Although various treatment methods have been used in the clinic, the prognosis of GBM patients is always poor due to high treatment resistance and high recurrence rates(35, 36). One important factor in drug resistance and recurrence is the TME, which is formed by various cells influenced by tumors, such as tumor-associated macrophages (TAMs)(37), neurons, astrocytes and microglia(38). Astrocytes, which have been confirmed to be one of the major origins of glioma according to the International Classification of Diseases–Oncology, version 3 and World Health Organization grade(4, 39, 40), play an essential role in remodeling the TME. Indeed, studies have reported that astrocytes directly or indirectly regulate the progression and drug resistance of glioma(41, 42). However, whether and how the crosstalk between glioma cells and astrocytes contributes to malignant changes in the TME is less well illustrated. Exosomes acting as mediators of intercellular communication in the TME have been reported to promote tumor cell progression in various cancers(43, 44). We therefore first examined whether glioma cells could induce malignant phenotypes in astrocytes through exosomes. Our results showed that treating NHAs with GDEs led to strong activation of NHAs. GDE-induced reactivated astrocytes exhibited a series of characteristics, including an increase in proliferation, migration, and the ability to secrete the proinflammatory cytokines IL-6 and IL-8. Indeed, GDE-induced RAs also showed elevated levels of GFAP expression, suggesting strong activation of astrocytes. Moreover, when we implanted GDE-treated astrocytes together with glioma cells into the brains of mice, a significantly larger volume of tumors was observed when compared to those implanted with NHAs, confirming that GDE-induced RAs indeed exerted a malignant effect on glioma progression. In support of our findings, a series of studies have confirmed the function of exosomes as mediators of intracellular communication in the TME. For example, tumor cell-secreted exosomes promoted fibroblasts to differentiate into myofibroblasts(45), which further remodeled the extracellular matrix in the TME. In glioma, it has been reported that GDEs can induce the M2-polarization of macrophages by sending IL-6 into macrophages(19). Through the ATXN1/STAT3 pathway, GDEs can promote the tube formation of vascular endothelial cells and thus increase angiogenesis in the TME(46). Exosomes have also been reported to induce the activation of astrocytes. Exosomes containing miR-21 secreted from neurons contributed to the overactivation of astrocytes in a mouse model of Alzheimer’s disease(47). This evidence further confirmed the vital role of exosomes in mediating the formation of RAs.
Hypoxia, resulting from the uneven growth rate of tumor cells and vessels, is one of the key characteristics of glioma. Hypoxia promotes tumor progression(48), and more importantly, it closely influences the formation and heterogeneity of tumor-derived exosomes and subsequently contributes to the formation of an immunosuppressive tumor microenvironment(49). Based on this notion, we compared the effects of exosomes derived from glioma under normoxic (N-GDEs) and hypoxic (H-GDEs) circumstances on the induction of RAs. We found that H-GDEs induced a much stronger malignant phenotype of astrocytes, accompanied by an increase in proliferation, migration, cytokine secretion and GFAP expression. Furthermore, when H-GDE-treated astrocytes were coimplanted with glioma cells in vivo, we observed a significantly higher proliferation rate of glioma than that of the N-GDE group. In line with our results, Qiu et al reported that H-GDEs lead to increased differentiation and activation in myeloid-derived suppressor cells by increasing the content of miR-1246 in exosomes(50). H-GDEs are also reported to possess higher levels of connexin 43 and promote the angiogenesis of glioma(51) These results together confirmed the role of exosomes not only in mediating the intracellular communication between glioma and astrocytes but also in transforming the detrimental effects of hypoxic conditions on the remodeling of the TME.
