Treatment-induced shrinking of tumour aggregates: a nonlinear volume-filling chemotactic approach.

Motivated by experimental observations in 3D/organoid cultures derived from glioblastoma, we propose a novel mechano-transduction mechanism where the introduction of a chemotherapeutic treatment induces mechanical changes at the cell level. We analyse the influence of these individual mechanical changes on the properties of the aggregates obtained at the population level. We employ a nonlinear volume-filling chemotactic system of partial differential equations, where the elastic properties of the cells are taken into account through the so-called squeezing probability, which depends on the concentration of the treatment in the extracellular microenvironment. We explore two scenarios for the effect of the treatment: first, we suppose that the treatment acts only on the mechanical properties of the cells and, in the second one, we assume it also prevents cell proliferation. We perform a linear stability analysis which enables us to identify the ability of the system to create patterns and fully characterize their size. Moreover, we provide numerical simulations in 1D and 2D that illustrate the shrinking of the aggregates due to the presence of the treatment.

Sphingolipid distribution at mitochondria-associated membranes (MAMs) upon induction of apoptosis.

The levels and composition of sphingolipids and related metabolites are altered in aging and in common disorders such as diabetes and cancers, as well as in neurodegenerative, cardiovascular, and respiratory diseases. Changes in sphingolipids have been implicated as being an essential step in mitochondria-driven cell death. However, little is known about the precise sphingolipid composition and modulation in mitochondria or related organelles. Here, we used LC-MS/MS to analyze the presence of key components of the ceramide metabolic pathway in vivo and in vitro in purified ER, mitochondria-associated membranes (MAMs), and mitochondria. Specifically, we analyzed the sphingolipids in the three pathways that generate ceramide: sphinganine in the de novo ceramide pathway, SM in the breakdown pathway, and sphingosine in the salvage pathway. We observed sphingolipid profiles in mouse liver, mouse brain, and a human glioma cell line (U251). We analyzed the quantitative and qualitative changes of these sphingolipids during staurosporine-induced apoptosis in U251 cells. Ceramide (especially C16-ceramide) levels increased during early apoptosis possibly through a conversion from mitochondrial sphinganine and SM, but sphingosine and lactosyl- and glycosyl-ceramide levels were unaffected. We also found that ceramide generation is enhanced in mitochondria when SM levels are decreased in the MAM. This decrease was associated with an increase in acid sphingomyelinase activity in MAM. We conclude that meaningful sphingolipid modifications occur in MAM, the mitochondria, and the ER during the early steps of apoptosis.

Cytosine methylation of mature microRNAs inhibits their functions and is associated with poor prognosis in glioblastoma multiforme.

BACKGROUND: Literature reports that mature microRNA (miRNA) can be methylated at adenosine, guanosine and cytosine. However, the molecular mechanisms involved in cytosine methylation of miRNAs have not yet been fully elucidated. Here we investigated the biological role and underlying mechanism of cytosine methylation in miRNAs in glioblastoma multiforme (GBM). METHODS: RNA immunoprecipitation with the anti-5methylcytosine (5mC) antibody followed by Array, ELISA, dot blot, incorporation of a radio-labelled methyl group in miRNA, and miRNA bisulfite sequencing were perfomred to detect the cytosine methylation in mature miRNA. Cross-Linking immunoprecipiation qPCR, transfection with methylation/unmethylated mimic miRNA, luciferase promoter reporter plasmid, Biotin-tagged 3'UTR/mRNA or miRNA experiments and in vivo assays were used to investigate the role of methylated miRNAs. Finally, the prognostic value of methylated miRNAs was analyzed in a cohorte of GBM pateints. RESULTS: Our study reveals that a significant fraction of miRNAs contains 5mC. Cellular experiments show that DNMT3A/AGO4 methylated miRNAs at cytosine residues inhibit the formation of miRNA/mRNA duplex and leading to the loss of their repressive function towards gene expression. In vivo experiments show that cytosine-methylation of miRNA abolishes the tumor suppressor function of miRNA-181a-5p miRNA for example. Our study also reveals that cytosine-methylation of miRNA-181a-5p results is associated a poor prognosis in GBM patients. CONCLUSION: Together, our results indicate that the DNMT3A/AGO4-mediated cytosine methylation of miRNA negatively.

