Global gene expression of canine bladder K9TCC cells and cancer stem cells after COX-2 inhibition. Global gene expression of canine bladder K9TCC cells and cancer stem cells after COX-2 inhibition

Cancer stem cells (CSCs) are fundamental to bladder cancer progression and the cyclooxygenase-2 (COX-2) pathway has a pro-tumorigenic role in CSC function. Naturally occurring bladder cancer in dogs has the potential to be a natural model of the human disease. Here, we used canine specific microarrays to determine changes in global gene expression of canine bladder cancer cells (the K9TCC cell line) and bladder cancer stem cells isolated from the K9TCC cell line after treatment with the selective COX-2 inhibitor mavacoxib or a COX-2 siRNA. Overall design: K9TCC cells were grown as aherent cells or in anchorage dependent conditions to form cancer stem cell spheres for at least 5 passages to maintain self-renwal properties. Both adherent cells (Adh) and cancer stem cell spheres (Sphs) were treated with either 0 or 50uM mavacoxib (0uM treatment was DMSO, the vehicle control for the drug) for 24 hours, or transfected with a COX-2 siRNA or negative control siRNA for 48 hours. Cells were harvested and RNA extracted for microarray analysis on the Affymetrix Canine Gene 1.1 ST microarray (by Edinburgh Genomics). 3 replicates of each condition and cell type were assessed.

癌症干细胞（Cancer Stem Cells, CSCs）是膀胱癌进展的核心驱动因素，环氧合酶-2（Cyclooxygenase-2, COX-2）通路在CSC功能中发挥促肿瘤生成作用。犬类自发性膀胱癌有望成为人类膀胱癌研究的天然疾病模型。本研究采用犬特异性微阵列技术，分析经选择性COX-2抑制剂马瓦昔布（mavacoxib）或COX-2小干扰RNA（small interfering RNA, siRNA）处理后，犬膀胱癌细胞（K9TCC细胞系）以及从该细胞系中分离得到的膀胱癌干细胞的全基因表达谱变化。实验整体设计如下：将K9TCC细胞以两种方式培养：其一为贴壁单层培养，其二为在锚定依赖条件下培养以形成癌症干细胞球，所有培养体系均至少传代5次以维持细胞的自我更新特性。分别对贴壁细胞（Adh）与癌症干细胞球（Sphs）开展两种处理：一是分别以0μM（以二甲基亚砜（Dimethyl sulfoxide, DMSO）作为药物溶剂对照）或50μM马瓦昔布处理24小时；二是使用COX-2 siRNA或阴性对照siRNA转染48小时。收集细胞并提取RNA，采用Affymetrix犬基因1.1 ST微阵列（Affymetrix Canine Gene 1.1 ST microarray）进行微阵列基因表达分析，实验由爱丁堡基因组学中心（Edinburgh Genomics）完成。每种实验条件与细胞类型均设置3次生物学重复并进行检测。