Overcoming Barriers in Glioblastoma-Advances in Drug Delivery Strategies.

The world of cancer treatment is evolving rapidly and has improved the prospects of many cancer patients. Yet, there are still many cancers where treatment prospects have not (or hardly) improved. Glioblastoma is the most common malignant primary brain tumor, and even though it is sensitive to many chemotherapeutics when tested under laboratory conditions, its clinical prospects are still very poor. The blood-brain barrier (BBB) is considered at least partly responsible for the high failure rate of many promising treatment strategies. We describe the workings of the BBB during healthy conditions and within the glioblastoma environment. How the BBB acts as a barrier for therapeutic options is described as well as various approaches developed and tested for passing or opening the BBB, with the ultimate aim to allow access to brain tumors and improve patient perspectives.

CRISPR-Cas knockout of miR21 reduces glioma growth.

Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.

Comparative Analysis Identifies Similarities between the Human and Murine Microglial Sensomes.

One of the essential functions of microglia is to continuously sense changes in their environment and adapt to those changes. For this purpose, they use a set of genes termed the sensome. This sensome is comprised of the most abundantly expressed receptors on the surface of microglia. In this study, we updated previously identified mouse microglial sensome by incorporating an additional published RNAseq dataset into the data-analysis pipeline. We also identified members of the human microglial sensome using two independent human microglia RNAseq data sources. Using both the mouse and human microglia sensomes, we identified a key set of genes conserved between the mouse and human microglial sensomes as well as some differences between the species. We found a key set of 57 genes to be conserved in both mouse and human microglial sensomes. We define these genes as the "microglia core sensome". We then analyzed expression of genes in this core sensome in five different datasets from two neurodegenerative disease models at various stages of the diseases and found that, overall, changes in the level of expression of microglial sensome genes are specific to the disease or condition studied. Our results highlight the relevance of data generated in mice for understanding the biology of human microglia, but also stress the importance of species-specific gene sets for the investigation of diseases involving microglia. Defining this microglial specific core sensome may help identify pathological changes in microglia in humans and mouse models of human disease.

Extracellular Vesicle-Mediated Bilateral Communication between Glioblastoma and Astrocytes.

Glioblastoma the most aggressive form of brain cancer, comprises a complex mixture of tumor cells and nonmalignant stromal cells, including neurons, astrocytes, microglia, infiltrating monocytes/macrophages, lymphocytes, and other cell types. All nonmalignant cells within and surrounding the tumor are affected by the presence of glioblastoma. Astrocytes use multiple modes of communication to interact with neighboring cells. Extracellular vesicle-directed intercellular communication has been found to be an important component of signaling between astrocytes and glioblastoma in tumor progression. In this review, we focus on recent findings on extracellular vesicle-mediated bilateral crosstalk, between glioblastoma cells and astrocytes, highlighting the protumor and antitumor roles of astrocytes in glioblastoma development.

Versatile Role of Rab27a in Glioma: Effects on Release of Extracellular Vesicles, Cell Viability, and Tumor Progression.