Exosomes participate in message exchange between tumor cells and other cell types in the TME by delivering tumor-derived content to recipient cells(52). Among all genetic contents delivered by exosomes, miRNAs have been reported to have specific and important effects on various biological activities of cells, such as proliferation, DNA repair and feedback regulation(53, 54). Therefore, we used a miRNA array to identify the potential miRNAs that mediated the effects of GDEs on the formation of RAs. After comparing the differentially regulated miRNAs in H-GDEs and N-GDEs, we found that miR-423-3p was significantly upregulated in the H-GDEs. We next examined the effects of miR-423-3p on the induction of RAs. Similar to what we found with the H-GDEs, miR-423-3p-transfected NHAs exhibited a significant increase in proliferation, migration, and secretion of proinflammatory cytokines, accompanied by elevated levels and atypia of GFAP. To further test whether miR-423-3p mediates the effects of H-GDEs on the transformation of RAs, we generated cells stably expressing miR-423-3p using lentivirus. Compared with the effect of N-GDEs from control cells, N-GDEs with stabilized expression of miR-423-3p resulted in a much more malignant phenotype of RAs, imitating the effect of H-GDEs. In contrast, when we inhibited miR-423-3p in NHAs treated with H-GDEs, the effect of H-GDEs was reduced. These results strongly suggest that miR-423-3p is the key component that mediates the effects of GDEs on the induction of RAs. Our xenograft model further confirmed our findings in vitro. The volume of glioma cells coimplanted with the astrocytes that were transfected with miR-423-3p grew significantly larger than that of the mice in the control group, leading to a greater reduction in the survival time of mice. In line with our findings, miR-423-3p has been reported to promote the progression of gliomas(19) as well as various other tumors, including gastric cancer, hepatocellular carcinoma, and colorectal cancer(55–57), indicating its functional role in promoting malignancy. These results together strongly suggest that miR-423-3p mediates the effects of GDEs on the induction of astrocyte activation.
Given that glioma induced RAs through miR423-3p-containing exosomes, we next explored the possible underlying cellular and molecular mechanisms. Autophagy is an important regulatory process that maintains cellular homeostasis. However, in tumor cells, its function may vary under different situations. In the early stage of tumor formation, autophagy can help eliminate tumor cells and inhibit their progression, but when autophagy is dysregulated, it helps the survival and growth of tumor cells(58, 59). Recent studies have reported that autophagy is engaged in crosstalk between tumors and other cell types in the TME(60), suggesting that autophagy can also contribute to the formation of a malignant TME. We detected the autophagy level in NHAs treated with H-GDEs and N-GDEs. Morphologically, we observed an increase in the formation of autophagosomes in the astrocytes of the H-GDE-treated group. We further confirmed this finding using western blotting and immunofluorescence to detect autophagy-related signaling molecules. The expression level of LC3B in the H-GDE group was higher than that in the N-GDE group, and the expression of P62 was opposite to that of LC3B, indicating an increase in autophagy. We next examined whether miR-423-3p is a direct messenger that induces autophagy in NHAs. We found that miR-423-3p induced autophagy in NHAs, including the formation of autophagosomes, an increase in LC3B and a decrease in P62. Similar to what we found, Zhang et al. reported that the upregulation of miR-2188-5p can increase the autophagic flux of tracheal cells in an ammonia environment(61). In another report, miR-122 was confirmed to induce protective autophagy in hepatocytes under arsenic stress(62). These studies confirmed the roles of miRNAs in regulating autophagy. However, whether autophagy promotes the activation of astrocytes has not yet been clarified. Using the autophagy inhibitor 3-MA or knocking down beclin1, we found that NHAs treated with H-GDEs or miR-423-3p mimicked the phenotype observed with autophagy inhibition. The typical properties of RAs were also attenuated, suggesting that H-GDEs or miR-423-3p could possibly induce RAs through pathological changes by inducing autophagy. However, most of the published research suggests that the activation of astrocytes is induced by the inhibition of autophagy, which is opposite to the results of our experiments. However, as we have clarified above, autophagy may have different effects on tumors under different periods and circumstances. Furthermore, astrocytes are considered one of the origins of glioma. When astrocytes are malignantly induced into glioma cells, the reactive procedure is deemed necessary. Therefore, in some situations, autophagy in RAs may be increased to induce RAs, which leads to tumor progression. In addition, a previous study reported that in the TME and distal tissue, tumor cells can induce the non-cell-autonomous autophagy of normal cells to promote the progression of tumors in the early stage(63), which is in line with our research. Moreover, we found that most of the research focusing on the formation of RAs by autophagic inhibition is nononcological research. Hence, the effect of autophagy in the TME and astrocytes may be different.
In summary, our experiments identified a positive feedback mechanism through which glioma triggers the activation of astrocytes, and RAs in turn promotes the malignancy of glioma. Furthermore, we discovered that glioma promotes the transformation of NHAs to RAs through the secretion of miR423-3p-containing exosomes, which subsequently induce astrocytic reactivity through autophagy. This study provides new biological insight into glioma treatment. Inhibiting miR-423-3p and autophagy may disrupt the formation of the TME and ultimately impair the progression of glioma.