifferentiated adult chondrocytes with a high number of cell passage (owing to the extensive amplification in culture). Cartibeads engineered from chondrocytes hold potential as an advanced therapy medicinal product for treating cartilage lesions with established efficacy. CLINICAL RELEVANCE: This successful preclinical study, combined with standardized manufacturing of Cartibeads according to good manufacturing practice guidelines, led to the approval of first-in-human clinical trial by the ethics committee and local medical authority. The generated data highlighted a promising therapy to treat cartilage lesions from a small amount of starting biopsy specimen. With our innovative cell amplification technology, very large lesions can be treated, and older active patients can benefit from it.

Modelling cancer metabolism in vitro: current improvements and future challenges.

Advances in cancer biology over the past decades have revealed that metabolic adaptation of cancer cells is an essential aspect of tumorigenesis. However, recent insights into tumour metabolism in vivo have revealed dissimilarities with results obtained in vitro. This is partly due to the reductionism of in vitro cancer models that struggle to reproduce the complexity of tumour tissues. This review describes some of the discrepancies in cancer cell metabolism between in vitro and in vivo conditions, and presents current methodological approaches and tools used to bridge the gap with the clinically relevant microenvironment. As such, these approaches should generate new knowledge that could be more effectively translated into therapeutic opportunities.

Hyaline Cartilage Microtissues Engineered from Adult Dedifferentiated Chondrocytes: Safety and Role of WNT Signaling.

The repair of damaged articular cartilage is an unmet medical need. Chondrocyte-based cell therapy has been used to repair cartilage for over 20 years despite current limitations. Chondrocyte dedifferentiation upon expansion in monolayer is well known and is the main obstacle to their use as cell source for cartilage repair. Consequently, current approaches often lead to fibrocartilage, which is biomechanically different from hyaline cartilage and not effective as a long-lasting treatment. Here, we describe an innovative 3-step method to engineer hyaline-like cartilage microtissues, named Cartibeads, from high passage dedifferentiated chondrocytes. We show that WNT5A/5B/7B genes were highly expressed in dedifferentiated chondrocytes and that a decrease of the WNT signaling pathway was instrumental for full re-differentiation of chondrocytes, enabling production of hyaline matrix instead of fibrocartilage matrix. Cartibeads showed hyaline-like characteristics based on GAG quantity and type II collagen expression independently of donor age and cartilage quality. In vivo, Cartibeads were not tumorigenic when transplanted into SCID mice. This simple 3-step method allowed a standardized production of hyaline-like cartilage microtissues from a small cartilage sample, making Cartibeads a promising candidate for the treatment of cartilage lesions.

Local Sustained GM-CSF Delivery by Genetically Engineered Encapsulated Cells Enhanced Both Cellular and Humoral SARS-CoV-2 Spike-Specific Immune Response in an Experimental Murine Spike DNA Vaccination Model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide pandemic with recurrences. Therefore, finding a vaccine for this virus became a priority for the scientific community. The SARS-CoV-2 spike protein has been described as the keystone for viral entry into cells and effective immune protection against SARS-CoV-2 is elicited by this protein. Consequently, many commercialized vaccines focus on the spike protein and require the use of an optimal adjuvant during vaccination. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has demonstrated a powerful enhancement of acquired immunity against many pathogens when delivered in a sustained and local manner. In this context, we developed an encapsulated cell-based technology consisting of a biocompatible, semipermeable capsule for secretion of GM-CSF. In this study, we investigated whether murine GM-CSF (muGM-CSF) represents a suitable adjuvant for SARS-CoV-2 immunization, and which delivery strategy for muGM-CSF could be most beneficial. To test this, different groups of mice were immunized with intra-dermal (i.d.) electroporated spike DNA in the absence or presence of recombinant or secreted muGM-CSF. Results demonstrated that adjuvanting a spike DNA vaccine with secreted muGM-CSF resulted in enhancement of specific cellular and humoral immune responses against SARS-CoV-2. Our data also highlighted the importance of delivery strategies to the induction of cellular and humoral-mediated responses.

Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells.

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.

