All cell lines were grown in DMEM supplemented with 10% fetal bovine serum, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, 1.5 g/L sodium bicarbonate, penicillin/streptomycin, and fungizone.
RNA was extracted from 4T1 parental and individual in vivo selected metastatic sub-populations using RNeasy Mini Kits and QIAshredder columns (Qiagen, Mississauga, ON, Canada).
Purified total RNA (40 ng) was subjected to two consecutive rounds of T7-based amplification using Amino Allyl MessageAmp II kits (Applied Biosystems, Streetsville, ON, Canada) and the resulting aRNA was conjugated to Cy5 dyes (GE Healthcare Bio-sciences, Bair d’Urfe, QC, Canada).
Hybridization to 44K whole mouse genome microarray gene expression chips (Agilent Technologies) was conducted following manufacturer’s protocol (Agilent Technologies).
Microarray chips were then washed and immediately scanned using a DNA Microarray Scanner (Model G2565BA, Agilent Technologies).
Microarray data were feature extracted using Feature Extraction Software (v. 18.104.22.168) available from Agilent, using the default variables. Microarray expression data were normalized and analyzed using GeneSpring software (V7.3, Agilent Technologies). Each group (parental cell line and the explants derived form mammary fat pad, bone, lung and liver metastasis) was individually compared to all the other groups. A two-fold change cut off was determined and differentially expressed genes were selected using a non-parametric test coupled with a false discovery rate of 0.05.