*3.6. In Vivo Evaluation of Pacritinib*

Finally, we evaluated the potential of using pacritinib as a treatment for GBM using a preclinical mouse model bearing TMZ-resistant LN18 cells (cocultured with exosomes isolated from GAMs). Representative brain slices showed that a single treatment of pacritinib suppressed the tumorigenesis of TMZ-resistant LN18 cells compared to TMZ single treatment and vehicle control (Figure 6A). Notably, there was no significant difference in tumor size between vehicle control and TMZ single treatment groups (Figure 6B), while the combination of pacritinib and TMZ appeared to produce the most significant inhibitory effect on tumor progression (right panel, Figure 6B). In support, tumor samples harvested from the combination of pacritinib and TMZ showed the lowest level of STAT3, Sox2, PDCD4, and miR-21-5p and an increased level of GFAP (Figure 6C). Microglial cells isolated from the single pacritinib treatment and the combination of pacritinib and TMZ groups also demonstrated a significantly reduced CD206 mRNA level and an increased TNF-α level (Figure 6D). The overall median survival was significantly increased in each treatment group compared with vehicle control (Figure 6E). Median survival was 19 days for vehicle control, 24 days for TMZ (*p* = 0.024, compared with control ), 26.5 days for pacritinib (*p* = 0.0098, compared with control ), and 32.5 day for combination of pacritinib and TMZ (*p* = 0.0006, compared with control, *p* = 0.0092, compared with TMZ, *p* = 0.0219, compared with pacritinib) (Figure 6F).

**Figure 6.** In vivo evaluation of pacritinib for treating GBM and reducing M2 GAMs in TMZ-resistant LN18 bearing mice. (**A**) Immunohistochemical staining in TMZ-resistant LN18-bearing mice showed that treatment in the pacritinib group and pacritinib/TMZ combination group suppressed tumorigenesis. (**B**) The tumor size showed that the significantly reduced tumor size in the pacritinib group and the combination of pacritinib and TMZ group led to the most significantly reduced tumor size. NS, statistically nonsignificant. (**C**) Comparative real-time PCR analyses showed the reduced mRNA level of STAT3, Sox2, PDCD4, and miR-21-5p and the increased GFAP expression in the pacritinib group and pacritinib/TMZ combination group (lane 1, control; lane 2, TMZ alone; lane 3, pacritinib alone; lane 4, pacritinib/TMZ combination). (**D**) M2 GAMs from tumor samples showed a significantly reduced CD206 (M2 marker) mRNA level (lane 3, pacritinib alone; lane 4, pacritinib/TMZ combination) and an increase in TNF-α (lanes 3 and 4). (**E**) Kaplan–Meier survival curve and (**F**) statistical comparisons showed increased median overall survival in TMZ, pacritinib, and pacritinib/TMZ combination groups. Scale lengths = 50 μm, \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001.