Impairing temozolomide resistance driven by glioma stem-like cells with adjuvant immunotherapy targeting O-acetyl GD2 ganglioside.

Stem cell chemoresistance remains challenging the efficacy of the front-line temozolomide against glioblastoma. Novel therapies are urgently needed to fight those cells in order to control tumor relapse. Here, we report that anti-O-acetyl-GD2 adjuvant immunotherapy controls glioma stem-like cell-driven chemoresistance. Using patient-derived glioblastoma cells, we found that glioma stem-like cells overexpressed O-acetyl-GD2. As a result, monoclonal antibody 8B6 immunotherapy significantly increased temozolomide genotoxicity and tumor cell death in vitro by enhancing temozolomide tumor uptake. Furthermore, the combination therapy decreased the expression of the glioma stem-like cell markers CD133 and Nestin and compromised glioma stem-like cell self-renewal capabilities. When tested in vivo, adjuvant 8B6 immunotherapy prevented the extension of the temozolomide-resistant glioma stem-like cell pool within the tumor bulk in vivo and was more effective than the single agent therapies. This is the first report demonstrating that anti-O-acetyl-GD2 monoclonal antibody 8B6 targets glioblastoma in a manner that control temozolomide-resistance driven by glioma stem-like cells. Together our results offer a proof of concept for using anti-O-acetyl GD2 reagents in glioblastoma to develop more efficient combination therapies for malignant gliomas.

Mitochondria transfer from tumor-activated stromal cells (TASC) to primary Glioblastoma cells.

The tumor microenvironment (TME) controls many aspects of cancer development but little is known about its effect in Glioblastoma (GBM), the main brain tumor in adults. Tumor-activated stromal cell (TASC) population, a component of TME in GBM, was induced in vitro by incubation of MSCs with culture media conditioned by primary cultures of GBM under 3D/organoid conditions. We observed mitochondrial transfer by Tunneling Nanotubes (TNT), extracellular vesicles (EV) and cannibalism from the TASC to GBM and analyzed its effect on both proliferation and survival. We created primary cultures of GBM or TASC in which we have eliminated mitochondrial DNA [Rho 0 (ρ0) cells]. We found that TASC, as described in other cancers, increased GBM proliferation and resistance to standard treatments (radiotherapy and chemotherapy). We analyzed the incorporation of purified mitochondria by ρ0 and ρ+ cells and a derived mathematical model taught us that ρ+ cells incorporate more rapidly pure mitochondria than ρ0 cells.

Low-Dose Pesticide Mixture Induces Accelerated Mesenchymal Stem Cell Aging In Vitro.

The general population is chronically exposed to multiple environmental contaminants such as pesticides. We have previously demonstrated that human mesenchymal stem cells (MSCs) exposed in vitro to low doses of a mixture of seven common pesticides showed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation toward adipogenesis. Thus, we hypothesized that common combination of pesticides may induce a premature cellular aging of adult MSCs. Our goal was to evaluate if the prolonged exposure to pesticide mixture could accelerate aging-related markers and in particular deteriorate the immunosuppressive properties of MSCs. MSCs exposed to pesticide mixture, under long-term culture and obtained from aging donor, were compared by bulk RNA sequencing analysis. Aging, senescence, and immunomodulatory markers were compared. The protein expression of cellular aging-associated metabolic markers and immune function of MSCs were analyzed. Functional analysis of the secretome impacts on immunomodulatory properties of MSCs was realized after 21 days' exposure to pesticide mixture. The RNA sequencing analysis of MSCs exposed to pesticide showed some similarities with cells from prolonged culture, but also with the MSCs of an aged donor. Changes in the metabolic markers MDH1, GOT and SIRT3, as well as an alteration in the modulation of active T cells and modifications in cytokine production are all associated with cellular aging. A modified functional profile was found with similarities to aging process. Stem Cells 2019;37:1083-1094.

