Despite the multidisciplinary management in the treatment of glioblastomas, the average survival of GBM patients is still 15 months. In recent years, molecular biomarkers have gained more and more importance both in the diagnosis and therapy of glial tumors. At the same time, it has become clear that non neoplastic cells, which constitute about 30% of glioma mass, dramatically influence tumor growth, spread, and recurrence. This is the main reason why, in recent years, scientific research has been focused on understanding the function and the composition of tumor microenvironment and its role in gliomagenesis and recurrence. The aim of this review is to summarize the most recent discovery about resident microglia, tumor-associated macrophages, lymphocytes, and the role of extracellular vesicles and their bijective interaction with glioma cells. Moreover, we reported the most recent updates about new therapeutic strategies targeting immune system receptors and soluble factors. Understanding how glioma cells interact with non-neoplastic cells in tumor microenvironment is an essential step to comprehend mechanisms at the base of disease progression and to find new therapeutic strategies for GBM patients. However, no significant results have yet been obtained in studies targeting single molecules/pathways; considering the complex microenvironment, it is likely that only by using multiple therapeutic agents acting on multiple molecular targets can significant results be achieved.

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Systematic Review on Tumor Microenvironment in Glial Neoplasm: From Understanding Pathogenesis to Future Therapeutic Perspectives

Extracellular vesicles are secreted by a wide variety of cells, and their primary functions include intercellular communication, immune responses, human reproduction, and synaptic plasticity. Their molecular cargo reflects the physiological processes that their cells of origin are undergoing. Thus, many studies have suggested that extracellular vesicles could be a promising biomarker tool for many diseases, mainly due to their biological relevance and easy accessibility to a broad range of body fluids. Moreover, since their biological composition leads them to cross the blood-brain barrier bidirectionally, growing evidence points to extracellular vesicles as emerging mirrors of brain diseases processes. In this regard, this review explores the biogenesis and biological functions of extracellular vesicles, their role in different physiological and pathological processes, their potential in clinical practice, and the recent outstanding studies about the role of exosomes in major human brain diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), or brain tumors.

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Extracellular vesicles, the emerging mirrors of brain physiopathology

Drug delivery of extracellular vesicles: preparation, delivery strategies and applications.

Extracellular vesicles (EVs) are membrane-bound particles released by all cells. Previous research has found that these microscopic vesicles contribute to intercellular signaling and communication. EVs carry a variety of cargo, including nucleic acids, proteins, metabolites, and lipids. The composition of EVs varies based on cell of origin. Therefore, EVs can serve as an important biomarker in the diagnosis and treatment of various cancers. EVs derived from glioblastoma (GBM) cells carry biomarkers, which could serve as the basis for a potential diagnostic strategy known as liquid biopsy. Multiple EV isolation techniques exist, including ultrafiltration, size exclusion chromatography, flow field-flow fractionation, sequential filtration, differential ultracentrifugation, and density-gradient ultracentrifugation. Recent and ongoing work aims to identify cellular markers to distinguish GBM-derived EVs from those released by noncancerous cells. Strategies include proteomic analysis of GBM EVs, identification of GBM-specific metabolites, and use of Food and Drug Administration-approved 5-aminolevulinic acid—an oral agent that causes fluorescence of GBM cells—to recognize GBM EVs in a patient's blood. In addition, accurately and precisely monitoring changes in EV cargo concentrations could help differentiate between pseudoprogression and GBM recurrence, thus preventing unnecessary surgical interventions.

Advancements and Technical Considerations for Extracellular Vesicle Isolation and Biomarker Identification in Glioblastoma

Glioblastoma is simultaneously the most common and most aggressive primary brain tumor in the central nervous system, with poor patient survival and scarce treatment options. Most primary glioblastomas reoccur and evolve radio- and chemoresistant properties which make them resistant to further treatments. Based on gene mutations and expression profiles, glioblastoma is relatively well classified; however, research shows that there is more to glioblastoma biology than that defined solely by its genetic component. Specifically, the overall malignancy of the tumor is also influenced by the dynamic communication to its immediate and distant environment, as important messengers to neighboring cells in the tumor microenvironment extracellular vesicles (EVs) have been identified. EVs and their cargo can modulate the immune microenvironment and other physiological processes, and can interact with the host immune system. They are involved in tumor cell survival and metabolism, tumor initiation, progression, and therapy resistance. However, on the other hand EVs are thought to become an effective treatment alternative, since they can cross the blood–brain barrier, are able of specific cell-targeting and can be loaded with various therapeutic molecules.

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Dynamic Intercell Communication between Glioblastoma and Microenvironment through Extracellular Vesicles


 Introduction: Glioblastoma is resistant to standard treatment, leading to tumor recurrence and early death of patients. Using organoid models, we aim to explore the mechanisms of resistance to cancer therapies and identify novel therapeutic vulnerabilities, including stress protecting melanoma-associated antigens (MAGEs). MAGEs are normally restricted to expression in testis but are abnormally activated in cancer and are associated with therapy resistance.
 Methods: Organoids have been established from fresh tumor biopsies and are stored in Gliobank, a Slovenian collection of patient tumor samples with corresponding clinical data. Organoids were first characterized and compared with the corresponding parental tumor tissue by immunofluorescence and qPCR of selected markers. Organoids were then treated with a combination of irradiation, the chemotherapeutic agent temozolomide and/or a chemokine receptor antagonist, and response to therapy was assessed by immunofluorescence, qPCR, cell viability and invasion assays, multiplex ELISA and proximity ligation assay.
 Results: Organoids recapitulated the gene expression profile of parental glioblastoma tissues, including expression of genes related to cancer stem cells, DNA damage response, cell cycle progression, epithelial- to- mesenchymal transition, and cytokine signaling. In addition, the organoids preserved the cellular composition of the parental glioblastoma tissues, consisting of glioblastoma (stem) cells, macrophages, microglia, lymphocytes, and endothelial cells. Standard treatment decreased viability and invasion of organoids in only 2 of 6 patients, suggesting that organoids recapitulate tumor therapy resistance. The increased expression of MDM2 and CDKN1A in organoids after treatment with irradiation and temozolomide suggests that the p53 pathway and DNA damage response mechanisms may contribute to the therapy resistance. Next, we detected high abundance of secreted cytokines in the culture medium of the organoids, including CXCL12. However, treatment of the organoids with the CXCR4 antagonist Plerixafor, which blocked the interactions between CXCL12 and CXCR4 in the organoids, had no effect on organoid viability and invasion, suggesting resistance of glioblastoma to this immunotherapy. Since high expression of several type I MAGEs correlates with poor prognosis of glioma patients based on TCGA database analysis, we investigate the role of MAGEs in therapy resistance of glioblastoma organoids.
 Conclusions: Patient-derived tumor organoids provide a valuable tool to 1) identify novel therapeutic vulnerabilities in the context of the tumor microenvironment, 2) evaluate the effect of novel candidate treatments including immunotherapy, and 3) discover novel markers of therapy resistance.
 Citation Format: Barbara Breznik, Bernarda Majc, Anamarija Habič, Gloria Krapež, Andrej Porčnik, Jernej Mlakar, Tamara Lah Turnšek, Metka Novak, Klementina Fon Tacer. Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 170.

Abstract 170: Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens

Gloria Krapež

Papers

Dynamic Intercell Communication between Glioblastoma and Microenvironment through Extracellular Vesicles

Abstract 170: Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens