Transcriptomic profiling of SUM159 human breast cancer cells with ectopic expression of SCGB3A1

In cancer progression to metastasis, disseminated cancer cells frequently lodge near vasculature in secondary organs. However, our understanding of the cellular crosstalk evoked at perivascular sites is still rudimentary. In this study, we identified an inter-cellular machinery governing formation of a pro-metastatic vascular niche during breast cancer colonization in lungs. Transcriptomic analysis of endothelial cells (ECs) isolated from mouse lungs with metastases revealed a marked upregulation of genes linked to proliferation, inflammation and numerous secreted proteins. We showed that four secreted factors, INHBB, SCGB3A1, OPG and LAMA1, induced in ECs form a supportive niche that promotes metastasis in mice, by enhancing stem cell properties and survival ability of cancer cells. Interestingly, the blocking vascular endothelial cell growth factor (VEGF), a major cytokine regulating EC behaviors, dramatically suppressed EC proliferation whereas no impact was observed on the expression of the four vascular niche factors in lung ECs. We found that the formation of a vascular niche is correlated with inflammation, and revealed that metastasis-associated macrophages are essential for production of all of four niche factors in lung ECs. Macrophages are activated via TNC-TLR4 at perivasculature and sequentially stimulate ECs to produce the four niche factors. Thus, our findings provide mechanistic insights into the formation of a perivascular niche and offer the possibility that targeting macrophages may synergize with existing anti-angiogenic drugs to effectively suppress vascular function in metastatic colonization. We used microarrays to analyze the global changes of gene expression in SUM159 human breast cancer cells with ectopic expression of SCGB3A1. SUM159 human breast cancer cells were infected with lentivirus harboring pLVX-puro empty vector (control) or pLVX-puro-hSCGB3A1 and selected with 2 μg/ml puromycin (Invitrogen) for 7days. Cells were seeded into ultra-low attachment cell culture dishes with HuMEC-medium (Invitrogen) supplemented 0.1% BSA, B27 Supplement (Gibco), 50 U/ml penicillin (Sigma-Aldrich) and 50 μg/ml streptomycin (Sigma-Aldrich). After 5 days, cells were harvested and total RNA was isolated. RNA from three biological replicates was analyzed using Affymetrix Gene Chip human U133 Plus 2.0 Arrays.