*3.1. M2 Polarization of GAMs Promotes GBM Tumorigenesis*

Initially, we cocultured clinically isolated GAMs with human GBM cell lines U87MG and LN18, which showed increased colony (Figure 1A) and neurosphere (Figure 1B) forming abilities. Consistently, qPCR analysis demonstrated that the presence of GAMs was associated with an increased mRNA level of stemness markers (Figure 1C). The results from the Western blots were consistent where the expression of Sox2, Oct4, Wnt, and Nestin were elevated, while GFAP was decreased in the presence of GAMs (Figure 1D).

**Figure 1.** M2 glioblastoma multiforme (GBM)-associated macrophages (GAMs) promote GBM tumorigenesis. GBM cells U87MG and LN18 cocultured with M2 GAMs showed significantly increased colony forming ability (**A**) and tumor sphere generating ability (**B**) as compared to their parental controls. Comparative real-time PCR (**C**) and Western blots (**D**) showed that M2 GAM cocultured GBM cells expressed a significantly higher level of stemness markers, Sox2, Oct4, Wnt, and Nestin while GFAP was reduced. Scale lengths = 100 μm, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.