An Experimentally Defined Hypoxia Gene Signature in Glioblastoma and Its Modulation by Metformin.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor, characterized by a high degree of intertumoral heterogeneity. However, a common feature of the GBM microenvironment is hypoxia, which can promote radio- and chemotherapy resistance, immunosuppression, angiogenesis, and stemness. We experimentally defined common GBM adaptations to physiologically relevant oxygen gradients, and we assessed their modulation by the metabolic drug metformin. We directly exposed human GBM cell lines to hypoxia (1% O2) and to physioxia (5% O2). We then performed transcriptional profiling and compared our in vitro findings to predicted hypoxic areas in vivo using in silico analyses. We observed a heterogenous hypoxia response, but also a common gene signature that was induced by a physiologically relevant change in oxygenation from 5% O2 to 1% O2. In silico analyses showed that this hypoxia signature was highly correlated with a perinecrotic localization in GBM tumors, expression of certain glycolytic and immune-related genes, and poor prognosis of GBM patients. Metformin treatment of GBM cell lines under hypoxia and physioxia reduced viable cell number, oxygen consumption rate, and partially reversed the hypoxia gene signature, supporting further exploration of targeting tumor metabolism as a treatment component for hypoxic GBM.

Generation of human induced pluripotent stem cell line UNIGEi003-A from skin fibroblasts of an apparently healthy male donor.

Dermal fibroblasts isolated from an apparently healthy 50-year-old man were successfully transformed into induced pluripotent stem cells (iPSCs) by using the integration-free CytoTune-iPS Sendai Reprogramming method. The generated iPSC line has been expanded under feeder-free conditions and displayed all hallmarks of a standard pluripotent stem cell line such as a normal karyotype, expression of pluripotent factors and differentiation capacity into the three germ layers.

Modeling Poliovirus Infection Using Human Engineered Neural Tissue Enriched With Motor Neuron Derived From Embryonic Stem Cells.

Poliomyelitis is caused by poliovirus (PV), a positive strand non-enveloped virus. Since its discovery in the 1950s, several cell culture and molecular methods have been developed to detect and characterize the various strains of PV. Here, we provide an accurate and standardized protocol to differentiate human embryonic stem cells (hESCs) toward engineered neural tissue enriched with motor neurons (MN ENTs). These MN ENTs expressed markers of motor neuron CHAT and Hb-9 as revealed by immunofluorescence staining and quantitative RT-PCR. Interestingly, our results suggest that motor neurons are responsible for the permissiveness of poliovirus within the MN ENTs. Moreover, our study revealed the molecular events occurring upon PV-3 infection in the MN ENTs and highlighted the modulation of a set of genes involved in EGR-EP300 complex. Collectively, we report the development of a reliable in vitro model to investigate the pathophysiology of PV infection, allowing to both design and assess novel therapeutic approaches against PV infection.

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

Glut3 Addiction Is a Druggable Vulnerability for a Molecularly Defined Subpopulation of Glioblastoma.

While molecular subtypes of glioblastoma (GBM) are defined using gene expression and mutation profiles, we identify a unique subpopulation based on addiction to the high-affinity glucose transporter, Glut3. Although Glut3 is a known driver of a cancer stem cell phenotype, direct targeting is complicated by its expression in neurons. Using established GBM lines and patient-derived stem cells, we identify a subset of tumors within the "proneural" and "classical" subtypes that are addicted to aberrant signaling from integrin αvß3, which activates a PAK4-YAP/TAZ signaling axis to enhance Glut3 expression. This defined subpopulation of GBM is highly sensitive to agents that disrupt this pathway, including the integrin antagonist cilengitide, providing a targeted therapeutic strategy for this unique subset of GBM tumors.

Galectin-3, a Druggable Vulnerability for KRAS-Addicted Cancers.