Introduction: Glioma cells exert influence over the tumor-microenvironment in part through the release of extracellular vesicles (EVs), membrane-enclosed structures containing proteins, lipids, and RNAs. In this study, we evaluated the function of Ras-associated protein 27a (Rab27a) in glioma and evaluated the feasibility of assessing its role in EV release in glioma cells in vitro and in vivo. Methods: Rab27a was knocked down via a short hairpin RNA (shRNA) stably expressed in mouse glioma cell line GL261, with a scrambled shRNA as control. EVs were isolated by ultracentrifugation and quantified with Nanoparticle Tracking Analysis (NTA) and Tunable Resistive Pulse Sensing (TRPS). CellTiter-Glo viability assays and cytokine arrays were used to evaluate the impact of Rab27a knockdown. GL261.shRab27a cells and GL261.shControl were implanted into the left striatum of eight mice to assess tumor growth and changes in the tumor microenvironment. Results: Knockdown of Rab27a in GL261 glioma cells decreased the release of small EVs isolated at 100,000 × g in vitro (p = 0.005), but not the release of larger EVs, isolated at 10,000 × g. GL261.shRab27a cells were less viable compared to the scramble control in vitro (p < 0.005). A significant increase in CCL2 expression in shRab27a GL261 cells was also observed (p < 0.001). However, in vivo there was no difference in tumor growth or overall survival between the two groups, while shRab27a tumors showed lower proliferation at the tumor borders. Decreased infiltration of IBA1 positive macrophages and microglia, but not FoxP3 positive regulatory T cells was observed. Conclusion: Rab27a plays an important role in the release of small EVs from glioma cells, and also in their viability and expression of CCL2 in vitro. As interference in Rab27a expression influences glioma cell viability and expression profiles, future studies should be cautious in using the knockdown of Rab27a as a means of studying the role of small EVs in glioma growth.

Linking indirect effects of cytomegalovirus in transplantation to modulation of monocyte innate immune function.

Cytomegalovirus (CMV) is an important cause of morbidity and mortality in the immunocompromised host. In transplant recipients, a variety of clinically important "indirect effects" are attributed to immune modulation by CMV, including increased mortality from fungal disease, allograft dysfunction and rejection in solid organ transplantation, and graft-versus-host-disease in stem cell transplantation. Monocytes, key cellular targets of CMV, are permissive to primary, latent and reactivated CMV infection. Here, pairing unbiased bulk and single cell transcriptomics with functional analyses we demonstrate that human monocytes infected with CMV do not effectively phagocytose fungal pathogens, a functional deficit which occurs with decreased expression of fungal recognition receptors. Simultaneously, CMV-infected monocytes upregulate antiviral, pro-inflammatory chemokine, and inflammasome responses associated with allograft rejection and graft-versus-host disease. Our study demonstrates that CMV modulates both immunosuppressive and immunostimulatory monocyte phenotypes, explaining in part, its paradoxical "indirect effects" in transplantation. These data could provide innate immune targets for the stratification and treatment of CMV disease.

GlioM&M: Web-based tool for studying circulating and infiltrating monocytes and macrophages in glioma.

Monocytes, macrophages and microglia make up a large part of the glioma environment and have an important role in maintaining and propagating glioma progression. Targeting these cells to inhibit their tumor-promoting effect and reprogramming them into an anti-tumor phenotype is a potential therapeutic approach for glioma. In this study we analyzed the transcriptomes of eight different monocyte subgroups derived from the brain and the blood of glioma-bearing mice. We compared the expression profile of blood-derived monocytes versus tumor-infiltrating monocytes and found increased expression of both pro- and anti-inflammatory pathways in tumor infiltrating monocytes. To help disseminate these datasets, we created a user-friendly web-based tool accessible at www.glioma-monocytes.com. This tool can be used for validation purposes and to elucidate gene expression profiles of tumor-interacting monocytes and macrophages as well as blood-derived circulating monocytes. This tool can also be used to identify new markers and targets for therapy in these different cell populations.

Glioblastoma hijacks microglial gene expression to support tumor growth.

