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

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. 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. 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. 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.