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

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© 2017 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Figure S3 — Deregulated Myc Rapidly Re-programs Tumor Immunity, Related to Figure 2

(A) Quantification and representative immunostained images for B lymphocytes (anti-B220 aka CD45R) in mice treated with tamoxifen for 3 days.

(B) Quantification (by flow cytometry) of NKp46 positive cells as percentage of total live cells isolated from tumor-laden whole lungs of mice treated with oil (KRas^G12D-only) or 3 days tamoxifen (+ Myc). n = 6 mice per time point.

(C) Quantitative analysis (left) and representative immunostaining (right) of NK cell-activating signals showing rapid upregulation of the NKG2D-ligand Rae-1 (PAN Rae-1 antibody) and downregulation of MHC Class I on lung tumor cells following Myc activation. Bottom panels show MHC Cass I expression on adjacent normal lung epithelium (closed arrows). Scale bars apply across each row.

(D) Quantification of hypoxyprobe staining shown in Figure 2C (bottom row), confirming rapid transition from hypoxia to normoxia in adenomas following Myc activation.

(E) Immunohistochemistry and immunofluorescence analysis of serial sections of paraffin-embedded lung tumors of KM mice showing concordance of CD206 and F4/80 immunostaining. KM mice were treated for three days with Tamoxifen, lung tissue harvested and serial sections taken for immunostaining.

Top row: representative Immunostaining for macrophage marker CD206 with boxed region enlarged immediately to the right. Bottom row: Immunofluorescence staining for macrophage marker F4/80 with boxed region enlarged immediately to the right. Inset in bottom right panel shows non-overlap of staining for F4/80 and for the epithelial cell marker TTF1.

Right large panel: overlay of Immunofluorescence F4/80 and immunohistochemistry of CD206 showing overlap.

(F) Epithelial fluorescence in situ hybridization of MycER^T2 (red) combined with immunofluorescence for lung macrophage CD206 (green). Representative pictures are shown. Left panels: KRas^G12D-only mice (K) mice express no detectable MycER^T2 whereas KM mice show clear MycER^T2 nuclear staining confined to tumor masses that is independent of MycER^T2 activation (tamoxifen treatment). Right Panels: I and II and their progressive enlargements show MycER^T2 expression is specific to tumor cells and absent from normal lung epithelial cells. Dotted line indicates tumor boundary. III and IV confirm absence of any detectable MycER^T2 in CD206⁺ macrophages.

(G) MycER^T2 RNA expression in F4/80-affinity isolated lung macrophages in KM mice 16 weeks post AdV-CRE. Results are normalized to the average of the F4/80^- fraction. n = 6 mice per fraction (F4/80^- versus F4/80⁺).

Quantification graphs: FoV = Field of View. (A): n = 30 individual tumors (small symbols) from 6 mice (large symbols) per time point. (C): n = 4 mice per time point, n = 20 tumors analyzed; 5 per mouse. (D): n = 6 mice per time point, n = 30 tumors analyzed; 5 per mouse. Error bars represent the median with interquartile range (B220, NKp46) or mean ± SD (Rae-1, Hypoxyprobe). P values are based on Student’s t test (B220, NKp46) or two-way ANOVA (Rae-1, Hypoxyprobe). ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001.