Figure 4. Combined vaccine + axitinib therapy is more effective than single modality treatment in promoting a “Type-1” melanoma microenvironment.

A, Total RNA was purified from single cell suspensions of MO5 tumors harvested on the indicated days as described in Fig. 2. RT-PCR was then performed using primer pairs specific to the Type-1 associated markers Tbet, IFN-γ, CXCR3, and CXCL10. PCR products were resolved through electrophoresis, imaged, and band intensities were normalized to the intensity of β-actin-specific amplification from paired samples. Data is presented as the fold change in transcript levels (mean ± SD) compared to baseline values from the same cohort on the day of treatment initiation (i.e. day 7 post-tumor challenge). *p < 0.05 versus all groups. In B, RT-PCR analysis was performed using Tbet- and Foxp3-specific primer pairs as outlined in panel A and Materials and Methods, with the mean ratio of Tbet/Foxp3 transcript levels reported (+/− SD) on the indicated days after treatment initiation. In C, day 20 melanomas were harvested from all groups and tissue sections analyzed by immunofluorescence microscopy for expression of CD31 and VCAM-1 as outlined in Materials and Methods. Cell nuclei were imaged by staining with DAPI. Mean (+/− SD) absolute numbers of CD31⁺VCAM-1⁺ cells per high power field (HPF) over over 10 HPFs/slide are reported. *p < 0.05 versus all other groups.