Chen Hao, Ware Thomas M B, Iaria Josephine, Zhu Hong-Jian
Department of Surgery (RMH), University of Melbourne.
Department of Surgery (RMH), University of Melbourne;
J Vis Exp. 2018 Jul 30(137):57926. doi: 10.3791/57926.
Transforming Growth Factor β (TGF-β) signaling regulates many important functions required for cellular homeostasis and is commonly found overexpressed in many diseases, including cancer. TGF-β is strongly implicated in metastasis during late stage cancer progression, activating a subset of migratory and invasive tumor cells. Current methods for signaling pathway analysis focus on endpoint models, which often attempt to measure signaling post-hoc of the biological event and do not reflect the progressive nature of the disease. Here, we demonstrate a novel adenovirus reporter system specific for the TGF-β/Smad3 signaling pathway that can detect transcriptional activation in live cells. Utilizing an Ad-CAGA12-Td-Tom reporter, we can achieve a 100% infection rate of MDA-MB-231 cells within 24 h in vitro. The use of a fluorescent reporter allows for imaging of live single cells in real-time with direct identification of transcriptionally active cells. Stimulation of infected cells with TGF-β displays only a subset of cells that are transcriptionally active and involved in specific biological functions. This approach allows for high specificity and sensitivity at a single cell level to enhance understanding of biological functions related to TGF-β signaling in vitro. Smad3 transcriptional activity can also be reported in vivo in real-time through the application of an Ad-CAGA12-Luc reporter. Ad-CAGA12-Luc can be measured in the same manner as traditional stably transfected luciferase cell lines. Smad3 transcriptional activity of cells implanted in vivo can be analyzed through conventional IVIS imaging and monitored live during tumor progression, providing unique insight into the dynamics of the TGF-β signaling pathway. Our protocol describes an advantageous reporter delivery system allowing for quick high-throughput imaging of live cell signaling pathways both in vitro and in vivo. This method can be expanded to a range of image based assays and presents as a sensitive and reproducible approach for both basic biology and therapeutic development.
转化生长因子β（TGF-β）信号传导调节细胞稳态所需的许多重要功能，并且在包括癌症在内的许多疾病中通常过度表达。TGF-β在癌症晚期进展过程中与转移密切相关，可激活一部分具有迁移和侵袭能力的肿瘤细胞。目前用于信号通路分析的方法侧重于终点模型，这些模型通常试图在生物事件发生后测量信号传导，无法反映疾病的进展性质。在此，我们展示了一种针对TGF-β/Smad3信号通路的新型腺病毒报告系统，该系统可检测活细胞中的转录激活。利用Ad-CAGA12-Td-Tom报告基因，我们可以在体外24小时内使MDA-MB-231细胞的感染率达到100%。使用荧光报告基因可实时对活的单细胞进行成像，直接识别转录活性细胞。用TGF-β刺激感染的细胞，仅显示一部分具有转录活性并参与特定生物学功能的细胞。这种方法在单细胞水平上具有高特异性和高灵敏度，有助于增强对体外TGF-β信号传导相关生物学功能的理解。通过应用Ad-CAGA12-Luc报告基因，还可以在体内实时报告Smad3的转录活性。Ad-CAGA12-Luc可以与传统的稳定转染荧光素酶细胞系以相同方式进行测量。通过传统的IVIS成像可以分析体内植入细胞的Smad3转录活性，并在肿瘤进展过程中进行实时监测，从而深入了解TGF-β信号通路的动态变化。我们的方案描述了一种有利的报告基因递送系统，可对体外和体内的活细胞信号通路进行快速高通量成像。该方法可扩展到一系列基于图像的检测，是基础生物学和治疗开发的一种灵敏且可重复的方法。
