Fig. 5.

Heterozygous loss of Ptprd activates immune programs and influences the macrophage response. (A) Immune response gene-expression pathways are activated in Ptprd^+/−p16^−/− tumors. Gene-expression microarray analysis was performed on GFP⁺ tumor cells from Ptprd^+/+p16^−/− (n = 2), Ptprd^+/−p16^−/− (n = 2), and Ptprd^−/−p16^−/− (n = 3) mice. A selection of the top activated pathways enriched in the differentially expressed genes of Ptprd^+/−p16^−/− vs. Ptprd^+/+p16^−/− tumors are shown, false-discovery rate P < 0.05. (B) Expression of chemokines that promote M2 polarization of macrophages is increased in Ptprd^+/−p16^−/− tumors. Fold-change of chemokine expression normalized to mean of Ptprd^+/+p16^−/− expression from microarray analysis. Error bars = 1 SD. Ptprd^+/+p16^−/− (n = 2), Ptprd^+/−p16^−/− (n = 2), and Ptprd^−/−p16^−/− (n = 3). *Ptprd^+/−p16^−/− vs. Ptprd^+/+p16^−/− P < 0.05. (C) Examples of tumors stained with the Iba1 macrophage marker. Arrows indicate locations of amoeboid macrophages. Amoeboid morphology is enriched in large tumors and ramified macrophages mainly occur in smaller tumors (large tumors defined as >0.7 mm³ tumor volume). Examples from Ptprd^+/−p16^−/− mice (amoeboid) and Ptprd^+/+p16^−/− mice (ramified). (Scale bars, 100 μm.) (D) Representative images of Iba1 and p-Stat3 immunofluorescence on tumors from indicated genotypes. Green, Iba1; blue, DAPI; red, p-Stat3. (Large image scale bars, 20 μm.) White arrows indicate cells with nuclear p-Stat3 and cytoplasmic Iba1 and are enlarged (Insets) (Scale bars, 10 μm.) (E) Ptprd^+/−p16^−/− large tumors have greater p-Stat3 expression in Iba1⁺ macrophages. Metamorph quantification of Iba1⁺ and p-Stat3⁺ cells, *Ptprd^+/−p16^−/− vs. Ptprd^−/−p16^−/− (large tumors) P < 0.05.