Identifying the molecular basis for cancer cell dependence on oncogenes such as KRAS can provide new opportunities to target these addictions. Here, we identify a novel role for the carbohydrate-binding protein galectin-3 as a lynchpin for KRAS dependence. By directly binding to the cell surface receptor integrin αvß3, galectin-3 gives rise to KRAS addiction by enabling multiple functions of KRAS in anchorage-independent cells, including formation of macropinosomes that facilitate nutrient uptake and ability to maintain redox balance. Disrupting αvß3/galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvß3-expressing KRAS-mutant lung and pancreatic cancer patient-derived xenografts and spontaneous tumors in mice. Our work reveals galectin-3 as a druggable target for KRAS-addicted lung and pancreas cancers, and indicates integrin αvß3 as a biomarker to identify susceptible tumors.Significance: There is a significant unmet need for therapies targeting KRAS-mutant cancers. Here, we identify integrin αvß3 as a biomarker to identify mutant KRAS-addicted tumors that are highly sensitive to inhibition of galectin-3, a glycoprotein that binds to integrin αvß3 to promote KRAS-mediated activation of AKT. Cancer Discov; 7(12); 1464-79. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

Human three-dimensional engineered neural tissue reveals cellular and molecular events following cytomegalovirus infection.

Human cytomegalovirus (HCMV) is the most common cause of congenital infection of the central nervous system (CNS). To overcome the limited access to human neural tissue and stringent species specificity of HCMV, we used engineered neural tissues to: (i) provide a technical advance to mimick features of HCMV infection in a human neural fetal tissue in vitro and (ii) characterize the molecular and cellular phenomenon following HCMV infection in this tissue. Herein, we infected hESC-derived engineered neural tissues (ENTs) whose organization resembles fetal brain. Transcriptome analysis of ENTs demonstrated that HCMV infection displayed features of the infection with the expression of genes involved in lipid metabolism, growth and development, as well as stress and host-response in a time-dependent manner. Immunohistochemical analysis demonstrated that HCMV did not firstly infect neural tubes (i.e. radially organized, proliferating stem cell niches), but rather an adjacent side population of post-mitotic cells expressing nestin, doublecortin, Sox1, musashi and vimentin markers. Importantly, we observe the same tropism in naturally HCMV-infected fetal brain specimens. To the best of our knowledge this system represents the first human brain-like tissue able to provide a more physiologically model for studying HCMV infection.

Comprehensive metagenomic analysis of glioblastoma reveals absence of known virus despite antiviral-like type I interferon gene response.

Glioblastoma is a deadly malignant brain tumor and one of the most incurable forms of cancer in need of new therapeutic targets. As some cancers are known to be caused by a virus, the discovery of viruses could open the possibility to treat, and perhaps prevent, such a disease. Although an association with viruses such as cytomegalovirus or Simian virus 40 has been strongly suggested, involvement of these and other viruses in the initiation and/or propagation of glioblastoma remains vague, controversial and warrants elucidation. To exhaustively address the association of virus and glioblastoma, we developed and validated a robust metagenomic approach to analyze patient biopsies via high-throughput sequencing, a sensitive tool for virus screening. In addition to traditional clinical diagnostics, glioblastoma biopsies were deep-sequenced and analyzed with a multistage computational pipeline to identify known or potentially discover unknown viruses. In contrast to the studies reporting the presence of viral signatures in glioblastoma, no common or recurring active viruses were detected, despite finding an antiviral-like type I interferon response in some specimens. Our findings highlight a discrete and non-specific viral signature and uncharacterized short RNA sequences in glioblastoma. This study provides new insights into glioblastoma pathogenesis and defines a general methodology that can be used for high-resolution virus screening and discovery in human cancers.

The relationship between brain tumor cell invasion of engineered neural tissues and in vivo features of glioblastoma.

Glioblastoma is an aggressive brain tumor characterized by its high propensity for local invasion, formation of secondary foci within the brain, as well as areas of necrosis. This study aims to (i) provide a technical approach to reproduce features of the disease in vitro and (ii) characterize the tumor/host brain tissue interaction at the molecular level. Human engineered neural tissue (ENT) obtained from pluripotent stem cells was generated and co-cultured with human glioblastoma-initiating cells. Within two weeks, glioblastoma cells invaded the nervous tissue. This invasion displayed features of the disease in vivo: a primary tumor mass, diffuse migration of invading single cells into the nervous tissue, secondary foci, as well as peritumoral cell death. Through comparative molecular analyses, this model allowed the identification of more than 100 genes that are specifically induced and up-regulated by the nervous tissue/tumor interaction. Notably the type I interferon response, extracellular matrix-related genes were most highly represented and showed a significant correlation with patient survival. In conclusion, glioblastoma development within a nervous tissue can be engineered in vitro, providing a relevant model to study the disease and allows the identification of clinically-relevant genes induced by the tumor/host tissue interaction.

