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.

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.

Three-dimensional in vitro culture models in oncology research.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

Cellular Heterogeneity and Cooperativity in Glioma Persister Cells Under Temozolomide Treatment.

We have observed a drug-tolerant/persister state in a human glioblastoma (GBM) cell line after exposure to temozolomide, the standard-of-care chemotherapeutic agent for GBM. We used a multicolor lentiviral genetic barcode labeling to follow cell population evolution during temozolomide treatment. We observed no change in the distribution of the different colored populations of cells in persister or resistant cells suggesting that pre-existing minor subpopulations, which would be expected to be restricted to a single color, were not amplified/selected during the response to the drug. We have previously identified four genes (CHI3L1, FAT2, KLK5, and HB-EGF) that were over-expressed during the persister stage. Single-cell analysis of these four genes indicated that they were expressed in different individual cells ruling out the existence of a single persister-specific clone but suggesting rather a global answer. Even so, the transitory silencing of CHI3L1, FAT2, or KLK5 influenced the expression of the other three genes and the survival of U251 cells in absence of temozolomide. Since proteins encoded by the four genes are all localized in the extracellular matrix or interact within the extracellular compartment, we propose that cellular interactions and communications are important during the persister stage before the acquisition of chemo-resistance. Thus, persisters might be a new therapeutically relevant target in GBM.

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.

TOM20-mediated transfer of Bcl2 from ER to MAM and mitochondria upon induction of apoptosis.

In this work, we have explored the subcellular localization of Bcl2, a major antiapoptotic protein. In U251 glioma cells, we found that Bcl2 is localized mainly in the ER and is translocated to MAM and mitochondria upon induction of apoptosis; this mitochondrial transfer was not restricted to the demonstrator cell line, even if cell-specific modulations exist. We found that the Bcl2/mitochondria interaction is controlled by TOM20, a protein that belongs to the protein import machinery of the mitochondrial outer membrane. The expression of a small domain of interaction of TOM20 with Bcl2 potentiates its anti-apoptotic properties, which suggests that the Bcl2-TOM20 interaction is proapoptotic. The role of MAM and TOM20 in Bcl2 apoptotic mitochondrial localization and function has been confirmed in a yeast model in which the ER-mitochondria encounter structure (ERMES) complex (required for MAM stability in yeast) has been disrupted. Bcl2-TOM20 interaction is thus an additional player in the control of apoptosis.

Anti-PD1 therapy induces lymphocyte-derived exosomal miRNA-4315 release inhibiting Bim-mediated apoptosis of tumor cells.

Anti-PD1 immunotherapy, as a single agent or in combination with standard chemotherapies, has significantly improved the outcome of many patients with cancers. However, resistance to anti-PD1 antibodies often decreases the long-term therapeutic benefits. Despite this observation in clinical practice, the molecular mechanisms associated with resistance to anti-PD1 antibody therapy have not yet been elucidated. To identify the mechanisms of resistance associated with anti-PD1 antibody therapy, we developed cellular models including purified T cells and different cancer cell lines from glioblastoma, lung adenocarcinoma, breast cancer and ovarian carcinoma. A murine model of lung cancer was also used. Longitudinal blood samples of patients treated with anti-PD1 therapy were also used to perform a proof-of-concept study of our findings. We found that anti-PD1 exposure of T-cell promotes an enrichment of exosomal miRNA-4315. We also noted that exosomal miRNA-4315 induced a phenomenon of apopto-resistance to conventional chemotherapies in cancer cells receiving exosomal miRNA-4315. At molecular level, we discern that the apopto-resistance phenomenon was associated with the miRNA-4315-mediated downregulation of Bim, a proapoptotic protein. In cellular and mice models, we observed that the BH3 mimetic agent ABT263 circumvented this resistance. A longitudinal study using patient blood showed that miRNA-4315 and cytochrome c can be used to define the time period during which the addition of ABT263 therapy may effectively increase cancer cell death and bypass anti-PD1 resistance.This work provides a blood biomarker (exosomal miRNA-4315) for patient stratification developing a phenomenon of resistance to anti-PD1 antibody therapy and also identifies a therapeutic alternative (the use of a BH3 mimetic drug) to limit this resistance phenomenon.

