*3.2. Exosome Enriched with miR-21 from GAMs Promotes Tumorigenic Properties*

We further investigated the underlying tumorigenesis by isolating and characterizing exosomes secreted into the culture medium by GAMs. First, we used different markers for exosomes—CD9, CD63, and CD81—to verify the identity of the exosomes isolated from the GAMs (Figure 2A). Next, we showed that incubation of GAM-derived exosomes significantly increased TMZ resistance in both U87MG and LN18 cells (Figure 2B). For example, the estimated IC50 value for U87MG increased approximately 4-fold after incubation with GAM-derived exosomes, while this was even more significant in the LN18 cells after exosome treatment. This increased TMZ resistance was accompanied by increased colony-forming (Figure 2C) and tumor-sphere-forming (Figure 2D) abilities. We then screened a small cohort of microRNAs in two batches of GAM-derived exosomes and found that miR-21 appeared to be

the most abundant microRNA (Figure 2E). As shown in the heatmap, the miR-21 level appeared to be the most enriched in the exosomes collected from two samples of GAMs.

**Figure 2.** GAM-derived exosomes harbor miR-21, which promote GBM tumorigenesis. (**A**) Representative transmission electronic micrograph of exosomes isolated from clinical GAMs (left); Western blot validation of exosomes isolated from GAM culture medium showed the expression of CD9, CD63, and CD81. (**B**) Increased temozolomide (TMZ) resistance in U87MG and LN18 cells cocultured with exosomes (+exo). Enhanced colony-forming ability (**C**) and tumor-sphere-generating ability (**D**) in the presence GAM-derived exosomes. (**E**) MicroRNA profiling analyses showed that exosomes (two samples) isolated from M2 GAMs contained a high level of miR-21. Scale lengths = 100 μm, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.
