MSC-induced lncRNA expression changes in breast cancer cells

The development of triple-negative breast cancers (TNBCs) – a subset of tumors with particularly aggressive pathogenesis – is critically regulated by certain tumor-microenvironment-associated cells called mesenchymal stem/stromal cells (MSCs), which we and others have shown promote TNBC progression by activating a multitude of signaling nodes that propagate malignant traits in neighboring cancer cells. Characterization of these signaling cascades will better our understanding of TNBC biology, and stands to bring about novel therapeutics that can eliminate the morbidity and mortality associated with advanced disease. Here, we particularly focused on an emerging family of non-coding RNAs – called long non-coding RNAs or lncRNAs – and utilized a MSC-supported TNBC progression model to identify specific lncRNAs of functional relevance to TNBC pathogenesis. We used Affymetrix arrays to identify the gene expression changes that breast cancer cells (in this case, MDA-MB-231 cells) exhibit as they interact with admixed human MSCs Green fluorescent protein (GFP) -labeled MDA-MB-231 cells were admixed with human MSCs (at 1:3 ratio of cancer cells to MSCs) and cultured in complete media consisting of DMEM, 10%FBS, and Pen/Strep. After 3 days, cultures were trypsinized, filtered through 70 micometer mesh, suspended, and FACS-sorted to recover GFP-positive cells. GFP-MDA-MB-231 cells cultured alone and treated identically to the co-cultured counterparts were used as controls.

三阴性乳腺癌（triple-negative breast cancers, TNBCs）是一类发病机制具有高度侵袭性的肿瘤亚型，其发生发展受到一类与肿瘤微环境相关的细胞——间充质干细胞/基质细胞（mesenchymal stem/stromal cells, MSCs）的关键调控。我们及其他研究团队已证实，MSCs可通过激活众多信号节点，向邻近癌细胞传递恶性表型，进而促进TNBC的进展。解析这些信号级联反应，将加深我们对TNBC生物学特性的理解，并有望催生新型治疗手段，以消除晚期疾病相关的发病与死亡负担。本研究特别聚焦于一类新兴的非编码RNA家族——长链非编码RNA（long non-coding RNAs, lncRNAs），并利用MSCs支持的TNBC进展模型，筛选出与TNBC发病机制具有功能相关性的特异性lncRNAs。我们采用Affymetrix基因芯片，检测乳腺癌细胞（本研究中为MDA-MB-231细胞）与混合的人源MSCs共培养时的基因表达变化。具体实验方案如下：将绿色荧光蛋白（green fluorescent protein, GFP）标记的MDA-MB-231细胞与人源MSCs以癌细胞与MSCs 1:3的比例混合，接种于包含DMEM、10%胎牛血清（Fetal Bovine Serum, FBS）以及青霉素-链霉素（Penicillin-Streptomycin, Pen/Strep）的完全培养基中培养。培养3天后，使用胰酶消化细胞，经70μm滤网过滤后重悬细胞，通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）回收GFP阳性细胞。设置对照组：将单独培养且处理流程与共培养组完全一致的GFP-MDA-MB-231细胞作为对照。