Metastatic Breast Cancer Cell Culture and Characterization

E0.2 cells were maintained in DMEM supplemented with 10% FBS and 1% dual antibiotics. E0.2 cell line is a metastatic subclone of E0771 cell line generated in our laboratory [38 (link)]. MVT‐1 cells (derived from MMTV‐c‐Myc; MMTV‐VEGF bitransgenic mice) were obtained from Johnson [35 ] and were cultured as described previously [21 (link)]. The SUM159 cell line was kindly provided by S. Majumder (The Ohio State University) and cultured as described earlier [36 ]. The human breast carcinoma cell lines, MDA‐MB‐231 and MDA‐MB‐468, were obtained through ATCC. S100A7 overexpression or knockdown cells were generated from our previous studies [25 (link), 37 (link)]. PrestoBlue dye (Invitrogen, Eugene, OR, USA) was used to calculate cell viability. LPS and LPS blocker (polymyxin B, PMB) were purchased from Sigma‐Aldrich. All the cell lines were routinely checked for mycoplasma contamination and verified based on cell morphology. shRNAs targeting mouse Tlr4 (Locus ID 21898) were purchased from Origene and transfected with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) into MVT1 cells. Breast tumor microarrays (TMAs; BR1002b) were purchased from US Biomax, Inc (Rockville, MD, USA). The clinicopathological details of TMA are available in Table S1.