Involvement of the TGFß pathway in the regulation of α5 ß1 integrins by caveolin-1 in human glioblastoma.

Caveolin-1 plays a crucial role in the development of cancer and its progression. We previously reported that glioblastoma cells expressing low levels of caveolin-1 exerted a more aggressive phenotype than cells expressing high levels. Such phenotype was due to the induction of α(5) ß(1) integrin subsequent to the depletion of caveolin-1. Caveolin-1 was identified as a transcriptional repressor of α(5) ß(1) integrin. The current study was designed to identify in vitro, the molecular mechanisms by which caveolin-1 controls α(5) ß(1) integrin expression and to determine if a negative correlation between caveolin-1 and α(5) ß(1) integrins also exists in biopsies and xenografted human brain tumors. We showed that depletion of caveolin-1 lead to the activation of the TGFß/TGFßRI/Smad2 pathway which in turn induced the expression of α(5) ß(1) integrins. We showed that cells expressing the lowest levels of caveolin-1 but the highest levels of α(5) ß(1) integrins and TGFßRI were the most sensitive to a α(5) ß(1) integrin antagonist and a TGFßRI inhibitor. Screening human glioma biopsies and human glioblastoma xenografts, we isolated subgroups with either low levels of caveolin-1 but high levels of α(5) ß(1) integrin and TGFßRI or high levels of caveolin-1 but low levels of α(5) ß(1) integrin and TGFßRI. In conclusion, caveolin-1 controls α(5) ß(1) integrin expression through the TGFß/TGFßRI/Smad2 pathway. The status of caveolin-1/α(5) ß(1) integrins/TGFßRI might be a useful marker of the tumor evolution/prognosis as well as a predictor of anti-TGFß or anti-α(5) ß(1) integrin therapies.

Deregulation of TWIST-1 in the CD34+ compartment represents a novel prognostic factor in chronic myeloid leukemia.

The mechanisms of resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) often remain obscure. Analysis of patient samples during disease progression revealed the up-regulation of the oncogene TWIST-1, also measured in primary samples from TKI-resistant patients. Moreover, we found that TWIST-1 was overexpressed in CML diagnostic samples of patients who later developed cytogenetic resistance to imatinib, even those without any detectable resistance mechanism. We confirmed the up-regulation of TWIST-1 at both RNA and protein levels in imatinib-resistant cell lines, irrespective of any other resistance mechanism. Analysis with specific small interfering RNA suggested TWIST-1 involvement in the resistance phenotype. Finally, the kinetics of TWIST-1 expression during the individual medical histories of CML patients indicated that TWIST-1 expression is down-regulated by TKIs and up-regulated with TKI resistance. We hypothesize that the overexpression of the TWIST-1 oncogene represents a novel key prognostic factor potentially useful for optimizing CML management in the TKI era.

Caveolin-1 regulates glioblastoma aggressiveness through the control of alpha(5)beta(1) integrin expression and modulates glioblastoma responsiveness to SJ749, an alpha(5)beta(1) integrin antagonist.

Caveolin-1 plays a checkpoint function in the regulation of processes often altered in cancer. Although increased expression of caveolin-1 seems to be the norm in the glioma family of malignancies, populations of caveolin-1 positive and negative cells coexist among glioblastoma specimens. As no data are available to date on the contribution of such cells to the phenotype of glioblastoma, we manipulated caveolin-1 in the glioblastoma cell line U87MG. We showed that caveolin-1 plays a critical role in the aggressiveness of glioblastoma. We identified integrins as the main set of genes affected by caveolin-1. We reported here that the phenotypic changes observed after caveolin-1 modulation were mediated by alpha(5)beta(1) integrins. As a consequence of the regulation of alpha(5)beta(1) levels by caveolin-1, the sensitivity of cells to the specific alpha(5)beta(1) integrin antagonist, SJ749, was affected. Mediator of caveolin-1 effects, alpha(5)beta(1) integrin, is also a marker for glioma aggressiveness and an efficient target for the treatment of glioma especially the ones exerting the highest aggressive phenotype.