BACKGROUND: Glioblastomas are the most common and lethal primary brain tumors. Microglia, the resident immune cells of the brain, survey their environment and respond to pathogens, toxins, and tumors. Glioblastoma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Despite the presence of large numbers of microglia in glioblastoma, the tumors continue to grow, and these neuroimmune cells appear incapable of keeping the tumor in check. To understand this process, we analyzed gene expression in microglia interacting with glioblastoma cells. METHODS: We used RNASeq of isolated microglia to analyze the expression patterns of genes involved in key microglial functions in mice with glioblastoma. We focused on microglia that had taken up tumor-derived EVs and therefore were within and immediately adjacent to the tumor. RESULTS: We show that these microglia have downregulated expression of genes involved in sensing tumor cells and tumor-derived danger signals, as well as genes used for tumor killing. In contrast, expression of genes involved in facilitating tumor spread was upregulated. These changes appear to be in part EV-mediated, since intracranial injection of EVs in normal mice led to similar transcriptional changes in microglia. We observed a similar microglial transcriptomic signature when we analyzed datasets from human patients with glioblastoma. CONCLUSION: Our data define a microgliaGlioblastoma specific phenotype, whereby glioblastomas have hijacked gene expression in the neuroimmune system to favor avoiding tumor sensing, suppressing the immune response, clearing a path for invasion, and enhancing tumor propagation. For further exploration, we developed an interactive online tool at http://www.glioma-microglia.com with all expression data and additional functional and pathway information for each gene.

Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21.

Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression.

Glioma EVs Contribute to Immune Privilege in the Brain.

Glioblastoma cells release extracellular vesicles (EVs), sometimes referred to as microvesicles and exosomes, to transfer immune modulating molecules to immune cells, resulting in an immune privileged microenvironment. Here we discuss the potential EV-mediated mechanisms underlying glioma immune modulation, as well as the technical difficulties in studying these interactions.

Multidimensional communication in the microenvirons of glioblastoma.

Glioblastomas are heterogeneous and invariably lethal tumours. They are characterized by genetic and epigenetic variations among tumour cells, which makes the development of therapies that eradicate all tumour cells challenging and currently impossible. An important component of glioblastoma growth is communication with and manipulation of other cells in the brain environs, which supports tumour progression and resistance to therapy. Glioblastoma cells recruit innate immune cells and change their phenotype to support tumour growth. Tumour cells also suppress adaptive immune responses, and our increasing understanding of how T cells access the brain and how the tumour thwarts the immune response offers new strategies for mobilizing an antitumour response. Tumours also subvert normal brain cells - including endothelial cells, neurons and astrocytes - to create a microenviron that favours tumour success. Overall, after glioblastoma-induced phenotypic modifications, normal cells cooperate with tumour cells to promote tumour proliferation, invasion of the brain, immune suppression and angiogenesis. This glioblastoma takeover of the brain involves multiple modes of communication, including soluble factors such as chemokines and cytokines, direct cell-cell contact, extracellular vesicles (including exosomes and microvesicles) and connecting nanotubes and microtubes. Understanding these multidimensional communications between the tumour and the cells in its environs could open new avenues for therapy.

Preventing inflammation inhibits biopsy-mediated changes in tumor cell behavior.

Although biopsies and tumor resection are prognostically beneficial for glioblastomas (GBM), potential negative effects have also been suggested. Here, using retrospective study of patients and intravital imaging of mice, we identify some of these negative aspects, including stimulation of proliferation and migration of non-resected tumor cells, and provide a strategy to prevent these adverse effects. By repeated high-resolution intravital microscopy, we show that biopsy-like injury in GBM induces migration and proliferation of tumor cells through chemokine (C-C motif) ligand 2 (CCL-2)-dependent recruitment of macrophages. Blocking macrophage recruitment or administrating dexamethasone, a commonly used glucocorticoid to prevent brain edema in GBM patients, suppressed the observed inflammatory response and subsequent tumor growth upon biopsy both in mice and in multifocal GBM patients. Taken together, our study suggests that inhibiting CCL-2-dependent recruitment of macrophages may further increase the clinical benefits from surgical and biopsy procedures.

Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake.

Extracellular vesicles are a heterogeneous group of membrane-limited vesicles loaded with various proteins, lipids, and nucleic acids. Release of extracellular vesicles from its cell of origin occurs either through the outward budding of the plasma membrane or through the inward budding of the endosomal membrane, resulting in the formation of multivesicular bodies, which release vesicles upon fusion with the plasma membrane. The release of vesicles can facilitate intercellular communication by contact with or by internalization of contents, either by fusion with the plasma membrane or by endocytosis into "recipient" cells. Although the interest in extracellular vesicle research is increasing, there are still no real standards in place to separate or classify the different types of vesicles. This review provides an introduction into this expanding and complex field of research focusing on the biogenesis, nucleic acid cargo loading, content, release, and uptake of extracellular vesicles.

