Live Cell Imaging of the TGF- &beta;/Smad3 Signaling Pathway <em>In Vitro</em> and <em>In Vivo</em> Using an Adenovirus Reporter System

Abstract

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.

Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

“…To study TGF-β/SMAD-induced transcription, luciferase-based reporters coupled with multimerized SMAD3/4 binding elements have been used extensively in the literature, both in vitro and in vivo [11,12,15,[44][45][46][47]. Zhu and colleagues used the CAGA 12 -luciferase reporter in breast cancer cells to study TGF-β/SMAD3 activity by measuring luminescence during tumorigenesis in vivo, whereas others created a transgenic CAGA 12 -luciferase mouse, studying TGF-β/SMAD3 response to injury [45,48].…”

“…To study TGF-β/SMAD-induced transcription, luciferase-based reporters coupled with multimerized SMAD3/4 binding elements have been used extensively in the literature, both in vitro and in vivo [11,12,15,[44][45][46][47]. Zhu and colleagues used the CAGA 12 -luciferase reporter in breast cancer cells to study TGF-β/SMAD3 activity by measuring luminescence during tumorigenesis in vivo, whereas others created a transgenic CAGA 12 -luciferase mouse, studying TGF-β/SMAD3 response to injury [45,48]. To achieve a more dynamic visualization with the CAGA 12 -luciferase reporter, Sorre and colleagues coupled a nano luciferase (NLuc) as reporter to the CAGA 12 box and used microfluidic technology to apply an unstable Nluc substrate prior to image signal readout [11].…”

On-Target Anti-TGF-β Therapies Are Not Succeeding in Clinical Cancer Treatments: What Are Remaining Challenges?

“…New in vitro and in vivo models exploiting these points will deepen our knowledge about the real complexity of TGF-β signaling in carcinogenesis. To do so, sensitive technologies such as single cell signaling and live signaling in vitro and in vivo are to be employed (Zhou et al, 2014;Luwor et al, 2015;Chen et al, 2018) and further developed. Moreover, clinical trials combining anti-TGF-β therapies to other target therapies such as immune checkpoint inhibitors, or strategies allowing a more spatio-temporal controlled intervention using nanocarriers, may allow for an improved treatment and perhaps even cure of cancer patients.…”

Tracking leukemic T‐cell transcriptional dynamics in vivo with a blood‐based reporter assay

“…At the cellular level, cancer cells hijack normal signaling pathways to enhance proliferation, angiogenesis, and migration, often by activating or repressing key transcription factors [6]. While technically challenging, real-time monitoring of signaling pathways in situ provides a valuable window into malignant progression that is difficult if not impossible to capture in vitro [7,8].…”

“…(C) Survival curve of mice injected with sham (PBS vehicle), EF1α-GLUC, or unmodified (untransduced) Jurkat cells over a period of 61 days. (D) Spleens were collected from mice injected with sham (1,2), EF1α-GLUC(3,4,5), and untransduced(6,7,8) Jurkat cells. (E) Blood was collected approximately every 4 h from mice injected with either EF1α-GLUC or Circa2-GLUC Jurkat cells and assayed for GLUC.…”