Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Summary

Here, we present a protocol for live cell imaging of TGF-β/Smad3 signaling activity using an adenovirus reporter system. This system tracks transcriptional activity in real-time and can be applied to both single cells in vitro and in live animalmodels.

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.

Introduction

Transforming Growth Factor β (TGF-β) is an essential cytokine implicated in human development that signals through a heterodimeric complex consisting of type II and type I receptors¹. Binding to type II receptor results in the recruitment and phosphorylation of type I receptor, which in turn phosphorylates downstream Smad2/3 proteins^2,3. These activated Smad2/3 proteins bind to Smad4, forming a complex that translocates into the nucleus and regulates gene transcription⁴. Under homeostatic conditions TGF-β/Smad signaling is tightly regulated; howeve....

Protocol

All animal experiments were approved by the University of Melbourne Animal Ethics Committee.

Note: The sequence, construction and generation protocol for the adenoviral vectors pAd-CMV-Td-Tom, pAd-CMV-GFP and pAd-CAGA₁₂-Luc/Td-Tom have been previously described^11,12,13. All vectors are commercially available.

1. Virus Titer Determination Using 50% Tissue-culture Infectious Dose (TCID₅₀)

1. Prepare 1 x 10⁶ HEK293A cells in 10 mL of DMEM + 10% FBS media.<....

Results

Live single cell imaging in vitro

To accurately assess the activation of TGF-β/Smad signaling in single cells using adenovirus based reagents, it is important to first determine the optimal Multiplicity of Infection (MOI) for each cell line. The optimal MOI is determined when 100% of cells are positive for adenoviral infection with no present cytopathic or cytotoxic effects. To determine this, we used a constitutively acti.......

Discussion

We have developed a technique to allow for real-time imaging of TGF-β/Smad3 signaling in single live cells. Using this novel method, we have previously identified a sub-population of cells with dynamic TGF-β/Smad3 transcriptional activity that was associated with enhanced invasion and migration⁸. This method improves on traditional assays for TGF-β signaling, such as western blots of Smad3 phosphorylation and TGF-β targeted gene expression, by capturing the heterogeneity of TGF.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	ThermoFisher	1881024	Warm in 37 °C waterbath before use
Foetal Bovine Serum	Scientifix Life	FBS500-S	Heat inactivated before use
Recombinant Human TGF-β1	PEPROTECH	100-21	Aliquot in DDW to make final concentration at 10 mg/mL
Hoechst-33258	Tocris Bioscience	5117	Dilute in to PBS to make final concentration at 1 μL/mL
Luciferase Reporter Assay Kit	Promega	197897	Dilute 5x in PBS before use
Luminometer	Promega	9100-002
Phase contrast fluorescence microscopy	OLYMPUS	IX50
Centrifuge	eppendorf	5810 R
VivoGl Luciferin	Promega	P1041
IVIS Lumina III In Vivo Imaging System	PerkinElmer	CLS136334
0.5% Trypsin-EDTA (10x)	ThermoFisher	15400-054	Diltue to 0.05% (1x) in PBS
Cell Culture Lysis 5x Reagent	Promega	E153A	Dilute to 1x in DDW
10% Formalin	Sigma-Aldrich	F5554-4L
HEK 293A	ThermoFisher	R70507
MDA-MB-231	ATCC	CRM-HTB-26
PRKDC-SCID	Animal Resources Centre	SCIDF6
Matrigel	Corning	354234
Isoflurane	Zoetis	26675-46-7
Ethanol	Chem-supply	EA043-10L-P
Refresh Night Time	Allergan	1750D	Lubricating Eye Ointment
Solution			Composition
Phosphate-Buffered Saline (PBS)			NaH2PO4.2H2O (4 mM); NaHPO4 (16 mM); NaCl (0.12M)
FBS-DMEM			5% heat inactivated FBS; 10 μg/mL penicillin; 100 μg/mL streptomycin