Low-Dose Pesticide Mixture Induces Senescence in Normal Mesenchymal Stem Cells (MSC) and Promotes Tumorigenic Phenotype in Premalignant MSC.

Humans are chronically exposed to multiple environmental pollutants such as pesticides with no significant evidence about the safety of such poly-exposures. We exposed mesenchymal stem cells (MSC) to very low doses of mixture of seven pesticides frequently detected in food samples for 21 days in vitro. We observed a permanent phenotype modification with a specific induction of an oxidative stress-related senescence. Pesticide mixture also induced a shift in MSC differentiation towards adipogenesis but did not initiate a tumorigenic transformation. In modified MSC in which a premalignant phenotype was induced, the exposure to pesticide mixture promoted tumorigenic phenotype both in vitro and in vivo after cell implantation, in all nude mice. Our results suggest that a common combination of pesticides can induce a premature ageing of adult MSC, and as such could accelerate age-related diseases. Exposure to pesticide mixture may also promote the tumorigenic transformation in a predisposed stromal environment. Abstract Video Link: https://youtu.be/mfSVPTol-Gk Stem Cells 2017;35:800-811.

Pharmacological targeting of apelin impairs glioblastoma growth.

Glioblastoma are highly aggressive brain tumours that are associated with an extremely poor prognosis. Within these tumours exists a subpopulation of highly plastic self-renewing cancer cells that retain the ability to expand ex vivo as tumourspheres, induce tumour growth in mice, and have been implicated in radio- and chemo-resistance. Although their identity and fate are regulated by external cues emanating from endothelial cells, the nature of such signals remains unknown. Here, we used a mass spectrometry proteomic approach to characterize the factors released by brain endothelial cells. We report the identification of the vasoactive peptide apelin as a central regulator for endothelial-mediated maintenance of glioblastoma patient-derived cells with stem-like properties. Genetic and pharmacological targeting of apelin cognate receptor abrogates apelin- and endothelial-mediated expansion of glioblastoma patient-derived cells with stem-like properties in vitro and suppresses tumour growth in vivo. Functionally, selective competitive antagonists of apelin receptor were shown to be safe and effective in reducing tumour expansion and lengthening the survival of intracranially xenografted mice. Therefore, the apelin/apelin receptor signalling nexus may operate as a paracrine signal that sustains tumour cell expansion and progression, suggesting that apelin is a druggable factor in glioblastoma.

HB-EGF is associated with DNA damage and Mcl-1 turnover in human glioma cell lines treated by Temozolomide.

Temozolomide (TMZ) is the main chemotherapeutic agent used for treating newly diagnosed Glioblastoma Multiforme (GBM), the most frequent malignant brain tumors in adults. This alkylating agent induces DNA double strand breaks (DSBs) which in turn lead to apoptosis by activating the Bcl-2 controlled mitochondrial pathway. However, GBM invariably recur as tumors become resistant to TMZ. We investigated the implication of EGFR ligands in this resistance and we found that the pro Heparin Binding Epidermal Growth Factor (proHB-EGF) expression is linked to the early response to TMZ in human glioma cell lines. However, HB-EGF does not affect apoptosis per se although its expression is associated with the degradation of Mcl-1. HB-EGF is implicated in DSBs repair as silencing of HB-EGF increased γH2AX foci half-life as well as USP9X expression, two features that could be linked to the observed effect on Mcl-1. Our data demonstrate a new role for HB-EGF in TMZ treated cell lines.

Differentiation-related response to DNA breaks in human mesenchymal stem cells.

