Transcriptomic profiling of metastasis-associated lung endothelial cells from mice treated with anti-VEGFR2 antibody (DC101)

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 metastasis-associated lung endothelial cells upon VEGFR2 inhibition For profiling of lung endothelial cells (ECs) associating with metastasis, NSG mice (6-8 weeks of age) were injected with MDA-MB-231-LM2 (MDA-LM2) breast cancer cells via the tail vein. On the day 21 and 24 post cancer cell injection, mice were treated with rat IgG or anti-VEGFR2 antibody (DC101, 40 mg/kg, i.p. injection), and lungs from mice were harvested on day 25. Lungs from age-matched healthy mice treated with rat IgG were used as control group. ECs were isolated from whole lungs by fluorescence-activated cell sorting (FACS) using a panel of negative selection markers and CD31 as positive endothelial cell marker. For each group, 3 biological replicates were analyzed.