Figure 5: Myc recruits pro-metastatic macrophages to the tumor microenvironment.

A. Representative immunofluorescence images (top) and quantification (bottom) of T cells (CD3+), Neutrophils (anti-neutrophil antibody+), and macrophages (F4/80+) in primary KPCXY tumors categorized as Met^Low or Met^High, with quantification below (n=3 mice for each subgroup and 4-5 random fields of view analyzed).

B. Representative immunofluorescence images (left) and quantification (right) of macrophages that have migrated across a transwell filter following co-culture with Met^High or Met^Low tumor cells (n=2 Met^Low and 2 Met^High cell lines used, 3 replicates per cell line with 3 20X images taken per transwell; each dot represents quantification of an independent image).

C. Quantification of tumor infiltrating macrophages (as a percentage of total CD45+ cells) in Met^Low or Met^High subcutaneous tumors assessed by flow cytometry (n=5 Met^High cell lines and 3 Met^Low cell lines; 2 NOD.SCID mice examined per cell line with 2 tumors per mouse; each dot represents an independent tumor).

D. Quantification of tumor infiltrating macrophages (as a percentage of total CD45+ cells) in Myc_OE or control (EV) subcutaneous tumors assessed by flow cytometry (n=2 Myc_OE cell lines and 2 EV cell lines; 2 NOD.SCID mice examined per cell line with 2 tumors per mouse; each dot represents an independent tumor).

E-F. Representative immunofluorescence images (left) and quantification (right) of Arg1+ (E) and CD206+ (F) tumor associated macrophages in primary KPCXY tumors categorized as Met^Low or Met^High (n=3 mice for each subgroup and 4-5 random fields of view analyzed).

G-H. Quantification of Arg1+ (G) and CD206+ (H) tumor associated macrophages in primary MYC_OE or control (EV) orthotopic tumors assessd by immunflourescence staining (n=2 Myc_OE cell lines and 2 EV cell lines; 2 NOD.SCID mice examined per cell line; 4-5 random fields of view analyzed).

I. Quantification of tumor cell intravasation from an in vitro trans-endothelial migration (iTEM) assay. MYC_OE or EV-transduced tumor cells were cultured in transwell filters seeded with an endothelial cell monolayer in the presence or absence of macrophages (see Methods). Tumor cells that traversed the endothelial layer were quantified and normalized to the EV control in the absence of macrophages for each of two Met^Low tumor lines.

J. Schematic outline of the macrophage depletion experiment. Mice were orthotopically implanted with Myc-OE cells (n=2 independent cell lines) and after 10d tumor-bearing animals were treated with a combination of CSFR inhibitor (GW2580) and liposomal clodronate (CLD) or vehicle. Metastases were quantified 14d later.

K. Quantification of total metastases (liver and lung) following the macrophage depletion strategy outlined in (J) (n=6 control mice and n=7 GW2580+CLD mice; each dot represents an independent mouse).

Statistical analysis (A-H, K) by Student’s unpaired t-test with significance indicated (*, p<0.05; **, p<0.005; ***, p<0.0001; ns, not significant); statistical analysis (I) by two-way ANOVA (*p<0.05, **p<0.01, ***p<0.001). Error bars indicate SEM. Scale bars = 10 μm (A, E-F) and 50 μm (B).