Transcriptomic profiling of lung endothelial cells at different stages of lung colonization by MDA-MB-231-LM2 cells

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 lung endothelial cells at different stages of lung colonization by MDA-MB-231-LM2 cells For profiling of lung endothelial cells (ECs) at different metastatic stages, NSG mice (6-8 weeks of age) were injected with MDA-MB-231-LM2 (MDA-LM2) breast cancer cells via the tail vein. At week 1, 2 and 3 post cancer cell injection, lungs from mice were harvested. Lungs from age-matched healthy mice were used as control group. Endothelial cells 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 time point, 3 biological replicates were analyzed.

在癌症进展至转移的过程中，播散的肿瘤细胞常会定植于继发器官的血管附近。然而，我们对血管周围位点诱发的细胞互作的认知仍较为粗浅。
本研究明确了一种调控肺脏乳腺癌定植过程中促转移血管微生态位形成的细胞间调控机制。对转移灶小鼠肺脏分离的内皮细胞（endothelial cells, ECs）进行转录组分析发现，与增殖、炎症及众多分泌蛋白相关的基因表达显著上调。我们证实，内皮细胞中诱导表达的四种分泌因子INHBB、SCGB3A1、OPG及LAMA1可形成支持性微生态位，通过增强肿瘤细胞的干细胞特性与存活能力，促进小鼠体内的转移进程。值得注意的是，阻断调控内皮细胞行为的主要细胞因子——血管内皮生长因子（vascular endothelial growth factor, VEGF），可显著抑制内皮细胞增殖，但对肺脏内皮细胞中上述四种微生态位因子的表达无明显影响。我们发现血管微生态位的形成与炎症反应相关，并揭示转移相关巨噬细胞对于肺脏内皮细胞中四种微生态位因子的产生必不可少。巨噬细胞通过血管周围的TNC-TLR4通路激活，并依次刺激内皮细胞产生四种微生态位因子。综上，本研究结果为血管周围微生态位的形成提供了机制层面的见解，并提示靶向巨噬细胞或可与现有抗血管生成药物协同作用，以有效抑制转移定植过程中的血管功能。
我们使用基因芯片分析MDA-MB-231-LM2细胞肺脏定植不同阶段的肺内皮细胞基因表达全局变化。为分析不同转移阶段的肺内皮细胞（ECs），我们向6-8周龄的NSG小鼠经尾静脉注射MDA-MB-231-LM2（简称MDA-LM2）乳腺癌细胞。于肿瘤细胞注射后第1、2、3周采集小鼠肺脏，以同周龄健康小鼠的肺脏作为对照组。通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS），以一系列阴性选择标记物结合CD31作为内皮细胞阳性标记物，从全肺组织中分离内皮细胞。每个时间点设置3次生物学重复进行分析。