Transcriptomic profiling of bone marrow-derived macrophages upon treatment with recombinant Tenascin-C and TLR4 inhibitor

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 bone marrow-derived macrophages upon treatment with Tenascin-C and TLR4 inhibitor For profiling of bone marrow-derived macrophages (BMDMs), bone marrow cells were obtained from BALB/c mice (8-10 weeks old) and cultured for 7 days in DMEM (ThermoFisher Scientific) supplemented with 10% FBS and 20 μg/ml macrophage-colony stimulating factor (M-CSF, Peprotech). Differentiated BMDMs were further cultured overnight with DMEM containing 1% FBS and 20 μg/ml M-CSF. BMDMs were then treated with 3 μM TAK-242 (TLR4i, Merck) or vehicle control (0.1% DMSO in final) for 1 hr, and stimulated with 2 μg/ml recombinant Tenascin-C (TNC, Merck) for 6 hrs. Total RNA from three biological replicates was isolated and analyzed using Affymetrix Gene Chip Mouse Genome U430 2.0 Arrays.