Extracellular vesicles derived from dendritic cells loaded with VEGF-A siRNA and doxorubicin reduce glioma angiogenesis in vitro.

BACKGROUND: Numerous attempts have been devoted to designing anti-angiogenic agents as a strategy to slow tumor growth and progression. Clinical applications of conventional anti-angiogenic agents face some challenges, e.g., off-target effects for TKIs and also low solid tumor penetration for mAbs. Furthermore, although anti-angiogenic therapy provides a normalization window for better chemo-RT response, in long-term treatments, tumor hypoxia as a result of total removal of VEGF-A by mAbs from the TME or complete blockade of TK receptors induces over-activation of compensatory angiogenic pathways, causing escape. Herein, we investigate the efficacy of si-DOX-DC-EVs to reduce glioma angiogenesis and invasiveness. METHODS: Mature DCs were generated from PBMC and EVs were isolated from the DCs culture media. siRNA and Doxorubicin were loaded into EVs by EP and incubation. Afterward, the uptake of DC-EVs was assessed by flow cytometry, and the subcellular localization of EVs was tested by confocal imaging. Tube formation assay was performed to assess the efficacy of si-DOX-DC-EVs to reduce tumor angiogenesis which was analyzed by DHM. Morphometric analysis of apoptotic cells was performed by DHM and confocal imaging and further, ELISA was performed for hypoxia-related and angiogenic cytokines. The impact of our theranostic system "si-DOX-DC-MVs" on the formation of vascular mimics, colonies, and invasion of C6 cells was checked in vitro. Afterward, orthotropic rat models of glioma were generated and the optimal administration route was selected by in vivo fluorescent analysis. Then, the microvessel density, vimentin expression, and accumulation of immune cells in tumoral tissues were assessed by IHC. Finally, necropsy and autopsy analyses were performed to check the safety of our theranostic agent. RESULTS: DC-EVs loaded with si-DOX-DC-EVs were successfully uptaken by cells with different subcellular trafficking for MVs and exosomes, reduced tumor angiogenesis in DHM analysis, and induced apoptosis in tumoral cells. Moreover, using DHM, we performed a detailed label-free analysis of tip cells which suggested that the tip cells in si-DC-MV treatments lost their geometrical migration capacity to form tube-like structures. Furthermore, the ELISAs performed highlighted that there is a mild overactivation of compensatory Tie2/Ang2 pathway after VEGF-A blockade which confers with severe hypoxia and sustains normal angiogenesis which is the optimal goal of anti-angiogenesis therapy for cancer to avoid resistance.The results of our VM analyses indicated that si-DOX-DC-MVs completely inhibited VM process. Moreover, the invasion, migration, and colony formation of the C6 cells treated with si-DOX-MVs were the least among all treatments. IN was the optimal route of administration. The MVD analyses indicated that si-DOX-DC-MVs reduced the number of tumoral microvessels and normalized vessel morphology. Intense CD8+ T cells were observed near the tumoral vessels in the si-DOX-DC-MVs group and with minimal activation of MT (low Vimentin expression). Necropsy and toxicology results proved that the theranostic system proposed is safe. CONCLUSIONS: DC-EVs loaded with VEGF-A siRNA and Doxorubicin were more potent than BV alone as a multi-disciplinary strategy that combats glioma growth by cytotoxic impacts of DOX and inhibits angiogenesis by VEGF-A siRNAs with excess immunologic benefits from DC-EVs. This next-generation anti-angiogenic agent normalizes tumor vessel density rather than extensively eliminating tumor vessels causing hypoxia and mesenchymal transition.

A dynamic study of VEGF-A siDOX-EVs trafficking through the in-vitro insert co-culture blood-brain barrier model by digital holographic microscopy.

Introduction: Modeling the blood-brain barrier has long been a challenge for pharmacological studies. Up to the present, numerous attempts have been devoted to recapitulating the endothelial barrier in vitro to assess drug delivery vehicles' efficiency for brain disorders. In the current work, we presented a new approach for analyzing the morphometric parameters of the cells of an insert co-culture blood-brain barrier model using rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes. This analytical approach could aid in getting further information on drug trafficking through the blood-brain barrier and its impact on the brain indirectly. Methods: In the current work, we cultured rat brain astrocytes, rat brain microvascular endothelial cells, and rat brain pericytes and then used an insert well to culture the cells in contact with each other to model the blood-brain barrier. Then, the morphometric parameters of the porous membrane of the insert well, as well as each cell type were imaged by digital holographic microscopy before and after cell seeding. At last, we performed folate conjugation on the surface of the EVs we have previously tested for glioma therapy in our previous work called VEGF-A siDOX-EVs and checked how the trafficking of EVs improves after folate conjugation as a clathrin-mediated delivery setup. the trafficking and passage of EVs were assessed by flow cytometry and morphometric analysis of the digital holographic microscopy holograms. Results: Our results indicated that EVs successfully entered through the proposed endothelial barrier assessed by flow cytometry analysis and furthermore, folate conjugation significantly improved EV passage through the blood-brain barrier. Moreover, our results indicated that the VEGF-A siDOX-EVs insert cytotoxic impact on the cells of the bottom of the culture plate. Conclusion: folate-conjugation on the surface of EVs improves their trafficking through the blood-brain barrier and by using digital holographic microscopy analysis, we could directly assess the morphometric changes of the blood-brain barrier cells for pharmacological purposes as an easy, label-free, and real-time analysis.

Efficacy of cell-based immunotherapies on patients with glioma: an umbrella review of systematic reviews and meta-analysis protocol.

INTRODUCTION: Glial brain tumours are highly mortal and are noted as major neurosurgical challenges due to frequent recurrence or progression. Despite standard-of-care treatment for gliomas, the prognosis of patients with higher-grade glial tumours is still poor, and hence empowering antitumour immunity against glioma is a potential future oncological prospect. This review is designed to improve our understanding of the efficacy of cell-based immunotherapies for glioma. METHODS AND ANALYSIS: This systematic review will be performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search of main electronic databases: PubMed/MEDLINE, Scopus, ISI Web of Science EMBASE and ProQuest will be done on original articles, followed by a manual review of review articles. Only records in English and only clinical trials will be encountered for full-text review. All the appropriate studies that encountered the inclusion criteria will be screened, selected and then will undergo data extraction step by two independent authors. For meta-analyses, data heterogeneity for each parameter will be first evaluated by Cochran's Q and I2 statistics. In case of possible heterogeneity, a random-effects meta-analysis will be performed and for homogenous data, fixed-effects models will be selected for reporting the results of the proportional meta-analysis. Bias risk will be assessed through Begg's and Egger's tests and will also be visualised by Funnel plots. ETHICS AND DISSEMINATION: As this study will be a systematic review without human participants' involvement, no ethical registration is required and meta-analysis will be presented at a peer-reviewed journal. PROSPERO REGISTRATION NUMBER: CRD42022373297.