We have recently shown that the in vitro differentiation of human mesenchymal stem cells (hMSCs) was accompanied by an increased sensitivity toward apoptosis; however, the mechanism responsible for this shift is not known. Here, we show that the repair of DNA double-strand breaks (DSBs) was more rapid in undifferentiated hMSCs than in differentiated osteoblasts by quantification of the disappearance of γ-H2AX foci in the nuclei after γ-irradiation-induced DNA damage. In addition, there was a marked and prolonged increase in the level of nuclear Ku70 and an increased phosphorylation of DNA-PKcs. This was accompanied by an augmentation in the phosphorylation of ATM in hMSCs post-irradiation suggesting the nonhomologous end joining repair mechanism. However, when hMSCs were induced to differentiate along the osteogenic or adipogenic pathways; irradiation of these cells caused an expeditious and robust cell death, which was primarily apoptotic. This was in sharp contrast to undifferentiated hMSCs, which were highly resistant to irradiation and/or temozolomide-induced DSBs. In addition, we observed a 95% recovery from DSB in these cells. Our results suggest that apoptosis and DNA repair are major safeguard mechanisms in the control of hMSCs differentiation after DNA damage.

Identification of MCM4 and PRKDC as new regulators of osteosarcoma cell dormancy based on 3D cell cultures.

Dormancy is a potential way for tumors to develop drug resistance and escape treatment. However, the mechanisms involved in cancer dormancy remain poorly understood. This is mainly because there is no in vitro culture model making it possible to spontaneously induce dormancy. In this context, the present work proposes the use of three-dimensional (3D) spheroids developed from osteosarcoma cell lines as a relevant model for studying cancer dormancy. MNNG-HOS, SaOS-2, 143B, MG-63, U2OS and SJSA-1 cell lines were cultured in 3D using the Liquid Overlay Technique (LOT). Dormancy was studied by staining cancer cells with a lipophilic dye (DiD), and long-term DiD+ cells were considered as dormant cancer cells. The role of the extracellular matrix in inducing dormancy was investigated by embedding cells into methylcellulose or Geltrex™. Gene expression of DiD+ cells was assessed with a Nanostring™ approach and the role of the genes detected in dormancy was validated by a transient down-expression model using siRNA treatment. Proliferation was measured using fluorescence microscopy and the xCELLigence technology. We observed that MNNG-HOS, 143B and MG-G3 cell lines had a reduced proliferation rate in 3D compared to 2D. U2OS cells had an increased proliferation rate when they were cultured in Geltrex™ compared to other 3D culture methods. Using 3D cultures, a transcriptomic signature of dormancy was obtained and showed a decreased expression of 18 genes including ETV4, HELLS, ITGA6, MCM4, PRKDC, RAD21 and UBE2T. The treatment with siRNA targeting these genes showed that cancer cell proliferation was reduced when the expression of ETV4 and MCM4 were decreased, whereas proliferation was increased when the expression of RAD21 was decreased. 3D culture facilitates the maintenance of dormant cancer cells characterized by a reduced proliferation and less differential gene expression as compared to proliferative cells. Further studies of the genes involved has enabled us to envisage their role in regulating cell proliferation.

Comparison of spheroids formed by rat glioma stem cells and neural stem cells reveals differences in glucose metabolism and promising therapeutic applications.

Cancer stem cells (CSCs) are thought to be partially responsible for cancer resistance to current therapies and tumor recurrence. Dichloroacetate (DCA), a compound capable of shifting metabolism from glycolysis to glucose oxidation, via an inhibition of pyruvate dehydrogenase kinase was used. We show that DCA is able to shift the pyruvate metabolism in rat glioma CSCs but has no effect in rat neural stem cells. DCA forces CSCs into oxidative phosphorylation but does not trigger the production of reactive oxygen species and consecutive anti-cancer apoptosis. However, DCA, associated with etoposide or irradiation, induced a Bax-dependent apoptosis in CSCs in vitro and decreased their proliferation in vivo. The former phenomenon is related to DCA-induced Foxo3 and p53 expression, resulting in the overexpression of BH3-only proteins (Bad, Noxa, and Puma), which in turn facilitates Bax-dependent apoptosis. Our results demonstrate that a small drug available for clinical studies potentiates the induction of apoptosis in glioma CSCs.

Drug-tolerant persister cells in cancer: the cutting edges and future directions.

