Fig. 2.

KRAS mutant tumor cells have a selective advantage to reprogram macrophages to a TAM-like phenotype. a–c Macrophages were obtained by culture of monocytes in DMEM medium supplemented with 10% heat-inactivated human AB serum in the presence or absence of 30% CM from the indicated cell lines for 6 days. a Representative flow cytometry staining for CD206/HLA-DR in macrophages. The solid lines represent cells stained with monoclonal antibodies, and dotted lines represent those stained with isotype controls. Numerical values denote the relative mean fluorescence intensity (RelMFI) normalized to isotype controls (mean ± SD) (n = 3). b Immunofluorescence images of CD68/DAPI in macrophages. Scale bars: 30 μm. c Cytokine levels in the media obtained from macrophage cultures (n = 3). d, e Primary TAMs were isolated from fresh CRC tumor samples. d The levels of indicated cytokines by real-time PCR assays and e the M1 makers by immunoblots in TAMs were compared between KRAS mutant CRC tissues (n = 3) and KRAS wild-type tissues (n = 5). p-values are for comparison with “Control” (a, c). *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001, by one-way ANOVA (a, c) or by two-tailed Student’s t-test (d)