Fig 2: CCL2 stimulation and CCR2 expression enhance MET phosphorylation through SRC dependent mechanisms in breast cancer cells. A. Flow cytometry for CCR2/MET co-expression in breast cancer cell lines. B-E. Immunoblot analysis on the effects of CCL2 treatment (100 ng/ml) of DCIS.com and HCC1937 cells (B), DCIS.com with wildtype CCR2 (WT) or CRISPR CCR2 knockout (CCR2-KO) (C), SUM225 pHAGE control or CCR2 overexpressing cells (CCR2-H) (D), and DCIS.com breast cancer cells with DMSO control with/without CCL2 and/or 10 mM PP2 (E). F. Co-immunoprecipitation analysis of DCIS.com cells treated with CCL2 for up to 30 min. G. Proximity Ligation assay for DCIS.com breast cancer cells treated with CCL2 for 5 or 15 min. Arrows point to positive signals (green fluorescence spots). DAPI overlay. Scale bar=50 microns.

Fig 3: LY2801653 treatment inhibits growth and invasion of SUM225 CCR2-H MIND lesions. NOD- SCID mice bearing SUM225 CCR2-H MIND lesions were treated with 12 mg/kg LY2801653 or vehicle control for 4 weeks (n = 8/group). Mammary tissues were measured for A. changes in mass, B. PCNA expression, C. cleaved caspase-3 expression or D. number of DCIS, DCIS+ microinvasive (DCIS+Mi) and invasive lesions through co-staining for a-sma (green) and CK/19 (red). Total number of lesions scored/group: n = 527 (vehicle), n = 948 (LY2801653). Statistical analysis was determined by Two Tailed t-test (A-C) or ?² test (D). Statistical significance was determined by p < 0.05. *p < 0.05, ***p < 0.001, n.s=not significant. Mean+SEM are shown. Scale bar= 400 microns.

Fig 4: LY2801653 treatment affects glycolysis in SUM225 CCR2-H MIND lesions. A-B. SUM225 CCR2-H MIND lesions were treated with control vehicle or LY2801653 and immunostained for expression of HKII (A) or PKM1/2 (B). Scale bar= 400 microns. C-F. Normal mammary tissues or MIND lesions (n = 3 per group) were subject to Ion chromatography-mass spectrometry analysis of glycolytic metabolites. Graphs were plotted as area normalized to tissue mass. The following glycolytic metabolites are shown: D-Glucose 6-phosphate (C), Fructose 6-phosphate (D), Fructose 1,6-bisphosphate (E) and Pyruvic Acid (F). Mean+SEM are shown.

Fig 5: MET expression correlates to CCR2 expression in human breast carcinoma tissues. A. Spearman correlation test was performed for CCR2 and MET mRNA expression in IDC samples from TCGA datasets (cbioportal.org). n = 963 B. Co-immunofluorescence staining was performed in normal breast, pure DCIS, DCIS co-occurring with IDC (co-DCIS) and IDC tissues for CCR2 (red) and MET (green) expression, with DAPI counterstain, n = 9/group. Overlapping expression indicated by arrows. Scale bar=200 microns. C. MET and CCR2 expression in DCIS and IDC tissues were quantified by Image J and normalized to DAPI. C-D. CCR2 and MET protein expression were detected in pure DCIS (n = 33), Co-DCIS (n = 58) (C) and IDC (D) (n = 67) tissues by DAB immunostaining. Expression was quantified by Image J. Statistical analysis was performed using Spearman correlation test. Statistical significance was determined by p < 0.05.