*3.5. Pacritinib Suppresses GBM Tumorigenesis and M2 Polarization of GAMs*

Elevated STAT3 signaling has been attributed to the malignancy of GBM and the generation of glioma stem cells [16]. In addition, increased STAT3 signaling is associated with the increased miR-21 level in the promotion of tumorigenesis [17,18]. Based on these premises, we examined a clinical STAT3 inhibitor, pacritinib, for its potential GBM inhibitory effects. We found that pacritinib treatment significantly suppressed the cell viability of both U87MG and LN18 cells at low IC50 values (0.5 and 1.7 μM, respectively) (Figure 5A). Subsequently, we showed that pacritinib-treated U87MG and LN18 cells contained a significantly lower ability to generate M2-polarized GAMs (Figure 5B), as reflected by the reduced CD206 (M2 marker) and increased TNF-α (M1 marker). In addition, the addition of pacritinib prominently suppressed colony formation (Figure 5C) and tumor sphere generation (Figure 5D). Furthermore, pacritinib treatment led to a decreased expression of Sox2, PDCD4, and STAT3; more importantly, the level of miR-21-5p in both GBM cell lines was suppressed as well (Figure 5E). Notably, pacritinib treatment led to significantly reduced exosome release and a corresponding level of miR-21-5p from GAMs (Figure 5F).

**Figure 5.** Pacritinib treatment suppresses GBM tumorigenesis and glioma stem cell (GSC) properties. (**A**) Pacritinib treatment significantly suppressed both U87MG and LN18 cells (approximate IC50 values 0.5 and 1.5 μM, respectively). (**B**) Pacritinib treatment significantly reduced GBM cells' ability to induce M2 GAMs. CD206 mRNA in GAMs was significantly reduced, while TNF-α was increased. Pacritinib treatment significantly reduced colony formation (**C**) and tumor sphere generation (**D**) in both U87MG and LN18 cells. (**E**) Pacritinib treatment led to a significantly reduced mRNA level of STAT3, Akt, Sox2, PDCD4, and miR-21-5p and increased GFAP in both U87MG and LN18 cells. (**F**) GAMs treated with pacritinib resulted in the decreased release of exosomes. Western blot of exosomes collected from GAMs showed a significantly lower abundance of exosomes (CD63 and CD9, markers of exosomes). The exosomes collected showed a significantly lower miR-21-5p level. Scale lengths = 100 μm, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.
