Gliomas are major neuro-oncological concerns due to high mortality and morbidity rates. Despite current neurosurgical advances for optimal tumor resection, the prognosis is still poor and many patients experience recurrence/progression. Hence, there is a rising interest to develop multimodal therapeutic strategies to combat glioma growth. In light of previous evidence, anti-angiogenesis therapy as an adjuvant for standard therapy of glioma (surgical resection and concurrent chemoradiation) has attracted much attention. Numerous monoclonal antibodies (mAbs) [[1], [2], [3]] and Tyrosine Kinase Inhibitors (TKIs) [[3], [4], [5], [6], [7], [8]] were tested to combat glioma aberrant angiogenesis; however, there are major challenges to the clinical efficacy of using mAbs due to low tumor penetration, large molecular size, increased interstitial fluid pressure, peritumoral vasogenic edema, and tumor heterogeneity in expression of the targeting antigens. The primary goal for anti-angiogenic agents is to normalize the tumor vasculature rather than to achieve complete inhibition. The normalized vasculature provides a window for more efficient chemotherapy delivered by normal endothelia to tumoral cells rather than leaky and haphazard vessels which are incompetent for delivering chemo and nutrients. However, prolonged high dose consumption of mAbs to pass the diseased Blood-Brain barrier (BBB) at pathologic conditions, i.e., tumorigenesis, may cause dose-limiting toxicities and also resistance to anti-angiogenic therapy due to over-activation of compensatory mechanisms induced by excessive central hypoxia in the TME [[9], [10], [11]]. Further, because tyrosine kinases are also expressed in normal tissues, high dose consumption of TKIs may increase their off-target effects in non-target organs. In the current work, we hypothesized that Extracellular Vesicles Derived from DCs loaded with VEGF-A siRNA and Doxorubicin may act as multi-potent glioma therapy and reduce glioma angiogenesis and invasiveness.

Furthermore, as noted earlier, anti-angiogenic therapy provides a window period in which the vessels are normalized and are more capable of delivering chemo-agents to tumoral cells and hence enhance the efficacy of chemotherapy. Based on previous evidence, Doxorubicin has long been considered to have synergic impacts, with TMZ the standard chemo-agent for gliomas [11]; however, a major concern which has hampered its potential advantages for gliomas is the BBB. There is strong literature on passage of exosomes through the BBB by various mechanisms, making them robust potential nano-carriers for brain diseases with intrinsic therapeutic cargos [12], and investigation of passage of exosomal Doxorubicin through the BBB is a future goal for this study. Herein, we aimed to investigate the synergic impact of Doxorubicin and VEGF-A siRNA on combating glioma cell growth and reducing tumor angiogenesis.

Furthermore, previous evidence suggests that dendritic cells (DCs) hold considerable potential as an adjuvant immunotherapy for high grade gliomas [12]. In recent years, the Extracellular Vesicles (EVs), namely, exosomes and microvesicles, have been the target for considerable interest not only as nano-sized vehicles (30–1000 nm) for drug delivery purposes, but also as modulators of intercellular signaling extensively used for regenerative and immunotherapy purposes. As a preclinical hypothesis, DC-derived Exosomes (DEXs) can act as perfect drug delivery systems which are more advantageous than synthetic nano-carriers due to their human derived nature, minimal recognition by the mononuclear phagocytic system (MPS), and their unique proteo-lipid structure which causes robust blood stability [[11], [12], [13], [14]]. Moreover, since many immune function-associated molecules of DCs are enriched in DEXs, they can maximize anti-tumor immune responses in the tumor immune microenvironment [12].

siRNAs, known as 20–25 base pairs double stranded non-coding RNAs, act as a post-transcriptional silencer of specific gene targets by assembly of the RNA-induced silencing complex (RISC) which causes cleavage of target mRNA molecules further degraded by cellular exonucleases. SiRNA delivery to cancer cells by synthetic nano-carriers has yielded promising results but still faces major hurdles to overcome, including inflammatory responses (e.g., allergic reaction), recognition of nano-carriers by innate and adaptive immune systems and entrapment in non-target organs (e.g., liver and spleen) [11]. VEGF-A knockdown by siRNAs has previously been of particular interest as a new strategy to combat tumor angiogenesis [13,14]. Previously, fibroblast and Mesenchymal Stem Cells (MSC)-Extracellular Vesicles (EVs) were noted as potent vehicles to deliver KRAS siRNA to pancreatic tumors successfully; this is currently being tested in clinical pipelines as a future therapeutic prospect for pancreatic cancer [15]. In the current work, we tested the in-vitro efficacy of VEGF-A siRNA-Doxorubicin-loaded Extracelluar Vesicle-derived Dendritic Cells (VEGF-A si-DOX-DEXs) as a multimodal agent to combat glioma growth and angiogenesis and invasiveness and also assessed the morphometric changes of tube-like structures treated with VEGF-A si-DOX-DEXs. We aimed to investigate the potential advantages of VEGF-A si-DOX-DEXs compared to BV, the conventional mAb used to target high-grade glioma angiogenesis as a multi-potent strategy. Fig. 1 summarizes the research schema.