Drug-tolerant persister (DTP) cell populations were originally discovered in antibiotic-resistant bacterial biofilms. Similar populations with comparable features have since been identified among cancer cells and have been linked with treatment resistance that lacks an underlying genomic alteration. Research over the past decade has improved our understanding of the biological roles of DTP cells in cancer, although clinical knowledge of the role of these cells in treatment resistance remains limited. Nonetheless, targeting this population is anticipated to provide new treatment opportunities. In this Perspective, we aim to provide a clear definition of the DTP phenotype, discuss the underlying characteristics of these cells, their biomarkers and vulnerabilities, and encourage further research on DTP cells that might improve our understanding and enable the development of more effective anticancer therapies.

Spatial Distribution of Immune Cells in Primary and Recurrent Glioblastoma: A Small Case Study.

Only a minority of patients with glioblastoma (GBM) respond to immunotherapy, and always only partially. There is a lack of knowledge on immune distribution in GBM and in its tumor microenvironment (TME). To address the question, we used paired primary and recurrent tumors from GBM patients to study the composition and the evolution of the immune landscape upon treatment. We studied the expression of a handful of immune markers (CD3, CD8, CD68, PD-L1 and PD-1) in GBM tissues in 15 paired primary and recurrent GBM. In five selected patients, we used Nanostring Digital Spatial Profiling (DSP) to obtain simultaneous assessments of multiple biomarkers both within the tumor and the microenvironment in paired primary and recurrent GBM. Our results suggest that the evolution of the immune landscape between paired primary and recurrent GBM tumors is highly heterogeneous. However, our study identifies B3-H7 and HLA-DR as potential targets in primary and recurrent GBM. Spatial profiling of immune markers from matched primary and recurrent GBM shows a nonlinear complex evolution during the progression of cancer. Nonetheless, our study demonstrated a global increase in macrophages, and revealed differential localization of some markers, such as B7-H3 and HLA-DR, between GBM and its TME.

Technical report: liquid overlay technique allows the generation of homogeneous osteosarcoma, glioblastoma, lung and prostate adenocarcinoma spheroids that can be used for drug cytotoxicity measurements.

Introduction: The mechanisms involved in cancer initiation, progression, drug resistance, and disease recurrence are traditionally investigated through in vitro adherent monolayer (2D) cell models. However, solid malignant tumor growth is characterized by progression in three dimensions (3D), and an increasing amount of evidence suggests that 3D culture models, such as spheroids, are suitable for mimicking cancer development. The aim of this report was to reaffirm the relevance of simpler 3D culture methods to produce highly reproducible spheroids, especially in the context of drug cytotoxicity measurements. Methods: Human A549 lung adenocarcinoma, LnCaP prostate adenocarcinoma, MNNG/HOS osteosarcoma and U251 glioblastoma cell lines were grown into spheroids for 20 days using either Liquid Overlay Technique (LOT) or Hanging Drop (HD) in various culture plates. Their morphology was examined by microscopy. Sensitivity to doxorubicin was compared between MNNG/HOS cells grown in 2D and 3D. Results: For all cell lines studied, the morphology of spheroids generated in round-bottom multiwell plates was more repeatable than that of those generated in flat-bottom multiwell plates. HD had no significant advantage over LOT when the spheroids were cultured in round-bottom plates. Finally, the IC50 of doxorubicin on MNNG/HOS cultured in 3D was 18.8 times higher than in 2D cultures (3D IC50 = 15.07 ± 0.3 µM; 2D IC50 = 0.8 ± 0.4 µM; *p < 0.05). Discussion: In conclusion, we propose that the LOT method, despite and because of its simplicity, is a relevant 3D model for drug response measurements that could be scaled up for high throughput screening.

A Simple 3D Cell Culture Method for Studying the Interactions between Human Mesenchymal Stromal/Stem Cells and Patients Derived Glioblastoma.

