CN34 tumour cells were isolated from the pleural effusion of a breast cancer patient treated at our institution, after written consent in accordance with Institutional Review Board (IRB) regulations. Brain metastatic populations from these cells and MDA-MB-231cells were obtained by consecutive rounds of in vivo selection in 6–7-week-old beige nude and athymic mice, respectively. All animal work was done in accordance with the MSKCC Institutional Animal Care and Use Committee. Methods for RNA extraction, labelling and hybridization for DNA microarray analysis have been described previously17 (link). Bioinformatics analyses with detailed descriptions can be found in the Methods. Knockdown and overexpression of candidate genes, and cetuximab inhibitor studies were performed as previously described6 (link). The in vitro BBB model was set up as previously described25 (link), and modified to enable tumour cell counting. Sambucus nigra lectin staining was performed using standard histochemical techniques, and quantified using Metamorph software analysis. The Methods section provides further information, including malignant cell isolation from pleural fluids, tumour cell extraction and cell culture protocols, animal inoculation and bioluminescence imaging, generation of retroviral gene knockdown and overexpression vectors, transfections and infections, RNA and protein expression, in vitro BBB transmigration assay, endothelial cell adhesion assay, and metastatic tissue staining and quantification.