**4. Discussion**

Despite advances in therapeutics development over the past decade, GBM remains challenging to treat due to its heterogeneity and malignant nature. The tumor microenvironment plays a crucial role in promoting GBM tumorigenesis. GAMs have been shown to be one of the key players in the GBM microenvironment. We first demonstrated that clinical samples of GAMs promoted GBM tumorigenesis. For instance, U87MG and LN18 GBM cells cocultured with clinical M2 GAMs showed increased colony-forming and tumor-sphere-generating abilities in association with increased stemness markers Sox2, STAT3, Wnt, and Nestin in the GBM cells. Accumulating evidence has supported the observations where GAMs induced epithelial–mesenchymal transition (EMT) in GBM cells and subsequently generated properties of GSCs [19]. In addition, our observations were in agreement with previous studies, where interactions between GBM and GAMs increased CD133+ GSCs and malignant phenotypes [20,21]. GAM-mediated GBM-promoting effects were through different communicating molecules such as M2 cytokines (IL-6, VEGF, and TGF-β1) [2]. Here, we showed that the presence of GAMs promoted GBM tumorigenesis and stemness not only via the cytokines but also through the aid of exosomes. More specifically, we found that GBM cells incubated with exosomes derived from GAMs exhibited enhanced ability in colony and tumor sphere formation; more importantly, exosome-incubated GBM cells became more resistant against TMZ. Emerging evidence indicates the functional roles of exosomes in GBM tumorigenesis. A recent study showed that exosomes secreted from GBM cells promoted the oncogenic transformation of astrocytes in the tumor microenvironment [22]. This observation complements the results of our study, which demonstrated intimate communication between the tumor microenvironment and tumor cells via the exchange of exosomes.

We performed an array analysis on the exosomes secreted by GAMs and found that the most abundant microRNA species was miR-21. Notably, a recent review points out that miR-21 plays a pivotal role in GBM pathogenesis, where miR-21 functions through the modulation of the insulin-like-growth-factor-associated signaling pathway, RECK, and TIMP3 to promote GBM tumorigenesis [23]. Our results provided an added feature of miR-21 in GBM tumorigenesis, where miR-21 was enriched in the exosomes secreted by GAMs. It is very plausible that GAM-derived miR-21-enriched exosomes were incorporated into GBM cells and executed their tumor-promoting functions. It has been well demonstrated that the transfer and uptake of exosomes between donor and recipient cells represents one of the major routes for intercellular communications in many diseases, including cancer [24]. We provided support that increased miR-21-5p in GBM cells by miR-21-5p mimic molecules resulted in similar tumorigenic and stemness properties in GBM cells cocultured with GAM-derived exosomes; the reduction of miR-21-5p significantly reduced the tumorigenic properties in both GBM cell lines. Furthermore, GBM cells transfected with miR-21-5p inhibitor showed a significantly reduced ability to generate M2 GAMs, based on our coculture experiments; this was attributed to the decreased secretion of M2 cytokines such as IL-6 and VEGF by miR-21-5p-silenced GBM cells and an increased secretion of TNF-α, an M1 marker. More importantly, we provided evidence that miR-21-5p targets PDCD4, a tumor suppressor in both GBM cell lines. PDCD4 has been shown to be frequently suppressed in GBM cells and is associated with poor prognosis [25,26]. In agreement with our results, a previous study also demonstrated that PDCD4 was targeted by miR-21 in GBM [27].

According to our experimental results, miR-416a ranks as the second-most abundant microRNA species in the GAM-secreted exosomes. It has been shown that miR-416a plays a key role in the progression of malignant melanoma via the activation of notch signaling [28]. The activation of notch signaling has also been shown to be responsible for the generation of GSCs [29,30]. The fact that miR-21 and miR-416a, two powerful oncogenic microRNA molecules, were enriched in the GAM exosomes further supports our notion that GAMs play a key contributing role in GBM malignancy and should be targeted in treatment design. Currently, the role of exosomal miR-416a in GBM tumorigenesis is under intense investigation in our laboratory.

Since targeting microRNA for therapeutic purposes still remains challenging, miR-21-5p represents a potential therapeutic target. Thus, we evaluated the feasibility of using a small-molecule agent which may indirectly increase the miR-21 level to convey therapeutic functions in GBM. STAT3 signaling has been shown to be important in GBM tumorigenesis as well as linked to the expression of miR-21 [17,31,32]. Based on these premises, we evaluated pacritinib, a recent FDA-approved inhibitor of STAT3/JAK2 signaling for treating myelofibrosis [33,34]. We found pacritinib treatment suppressed cell viability and colony/tumor sphere formation in association with decreased expression of STAT3, Sox2, PDCD4, and miR-21-5p and increased GFAP expression. Equally important, GAMs cocultured with pacritinib-treated U87MG and LN18 GBM cells showed a significantly reduced M2 marker (CD206) and increased M1 marker (TNF-α), strongly suggesting pacritinib not only suppressed GBM tumorigenesis but also affected GAM polarization. These tumor inhibitory and tumor microenvironment normalizing effects of pacritinib could be attributed to the suppression of STAT3/JAK2 signaling. Our observations were supported by a recent report that the inhibition of the JAK/STAT3 pathway resulted in disrupted intercellular communications between microglia and GBM cells [35] and pronounced anti-GBM effects [36,37]. In addition, we found that pacritinib treatment was able to suppress the number of miR-21-enriched exosomes secreted by GAMs.

Finally, we provided support for combining pacritinib with TMZ using a TMZ-resistant GBM mouse model. A single treatment of pacritinib was sufficient to suppress GBM growth, while the combination of pacritinib and TMZ exerted the most significant inhibitory effect. Several studies have demonstrated the benefit of using a STAT3 inhibitor to overcome TMZ resistance [38,39]. Notably, one report showed that STAT3 inhibitor treatment promoted the infiltration of tumoricidal lymphocytes [40]. Another study also lends support to our results, where the sequential combination of STAT3 inhibition and TMZ led to the induction of GBM apoptosis with an increased level of miR-21 [41]. This is consistent with another previous study that combined treatment with pacritinib and TMZ to dramatically reduce the activity of the JAK2/STAT3 pathway. This highlights the potential for pacritinib to be a useful adjuvant therapy with the standard-of-care TMZ. Additionally, pacritinib could be used as a salvage therapy for patients with a TMZ-resistant recurrent disease, as STAT3 inhibition sensitizes TMZ-resistant, patient-derived brain-tumor-initiating cell (BTIC) cultures [42].
