Figure 6: Myc acts through Cxcl3 and Mif to promote macrophage recruitment and metastasis.

A. Expression of selected cytokines/chemokines in human PDAC. Samples from the COMPASS cohort (enriched for tumor cells by laser capture microdissection) were stratified into MYC high and low groups based on RNA-seq (n=373) and assessed for the expression of five chemokines/cytokines identified as significantly upregulated in Met^High vs. Met^Low tumors (Supplementary Fig. S8A).

B. Relative expression of Mif and Cxcl3 in control or Myc knockdown (shRNA) Met^High cell line 850_Met^High_4. Data is representative of 2 independent Myc shRNAs (n=3 biological replicates).

C. Bar graph showing fold-increase in Cxcl3 and Mif mRNA levels comparing Myc_OE to EV control cell lines. Data representative of 2 independent cell lines (n=3 biological replicates)

D. Quantification of total F4/80+ tumor infiltrating macrophages by immunoflourescence in cell lines that were stably transduced with either a Cxcl3 or Mif overexpression construct (Cxcl3_OE and Mif_OE, respectively) or empty vector (EV). (n=4 tumors examined from each group with 4-5 random fields of view analyzed).

E. Quantification of total metastases (liver and lung) following orthotopic transplantation of EV, Cxcl3_OE, or Mif_OE orthotopic tumors from (D). Data were pooled from 2 independent Met^Low lines transduced with either the Cxcl3_OE, Mif_OE, or EV construct transplanted into 5 NOD.SCID mice (for each cell line). Each dot represents an independent animal.

F. Quantification of macrophages that migrated across a transwell filter following co-culture with 832 Myc_OE tumor cells treated with either a Cxcr2 inhibitor (AZD-5069) or a Mif inhibitor (ISO-1). Data are representative of two independent experiments; 3 replicates with 4-5 20X images taken per transwell.

G. Schematic outline of the Cxcr2 and Mif inhibitor experiment. Mice were orthotopically implanted with 832 Myc_OE cells and after 10d were treated with a Cxcr2 inhibitor (AZD-5069), Mif inhibitor (ISO-1), combination (AZD-5069+ISO-1), or vehicle. Metastases and macrophages were quantified 14d later.

H-I. Quantification of F4/80+ (H) and CD206 (I) macrophages in orthtotopic tumors following the Cxcr2 and Mif strategy outlined in (G) (n=4 tumors per group, 4-5 random fields of view analyzed; each dot represents an independent animal).

J. Quantification of total metastases (liver and lung) following the Cxcr2 and Mif strategy outlined in (G) (n=4 control mice, n=4 AZD-5069 mice, n=4 ISO-1 mice, and n=4 AZD-5069+ISO-1 mice; each dot represents an independent animal).

Statistical analysis by Student’s t-test with significance indicated (*, p<0.05; **, p<0.01; ***, p<0.007; ****, p<0.0005; *****, p<0.0001; n.s., not significant). Error bars indicate SEM.