We have developed a 3D biosphere model using patient-derived cells (PDCs) from glioblastoma (GBM), the major form of primary brain tumors in adult, plus cancer-activated fibroblasts (CAFs), obtained by culturing mesenchymal stem cells with GBM conditioned media. The effect of MSC/CAFs on the proliferation, cell-cell interactions, and response to treatment of PDCs was evaluated. Proliferation in the presence of CAFs was statistically lower but the spheroids formed within the 3D-biosphere were larger. A treatment for 5 days with Temozolomide (TMZ) and irradiation, the standard therapy for GBM, had a marked effect on cell number in monocultures compared to co-cultures and influenced cancer stem cells composition, similar to that observed in GBM patients. Mathematical analyses of spheroids growth and morphology confirm the similarity with GBM patients. We, thus, provide a simple and reproducible method to obtain 3D cultures from patient-derived biopsies and co-cultures with MSC with a near 100% success. This method provides the basis for relevant in vitro functional models for a better comprehension of the role of tumor microenvironment and, for precision and/or personalized medicine, potentially to predict the response to treatments for each GBM patient.

Challenges in glioblastoma research: focus on the tumor microenvironment.

Glioblastoma (GBM) is the most deadly type of malignant brain tumor, despite extensive molecular analyses of GBM cells. In recent years, the tumor microenvironment (TME) has been recognized as an important player and therapeutic target in GBM. However, there is a need for a full and integrated understanding of the different cellular and molecular components involved in the GBM TME and their interactions for the development of more efficient therapies. In this review, we provide a comprehensive report of the GBM TME, which assembles the contributions of physicians and translational researchers working on brain tumor pathology and therapy in France. We propose a holistic view of the subject by delineating the specific features of the GBM TME at the cellular, molecular, and therapeutic levels.

The mitochondrial pathways of apoptosis.

Apoptosis is a process of programmed cell death that serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Studies in nematode, Drosophila and mammals have shown that, although regulation of the cell death machinery is somehow different from one species to another, it is controlled by homologous proteins and involves mitochondria. In mammals, activation of caspases (cysteine proteases that are the main executioners of apoptosis) is under the tight control of the Bcl-2 family proteins, named in reference to the first discovered mammalian cell death regulator. These proteins mainly act by regulating the release of caspases activators from mitochondria. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of apoptosis. In this chapter, we present the current view on the mitochondrial pathway of apoptosis with a particular attention to new aspects of the regulation of the Bcl-2 proteins family control of mitochondrial membrane permeabilization: the mechanisms implicated in their mitochondrial targeting and activation during apoptosis, the function(s) of the oncosuppressive protein p53 at the mitochondria and the role of the processes of mitochondrial fusion and fission.

Bcl-2 Family Members and the Mitochondrial Import Machineries: The Roads to Death.

The localization of Bcl-2 family members at the mitochondrial outer membrane (MOM) is a crucial step in the implementation of apoptosis. We review evidence showing the role of the components of the mitochondrial import machineries (translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM)) in the mitochondrial localization of Bcl-2 family members and how these machineries regulate the function of pro- and anti-apoptotic proteins in resting cells and in cells committed into apoptosis.

The Activation of Mesenchymal Stem Cells by Glioblastoma Microvesicles Alters Their Exosomal Secretion of miR-100-5p, miR-9-5p and let-7d-5p.

Glioblastoma (GBM) is the most aggressive brain tumor, and despite initial response to chemo- and radio-therapy, the persistence of glioblastoma stem cells (GSCs) unfortunately always results in tumor recurrence. It is now largely admitted that tumor cells recruit normal cells, including mesenchymal stem cells (MSCs), and components of their environment, to participate in tumor progression, building up what is called the tumor microenvironment (TME). While growth factors and cytokines constitute essential messengers to pass on signals between tumor and TME, recent uncovering of extracellular vesicles (EVs), composed of microvesicles (MVs) and exosomes, opened new perspectives to define the modalities of this communication. In the GBM context particularly, we investigated what could be the nature of the EV exchange between GSCs and MSCs. We show that GSCs MVs can activate MSCs into cancer-associated fibroblasts (CAFs)-like cells, that subsequently increase their secretion of exosomes. Moreover, a significant decrease in anti-tumoral miR-100-5p, miR-9-5p and let-7d-5p was observed in these exosomes. This clearly suggests a miRNA-mediated GBM tumor promotion by MSCs exosomes, after their activation by GBM MVs.