N6-Adenosine Methylation of miRNA-200b-3p Influences Its Functionality and Is a Theranostic Tool.

MicroRNAs (miRNAs or miRs) play crucial roles in biological and pathological processes. Some miRNAs also appear as promising biomarkers and therapeutic tools. However, the epitranscriptomic regulation of miRNAs is not yet fully elucidated in all of their fields of application. We report that adenosine methylation of miR-200b-3p inhibits its repressive function toward its mRNA targets such as XIAP by blocking the formation of the miRNA/3' UTRmRNA duplex. Our data indicate that the adenosine methylation of miR-200b-3p is associated with the survival of glioblastoma patients. Collectively, our data support the idea that the adenosine methylation of miR-200b-3p can be used as a prodrug having a selective cytotoxicity against cancer cells (while being harmless to peripheral blood mononuclear cells [PBMCs], astrocytes, neurons, and hepatocytes).

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.

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.

Radiotherapy-induced overexpression of exosomal miRNA-378a-3p in cancer cells limits natural killer cells cytotoxicity.

Aim: We here hypothesized that tumor-derived exosomal miRNA (TexomiR) released from irradiated tumors may play a role in the tumor cells escape to natural killer (NK) cells. Materials & methods: Our study included the use of different cancer cell lines, blood biopsies of xenograph mice model and patients treated with radiotherapy. Results: The irradiation of cancer cells promotes the TET2-mediated demethylation of miR-378 promoter, miR-378a-3p overexpression and its loading in exosomes, inducing the decrease of granzyme-B (GZMB) secretion by NK cells. An inverse correlation between TexomiR-378a-3p and GZMB was observed in murine and human blood samples. Conclusion: Our work identifies TexomiR-378a-3p as a molecular signature associated with the loss of NK cells cytotoxicity via the decrease of GZMB expression upon radiotherapy.

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.

Identification of a transient state during the acquisition of temozolomide resistance in glioblastoma.

Drug resistance limits the therapeutic efficacy in cancers and leads to tumor recurrence through ill-defined mechanisms. Glioblastoma (GBM) are the deadliest brain tumors in adults. GBM, at diagnosis or after treatment, are resistant to temozolomide (TMZ), the standard chemotherapy. To better understand the acquisition of this resistance, we performed a longitudinal study, using a combination of mathematical models, RNA sequencing, single cell analyses, functional and drug assays in a human glioma cell line (U251). After an initial response characterized by cell death induction, cells entered a transient state defined by slow growth, a distinct morphology and a shift of metabolism. Specific genes expression associated to this population revealed chromatin remodeling. Indeed, the histone deacetylase inhibitor trichostatin (TSA), specifically eliminated this population and thus prevented the appearance of fast growing TMZ-resistant cells. In conclusion, we have identified in glioblastoma a population with tolerant-like features, which could constitute a therapeutic target.

Cell-free circulating epimarks in cancer monitoring.

Cancer numbers increasing, cases heterogeneity and the drug resistance emergence have pushed scientists to search for innovative solutions for patients and epimutations can be one. Methylated DNA, modified nucleosomes and noncoding RNAs are found in all cells, including tumor cells. They are intracellular actors but also have intercellular communication roles, being released in extracellular environment and in different body fluids. Here, we reviewed current literature on the use of these blood circulating epimarks in cancer monitoring. What stands out is that epimarkers must be considered as 'real time' images of the tumor, and can be isolated without invasive methods. In the future, the real challenge lies in the development of specific, sensitive, fast and clinically applicable detection and analysis methods of epimarkers.

Drug Resistance in Glioblastoma: The Two Faces of Oxidative Stress.