Potential Transfer of Polyglutamine and CAG-Repeat RNA in Extracellular Vesicles in Huntington's Disease: Background and Evaluation in Cell Culture.

In Huntington's disease (HD) the imperfect expanded CAG repeat in the first exon of the HTT gene leads to the generation of a polyglutamine (polyQ) protein, which has some neuronal toxicity, potentially mollified by formation of aggregates. Accumulated research, reviewed here, implicates both the polyQ protein and the expanded repeat RNA in causing toxicity leading to neurodegeneration in HD. Different theories have emerged as to how the neurodegeneration spreads throughout the brain, with one possibility being the transport of toxic protein and RNA in extracellular vesicles (EVs). Most cell types in the brain release EVs and these have been shown to contain neurodegenerative proteins in the case of prion protein and amyloid-beta peptide. In this study, we used a model culture system with an overexpression of HTT-exon 1 polyQ-GFP constructs in human 293T cells and found that the EVs did incorporate both the polyQ-GFP protein and expanded repeat RNA. Striatal mouse neural cells were able to take up these EVs with a consequent increase in the green fluorescent protein (GFP) and polyQ-GFP RNAs, but with no evidence of uptake of polyQ-GFP protein or any apparent toxicity, at least over a relatively short period of exposure. A differentiated striatal cell line expressing endogenous levels of Hdh mRNA containing the expanded repeat incorporated more of this mRNA into EVs as compared to similar cells expressing this mRNA with a normal repeat length. These findings support the potential of EVs to deliver toxic expanded trinucleotide repeat RNAs from one cell to another, but further work will be needed to evaluate potential EV and cell-type specificity of transfer and effects of long-term exposure. It seems likely that expanded HD-associated repeat RNA may appear in biofluids and may have use as biomarkers of disease state and response to therapy.

Directly visualized glioblastoma-derived extracellular vesicles transfer RNA to microglia/macrophages in the brain.

BACKGROUND: To understand the ability of gliomas to manipulate their microenvironment, we visualized the transfer of vesicles and the effects of tumor-released extracellular RNA on the phenotype of microglia in culture and in vivo. METHODS: Extracellular vesicles (EVs) released from primary human glioblastoma (GBM) cells were isolated and microRNAs (miRNAs) were analyzed. Primary mouse microglia were exposed to GBM-EVs, and their uptake and effect on proliferation and levels of specific miRNAs, mRNAs, and proteins were analyzed. For in vivo analysis, mouse glioma cells were implanted in the brains of mice, and EV release and uptake by microglia and monocytes/macrophages were monitored by intravital 2-photon microscopy, immunohistochemistry, and fluorescence activated cell sorting analysis, as well as RNA and protein levels. RESULTS: Microglia avidly took up GBM-EVs, leading to increased proliferation and shifting of their cytokine profile toward immune suppression. High levels of miR-451/miR-21 in GBM-EVs were transferred to microglia with a decrease in the miR-451/miR-21 target c-Myc mRNA. In in vivo analysis, we directly visualized release of EVs from glioma cells and their uptake by microglia and monocytes/macrophages in brain. Dissociated microglia and monocytes/macrophages from tumor-bearing brains revealed increased levels of miR-21 and reduced levels of c-Myc mRNA. CONCLUSIONS: Intravital microscopy confirms the release of EVs from gliomas and their uptake into microglia and monocytes/macrophages within the brain. Our studies also support functional effects of GBM-released EVs following uptake into microglia, associated in part with increased miRNA levels, decreased target mRNAs, and encoded proteins, presumably as a means for the tumor to manipulate its environs.