Figure 3. Combinatorial DAS + VAC therapy reduces immunoregulatory cell populations and alters hypoxia-mediated signaling in the tumor microenvironment. (A–D) Day 34 M05 melanomas were isolated from host mice that were left untreated or were treated (starting on d10 post tumor cell s.c. injection) with either 0.1 mg/day dasatinib (DAS) orally administered for 7 consecutive days, or contralateral s.c. vaccination (VAC) consisting of 106 OVA257–264 peptide-pulsed dendritic cells (DCs) genetically modified to overexpress IL-12, or a combination of DAS + VAC. (A) Dissociated tumors from the various treatment groups were analyzed for their content of myeloid-derived suppressor cells (MDSC; CD11b+Gr1+) and regulatory T cells (Tregs, CD4+Foxp3+) by immunofluorescence staining and flow cytometry. The percentage of cells bearing the specified phenotype is reported in panel insets. (B and C) RT-PCR analysis of the levels of Foxp3, CXCL12, HIF1α, HIF2α, and β-actin mRNA transcripts (panels B and C) and western blot analysis for expression of HIF1α, HIF2α, and β-actin protein (panel C). (D) Densitometric analysis of western blots (shown in C) scanned to determine comparative protein levels in each tumor cohort relative to the untreated control that was assigned an arbitrary value of 1.0. Representative data from 1 of 3 independent experiments is depicted. Statistical analyses were performed by Student’s t test or 1-way ANOVA; *P < 0.05 vs. untreated (t test), **P < 0.05 vs. all other groups (ANOVA).