Figure 4.

TAMs become M2-like during imatinib therapy. (A) GIST mice were treated with imatinib or vehicle control and sacrificed at the indicated time points. Means of tumor weight (left) and TAM percentage of CD45⁺ cells (right) are shown normalized to matched vehicle controls. There were 8–16 mice per time point for tumor weight and at least 3 mice per time point for TAM percentage, but only 1 imatinib-treated mouse and 2 controls existed at 24 wk (marked by an x). (B) GIST mice were treated with vehicle or imatinib for 2 wk and TAM proliferation was assessed by flow cytometry of intracellular Ki67 staining (seven to eight mice/group). (C and D) Mean fluorescence intensity (MFI) of various proteins on TAMs was measured by flow cytometry (C; represents at least two experiments, n = 6–7 per group) or TAMs were sorted from GIST mice (n = 8 pooled per group) and subjected to gene expression array (D). Selected statistically significant genes with a false discovery rate of <0.05 and a fold change greater than two are shown. Color of box depicts fold change (scale on left). On the right, mean value of absolute expression is shown for each group. (E and F) Selected genes were validated by RT-PCR (E; pooled RNA from eight per group) or flow cytometry (F; representative of at least two experiments, n = 6–7 per group). (G) Western blot analysis of C/EBPβ isoform expression (C/EBPβ, LAP, and LIP) on isolated TAMs from vehicle- or imatinib-treated mice (at least five mice per group). MW, molecular weight. (H) GIST mice were treated with imatinib for 2 wk, and then 5 × 10⁴ freshly isolated TAMs were cultured with or without 1 µg/ml LPS overnight in a 96-well plate. Supernatant cytokines were then measured by cytometric bead array. Shown is a representative of two experiments, five to seven mice per group. Bar graphs show mean ± SEM. *, P < 0.05.