The ER+ and PR+ human breast cancer cell line MCF-7 was purchased from DSMZ and maintained in RPMI-1640 medium (PAA Laboratories Inc., Cölbe, Germany) supplemented with 10% fetal calf serum (FCS, Invitrogen, Karlsruhe, Germany). Human macrophages were derived from mononuclear peripheral blood cells. Briefly, monocytes were obtained from buffy coats of healthy donors through double-density-gradient isolation. Macrophages were differentiated by culturing monocytes in fluorinated ethylene propylene-coated cell culture bags (CellGenix, Freiburg, Germany) in the presence of M-CSF (Immunotools, Friesoythe, Germany) for 7 days. For indirect co-culture experiments, macrophages were seeded in hanging cell culture inserts (0.4µm pore size, PET; Millipore, Billerica, MA, USA) and co-cultured with MCF-7 cells at a ratio of 2:1 under normoxia for 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA for array experiments was isolated using TRIZOL reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). RNA integrity for each sample was confirmed with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). Samples used for microarray experiments exhibited a RIN number greater than 7.5. Each RNA sample was then split into two aliquots that were either processed for the miRNA or the mRNA microarray.
Label
Cy3
Label protocol
Global gene expression analyses utilized the “Human GE 4x44K v2 Microarray Kit” (Agilent, Böblingen, Germany) and the QuickAmp Labeling Kit Cy3 One-Color (Agilent) as well as the RNA Spike-In Kit (Agilent). 500ng of total RNA were used as starting material for the synthesis of cDNA. Following in-vitro transcription, the quality and quantity of labeled RNA was determined using the NanoDrop D-1000 UV-VIS Spectrophotometer (Peqlab, Erlangen, Germany). For human miRNA microarray analyses, 200ng of total RNA were labeled using the miRNA Complete Labeling and Hyb Kit (Agilent).
Hybridization protocol
1.65µg of each labeled cDNA sample were fragmented and applied. Hybridizations were performed for 17 hours at 10 rpm and 65°C in a hybridization oven (Agilent). Labeled RNA samples were hybridized to Human miRNA 8x15K V2 microarrays (#G4470B, Agilent). Hybridizations were performed for 20 hours at 10 rpm and 65°C in a hybridization oven (Agilent).
Scan protocol
Cy3 intensities were detected by one-color scanning using the G2505B Agilent DNA microarray scanner at 5µm or 3µm resolution.
Description
Macrophage from patient 4 after co-culture with MCF-7, second sample
Data processing
Raw Median signal intensities were log2-transformed and quantile normalized.
