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 MYC-low groups based on RNA-seq (n = 373) and assessed for the expression of five chemokines/cytokines identified as significantly upregulated in Met^High versus Met^Low tumors (Supplementary Fig. S8A). FPKM, fragments per kilobase of exon per million. B, Relative expression of Mif and Cxcl3 in control or Myc knockdown [short hairpin RNA (shRNA)] Met^High cell line 850_Met^High_4. Data are representative of two 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 are representative of two 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), with n = 4 tumors examined from each group with four to five 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 two independent Met^Low lines transduced with the Cxcl3_OE, Mif_OE, or EV construct transplanted into five NOD.SCID mice (for each cell line). Each dot represents an independent animal. F, Quantification of macrophages that migrated across a transwell filter following coculture with 832 Myc_OE tumor cells treated with either a Cxcr2 inhibitor (AZD5069) or a MIF inhibitor (ISO-1). Data are representative of two independent experiments, including three replicates with four to five 20× 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 10 days were treated with a CXCR2 inhibitor (AZD5069), MIF inhibitor (ISO-1), combination (AZD5069 + ISO-1), or vehicle. Metastases and macrophages were quantified 14 days later. H and 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; four to five 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 AZD5069 mice, n = 4 ISO-1 mice, and n = 4 AZD5069 + ISO-1 mice; each dot represents an independent animal). Statistical analysis by Student t test with significance indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.007; ****, P < 0.0005; *****, P < 0.0001; ns, not significant). Error bars indicate SEM.