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. 2017 Nov 30;171(6):1301–1315.e14. doi: 10.1016/j.cell.2017.11.013

Figure 4.

Figure 4

Macrophages Mediate Myc-Induced Angiogenesis and T Cell Exclusion

(A) Coincident immunostaining of Meca32 and receptor-bound VEGF (VEGF:VEGFR2) in tumors at indicated time points (1, 3, and 7 days) after activation of Myc by tamoxifen treatment compared to KRasG12D-only (control). Inset shows enlargement of regions boxed in white. Scale bars apply across each row.

(B) Quantitative RT-PCR for VEGFA and PD-L1 mRNA in F4/80+ macrophages and F4/80− lung cells derived from whole tumor-laden lungs following 3 days Myc activation (tam) versus to KRasG12D-only control (3d oil).

(C) Immunofluorescence analysis of F4/80 and PD-L1 in lung tumors following Myc activation. Scale bar applies to both large panels. White arrows indicate F4/80+ cells.

(D) Immunostaining and respective quantification of PD-L1-positive macrophages in tumors after Myc activation for indicated time points (tamoxifen). T = tumor. Scale bars apply across each row.

(E) Quantification of immunohistochemical analysis for CD3, B220, CD206, NKp46, CD31, and Ki67 of lung tumors after Myc activation for 2 weeks concurrently with systemic treatment of PD-L1 blocking antibody compared to KRasG12D-only tumors (oil control).

(F) H&E staining (left) and quantification (right) of lung tumor burden in mice treated concurrently with tamoxifen (to activate Myc) and either control IgG or PD-L1-blocking antibody. Each individual data point represents a single mouse (n = 6 mice per group).

Quantification graphs: FoV = field of view. Small symbols = individual tumors, large symbols = average per mouse. (B) Each individual data point represents a single mouse. n = 5 (F4/80−; VEGFA), or 6 (F4/80+; VEGFA, PD-L1), and shows the expression data normalized to the average of the respective oil control. (D) n = 20 individual tumors from 4 mice per treatment group. Error bars represent the median with interquartile range. (E) n = 30 individual tumors from 6 mice per treatment group. Error bars represent the median with interquartile range (CD3, CD206, B220, CD31, Ki67) or mean ± SD (NKp46). p values are based on Student’s t test (CD3, B220, CD206, CD31, Ki67) or two-way ANOVA (NKp46). NS = non-significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

See also Figures S1 and S7.