Glioblastomas (GBM) are the most common primary brain tumor with a median survival of 15 months. A population of cells with stem cell properties (glioblastoma stem cells, GSCs) drives the initiation and progression of GBM and is localized in specialized microenvironments which support their behavior. GBM are characterized as extremely resistant to therapy, resulting in tumor recurrence. Reactive oxygen species (ROS) control the cellular stability by influencing different signaling pathways. Normally, redox systems prevent cell oxidative damage; however, in gliomagenesis, the cellular redox mechanisms are highly impaired. Herein we review the dual nature of the redox status in drug resistance. ROS generation in tumor cells affects the cell cycle and is involved in tumor progression and drug resistance in GBM. However, excess ROS production has been found to induce cell death programs such as apoptosis and autophagy. Since GBM cells have a high metabolic rate and produce high levels of ROS, metabolic adaptation in these cells plays an essential role in resistance to oxidative stress-induced cell death. Finally, the microenvironment with the stromal components participates in the enhancement of the oxidative stress to promote tumor progression and drug resistance.

Drug resistance in glioblastoma: are persisters the key to therapy?

Glioblastoma (GBM) represents the main form of brain tumors in adults, and one of the most aggressive cancers overall. The treatment of GBM is a combination of surgery (when possible), chemotherapy (usually Temozolomide, TMZ) and radiotherapy (RT). However, despite this heavy treatment, GBM invariably recur and the median length of survival following diagnosis is 12 to 15 months, with less than 10% of people surviving longer than five years. GBM is extremely resistant to most treatments because of its heterogeneous nature, which is associated with extreme clonal plasticity and the presence of cancer stem cells, refractory to TMZ- and RT-induced cell death. In this review, we explore the mechanisms by which cancer cells, and especially GBM, can acquire resistance to treatment. We describe and discuss the concept of persister/tolerant cells that precede and/or accompany the acquisition of resistance. Persister/tolerant cells are cancer cells that are not eliminated by treatment(s) because of different mechanisms ranging from dormancy/quiescence to senescence. We discuss the possibility of targeting these mechanisms in new therapeutic regimen.

Diuron exposure and Akt overexpression promote glioma formation through DNA hypomethylation.

BACKGROUND: Diuron is an environmental component listed as a likely human carcinogen. Several other studies report that diuron can be oncogenic for bladder, urothelial, skin, and mammary cells. No study mentions the putative effect of diuron on the glioma occurrence. OBJECTIVES: We here wanted to investigate the effects of diuron exposure on the glioma occurrence while wishing to incriminate a putative implication of DNA methylation modulation in this process. METHODS: In in vivo model of glioma, diuron exposure was firstly compared or combined with oncogenic overexpressions already known to promote gliomagenesis. ELISA quantifying the 5-methylcytosine level on DNA was performed to examine the global DNA methylation level. Quantitative real-time polymerase chain reaction and proximity ligation in situ assay were performed to identify the molecular causes of the diuron-induced changes of DNA methylation. The signatures diuron-induced changes of DNA methylation were analyzed in a cohort of 23 GBM patients. RESULTS: Diuron exposure is not sufficient to promote glioma, such as the oncogenic overexpression of Akt or Ras. However, the combination of diuron exposure and Akt overexpression promotes glioma. We observed that the diuron/Akt-induced glioma is characterized by three phenotypic signatures characterizing cancer cells: a global DNA hypomethylation, a loss of sensitivity to cell death induction, and a gain of signals of immune escape. Our data associated these phenotypes with three aberrant DNA methylation signatures: the LLT1, PD-L1, and Bcl-w hypomethylations. Strikingly, we observed that these three concomitant hypomethylations were only observed in GBM patients having a potential exposure to diuron via their professional activity. CONCLUSIONS: As single player, diuron is not an oncogenic of glioma, but it can participate to the glioma formation in association with other events (also devoid of oncogenic property as single player) such as Akt overexpression.