Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Background: Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Methods: Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. Results: We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Conclusion: Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Analysis of the Necrosis-Inducing Components of the Venom of Naja atra and Assessment of the Neutralization Ability of Freeze-Dried Antivenom.

Patients bitten by Naja atra who are treated with bivalent freeze-dried neurotoxic antivenom in Taiwan have an improved survival rate but develop necrotic wound changes. The World Health Organization (WHO) has suggested using the minimum necrotizing dose (MND) of venom as a method of evaluating the neutralization effect of antivenom. The aim of this study was to evaluate the effectiveness of antivenom for the prevention of necrosis based on the MND and clarify which component of the venom of N. atra induces necrosis. The neurotoxins (NTXs) were removed from the crude venom (deNTXs), and different concentrations of deNTXs were injected intradermally into the dorsal skin of mice. After three days, the necrotic lesion diameter was found to be approximately 5 mm, and the MND was calculated. A reduction in the necrotic diameter of 50% was used to identify the MND50. Furthermore, both phospholipase A2 (PLA2) and cytotoxins (CTXs) were separately removed from the deNTXs to identify the major necrosis-inducing factor, and the necrotic lesions were scored. All mice injected with deNTXs survived for three days and developed necrotic wounds. The MND of the deNTXs for mice was 0.494 ± 0.029 µg/g, that of the deNTXs-dePLA2 (major component retained: CTXs) was 0.294 ± 0.05 µg/g, and that of the deNTX-deCTX (major component retained: PLA2) venom was greater than 1.25 µg/g. These values show that CTX is the major factor inducing necrosis. These results suggest that the use of the deNTXs is necessary to enable the mice to survive long enough to develop venom-induced cytolytic effects. CTXs play a major role in N. atra-related necrosis. However, the MND50 could not be identified in this study, which meant that the antivenom did not neutralize venom-induced necrosis.

Carvedilol Ameliorates Experimental Atherosclerosis by Regulating Cholesterol Efflux and Exosome Functions.

Carvedilol (Cav), a nonselective ß-blocker with α1 adrenoceptor blocking effect, has been used as a standard therapy for coronary artery disease. This study investigated the effects of Cav on exosome expression and function, ATP-binding cassette transporter A1 (ABCA1) expression, and cholesterol efflux that are relevant to the process of atherosclerosis. Human monocytic (THP-1) cell line and human hepatic (Huh-7) cells were treated with Cav, and cholesterol efflux was measured. Exosomes from cell culture medium or mice serum were isolated using glycan-coated recognition beads. Low-density lipoprotein receptor knockout (ldlr-/-) mice were fed with high-fat diet and treated with Cav. Cav accentuated cholesterol efflux and enhanced the expressions of ABCA1 protein and mRNA in both THP-1 and Huh-7 cells. In addition, Cav increased expression and function of exosomal ABCA1 in THP-1 macrophage exosomes. The mechanisms were associated with inhibition of nuclear factor-κB (NF-κB) and protein kinase B (Akt). In hypercholesterolemic ldlr-/- mice, Cav enhanced serum exosomal ABCA1 expression and suppressed atherosclerosis by inhibiting lipid deposition and macrophage accumulation. Cav halts atherosclerosis by enhancing cholesterol efflux and increasing ABCA1 expression in macrophages and in exosomes, possibly through NF-κB and Akt signaling, which provides mechanistic insights regarding the beneficial effects of Cav on atherosclerotic cardiovascular disease.

Nanoimaging granule dynamics and subcellular structures in activated mast cells using soft X-ray tomography.

Mast cells play an important role in allergic responses. During activation, these cells undergo degranulation, a process by which various kinds of mediators stored in the granules are released. Granule homeostasis in mast cells has mainly been studied by electron microscopy (EM), where the fine structures of subcellular organelles are partially destroyed during sample preparation. Migration and fusion of granules have not been studied in detail in three dimensions (3D) in unmodified samples. Here, we utilized soft X-ray tomography (SXT) coupled with fluorescence microscopy to study the detailed structures of organelles during mast cell activation. We observed granule fission, granule fusion to plasma membranes, and small vesicles budding from granules. We also detected lipid droplets, which became larger and more numerous as mast cells were activated. We observed dramatic morphological changes of mitochondria in activated mast cells and 3D-reconstruction revealed the highly folded cristae inner membrane, features of functionally active mitochondria. We also observed giant vesicles containing granules, mitochondria, and lipid droplets, which we designated as granule-containing vesicles (GCVs) and verified their presence by EM in samples prepared by cryo-substitution, albeit with a less clear morphology. Thus, our studies using SXT provide significant insights into mast cell activation at the organelle level.