Heparin interferes with the uptake of liposomes in glioma.

In glioblastoma, a malignant primary brain tumor, liposomes have shown promise in pre-clinical and early phase clinical trials as delivery vehicles for therapeutics. However, external factors influencing cellular uptake of liposomes in glioma cells are poorly understood. Heparin and heparin analogues are commonly used in glioma patients to decrease the risk of thrombo-embolic events. Our results show that heparin inhibits pegylated liposome uptake by U87 glioma and GL261 cells in a dose dependent manner in vitro, and that heparin-mediated inhibition of uptake required presence of fetal bovine serum in the media. In a subcutaneous model of glioma, Cy5.5 labeled liposomes could be detected with in vivo imaging after direct intra-tumoral injection. Ex-vivo analysis with flow cytometry showed a decreased uptake of liposomes into tumor cells in mice treated systemically with heparin compared to those treated with vehicle only.

Extracellular communication between brain cells through functional transfer of Cre mRNA mediated by extracellular vesicles.

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo over time. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain, exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEV transfer at physiological levels, which will shed light on the role of bdEVs in neural communication within the brain and beyond.

Illuminating cellular and extracellular vesicle-mediated communication via a split-Nanoluc reporter in vitro and in vivo.

Tools to effectively demonstrate and quantify functional delivery in cellular communication have been lacking. This study reports the use of a fluorescently labeled split Nanoluc reporter system to demonstrate and quantify functional transfer between cells in vitro and in a subcutaneous tumor mouse model. Our construct allows monitoring of direct, indirect, and specifically extracellular vesicle-mediated functional communication.

Extracellular communication between brain cells through functional transfer of Cre mRNA.

In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

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.

Advances in local therapy for glioblastoma - taking the fight to the tumour.

Despite advances in neurosurgery, chemotherapy and radiotherapy, glioblastoma remains one of the most treatment-resistant CNS malignancies, and the tumour inevitably recurs. The majority of recurrences appear in or near the resection cavity, usually within the area that received the highest dose of radiation. Many new therapies focus on combatting these local recurrences by implementing treatments directly in or near the tumour bed. In this Review, we discuss the latest developments in local therapy for glioblastoma, focusing on recent preclinical and clinical trials. The approaches that we discuss include novel intraoperative techniques, various treatments of the surgical cavity, stereotactic injections directly into the tumour, and new developments in convection-enhanced delivery and intra-arterial treatments.

Living Proof of Activity of Extracellular Vesicles in the Central Nervous System.

The central nervous system (CNS) consists of a heterogeneous population of cells with highly specialized functions. For optimal functioning of the CNS, in disease and in health, intricate communication between these cells is vital. One important mechanism of cellular communication is the release and uptake of extracellular vesicles (EVs). EVs are membrane enclosed particles actively released by cells, containing a wide array of proteins, lipids, RNA, and DNA. These EVs can be taken up by neighboring or distant cells, and influence a wide range of processes. Due to the complexity and relative inaccessibility of the CNS, our current understanding of the role of EVs is mainly derived in vitro work. However, recently new methods and techniques have opened the ability to study the role of EVs in the CNS in vivo. In this review, we discuss the current developments in our understanding of the role of EVs in the CNS in vivo.

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.

Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients.

Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

Fibrinolytics and Intraventricular Hemorrhage: A Systematic Review and Meta-analysis.

Intraventricular hemorrhage (IVH) is an independent poor prognostic factor in subarachnoid and intra-parenchymal hemorrhage. The use of intraventricular fibrinolytics (IVF) has long been debated, and its exact effects on outcomes are unknown. A systematic review and meta-analysis were performed in accordance with the PRISMA guidelines to assess the impact of IVF after non-traumatic IVH on mortality, functional outcome, intracranial bleeding, ventriculitis, time until clearance of third and fourth ventricles, obstruction of external ventricular drains (EVD), and shunt dependency. Nineteen studies were included in the meta-analysis, totaling 1020 patients. IVF was associated with lower mortality (relative risk [RR] 0.58; 95% confidence interval [CI] 0.47-0.72), fewer EVD obstructions (RR 0.41; 95% CI 0.22-0.74), and a shorter time until clearance of the ventricles (median difference [MD] - 4.05 days; 95% CI - 5.52 to - 2.57). There was no difference in good functional outcome, RR 1.41 (95% CI 0.98-2.03), or shunt dependency, RR 0.93 (95% CI 0.70-1.22). Correction for publication bias predicted an increased risk of intracranial bleeding, RR 1.67 (95% CI 1.01-2.74) and a lower risk of ventriculitis, RR 0.68 (95% CI 0.45-1.03) in IVH patients treated with IVF. IVF was associated with improved survival, faster clearance of blood from the ventricles and fewer drain obstructions, but further research is warranted to elucidate the effects on ventriculitis, long-term functional outcomes, and re-hemorrhage.

The interference of genetic associations in establishing the prognostic value of the immunophenotype in acute myeloid leukemia.

BACKGROUND: In acute myeloid leukemia controversy exists about the role of immunophenotyping of the blasts at diagnosis as a potential prognostic factor. METHODS: We retrospectively analyzed immunophenotypic marker expression on blasts in relation to genetic aberrancies and survival data of 684 patients. All patients were included in different studies from the HOVON/SAKK Consortium. RESULTS: Markers CD2, CD7, CD11b, CD19, CD22, and CD56 all appeared to be associated with one or more established prognostic genetic aberrancies. In the overall population, differences in univariate survival analyses were observed for CD2, CD11b, and CD22. After correcting these survival differences for currently used risk profile data, only CD11b expression remained of prognostic value for poor overall survival (OS) and event-free survival (EFS). CD11b expression turned out to be an independent factor for poor OS and EFS in the subgroup of patients who lacked cytogenetic and molecular aberrancies. CONCLUSIONS: In this study, we demonstrate that associations between immunophenotypic markers and genetic aberrancies interfere with the prognostic properties of immunophenotypic markers. Such may account for most of the previously reported prognostic impact of these markers. Only CD11b expression remained as an independent prognostic marker. © 2017 International Clinical Cytometry Society.