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Method Article

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

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DOI:

10.3791/57926

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July 30th, 2018

 ,  ,  , 
1Department of Surgery (RMH), University of Melbourne

* These authors contributed equally

In This Article

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 proteins2,3. 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; however, in many diseases, the signaling pathway is deregulated and often overexpressed leading to progression of disease5,6,7. Recent studies have demonstrated that cellular response to TGF-β is heterogenous and subpopulations of TGF-β/Smad active cells are responsible for biological function in a time-dependent manner8,9. Common cellular analysis of TGF-β/Smad signaling involves the use of fixed endpoint assays that provide only a snapshot of cellular activity and often quantitate the average TGF-β/Smad effect10. These methods, however, may not accurately represent the molecular behaviors of TGF-β/Smad signaling in the physiological state during disease progression. Image-based analysis of live cells capture the dynamics of cellular and biological processes with both a spatial and temporal understanding.

Our goal was to develop a sensitive high-throughput method for live cell imaging of TGF-β/Smad signaling using adenovirus-based reagents. Here, we infected the human breast cancer cell line MDA-MB-231 with an adenovirus expressing the Smad3 CAGA motif binding sequence and a luciferase (Luc) or Td-Tomato (Td-Tom) reporter gene. Adenoviral reporter systems provide a quick and cheap method for plasmid introduction that can result in a 100% infection rate in cancer cell lines. Adenoviral reporter systems have also been successfully applied to cell lines that are difficult to transfect with conventional plasmid11. In this protocol we will describe a reproducible and noninvasive process to achieve live cell imaging of the TGFβ/Smad signaling pathway both in vivo and in vitro.

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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 described11,12,13. 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.
  2. Seed 100 µL of cell suspension into each well of a 96-well flat-bottom cell culture plate.
  3. Prepare 1 mL of a 1:100 dilution of virus stock in complete medium. Add 11 µL of diluted virus to each well of column 1.
  4. Perform serial 10-fold dilutions directly in the 96 well plate by mixing 11 µL of diluted virus with 100 µL of cell suspension until a final dilution of 1 x1012 is reached. Although cells are non-adherent at this stage, the number of cells transferred between wells remains constant.
  5. Pipette 11 µL of each dilution into each column starting with the highest dilution of 104. Replace pipette tips with each dilution to limit cross-over of viral particles.
  6. Add 11 µL of virus-free complete media to column 12 as a negative control.
  7. Incubate plate for 7 - 10 days at 37 °C with 10% CO2.
  8. Using a microscope, score the number of wells in each column displaying signs of cytopathic or cytotoxic effects. Consider a well positive if any sign of infection is present.
  9. Calculate the fraction of positive wells for each dilution using the Reed and Muench (1938) statistical method14.
  10. Calculate the proportional distance (PD) using: (A-50)/(A-B), where A is the percentage response above or equal to 50% and B is the percentage response below 50%.
  11. Calculate TCID50 using: 10X-PD, where X is the dilution giving a response greater than 50%.
  12. Calculate viral titer from using the following: 0.69/(0.011 x TCID50) PFU/mL

2. Adenovirus MOI Determination

  1. Seed 1.0 - 1.5 x 105 MDA-MB-231 cells with 500 μL of DMEM culture media containing 5% FBS in each well of a 12 well plate (35% cell confluency, 6 wells in total).
  2. Calculate the volumes of Ad-CMV-Td-Tom stock required for 0, 250, 500, 2500, and 5000 MOI using following formula: MOI = (volume [μL] x PFU/μL)/number of cells. For Ad-CMV-Td-Tom with a titer of 1 x 1010 (PFU/mL), the volumes of virus infection required MOIs are 0, 3.75, 7.5, 37.5, and 75 μL respectively.
  3. Infect the cells with the calculated volumes of Ad-CMV-Td-Tom in Section 2.2 by directly pipetting viral stock into the wells with thorough mixing.
  4. Place the plate in the incubator at 37 °C with 10% CO2 for 24 h.
  5. Remove the media from the wells and immediately fix the cells with 500 μL of 10% formalin for 5 min.  
  6. Aspirate the formalin from wells and wash wells with 500 μL of PBS once.
  7. Stain the cell nucleus with 500 μL of Hoechst solution for 5 min.
  8. Remove the Hoechst solution and wash the wells with 500 μL of PBS 3 times.
  9. Image the Td-Tomato protein and cell nucleus signal using 200X magnification on a fluorescence microscope. Excite Td-Tomato protein at 554 nm and the Hoechst dye at 352 nm.
  10. Analyze images using ImageJ to determine the working MOI required for 100% infection of Td-Tomato15.

3. Dual-adenovirus Transduction in Live Single Cells

  1. Seed 1.0 - 1.5 x 105 MDA-MB-231 cells with 500 μL of DMEM culture media containing 5% FBS in each well of a 12 well plate (35% cell confluency, 2 wells in total).
  2. Infect cells with 75 μL of Ad-CMV-GFP (titer = 5 x 109 PFU/mL) and 7.5 μL of Ad-CAGA12-Td-Tom (titer = 5 x 1010 PFU/mL) to obtain a 2,500 MOI, which can achieve 100% infection positivity.
  3. Treat cells with or without 0.25 μL of TGF-β per well (10 mg/mL in stock, 5 ng/μL as the final concentration) immediately after adenovirus infection.
  4. Place the plate in the incubator at 37 °C with 10% CO2 for 24 h.
  5. Image live cells with phase contrast fluorescence microscope. Excite GFP at 470 nm and Td-Tomato protein at 554 nm.
  6. Fix cells and stain cell nucleus as described in Steps 1.6 - 1.9.
  7. Image the Td-Tomato protein and Hoechst stain using a fluorescence microscope. Excite the Hoechst dye at 352 nm.
  8. Using ImageJ, calculate the percentage of GFP and Td-Tomato positive cells15.

4. Live Single Cell Signaling in Wound Healing Assay

  1. Seed 4 - 5 x 105 MDA-MB-231 cells with 1 mL of DMEM culture media containing 5% FBS in each well of a 6 well plate (50% cell confluency, 2 wells in total).
  2. Infect the cells with 22.5 μL of Ad-CAGA12-Td-Tom (titer = 5 x 1010 PFU/mL, MOI = 2,500).
  3. Place the plate in the incubator at 37 °C with 10% CO2 for 24 h.
  4. Scratch two crossed lines on the cell layer of each well using a P200 pipette tip.
  5. Remove the old media from wells and replace with 2 mL of fresh 5% FBS media with or without 0.5 μL of TGF-β per well (10 mg/mL in stock, 5 ng/μL as final concentration).
  6. Place the plate in the incubator at 37 °C with 10% CO2 for 5 min and take images of selected wound areas using 100X magnification on a phase contrast microscope.
  7. Place the plate in the incubator at 37 °C with 10% CO2 for 24 h.
  8. Take images of the wound closure at the same areas as before.
  9. Fix the cells, stain cell nucleus as described in Steps 1.6 - 1.9 and visualize Td-Tomato signal in the wound area and non-wound area using 200X magnification on a fluorescence microscope.
  10. Quantify the percentage of Td-Tomato positive cells in the wound and non-wound area using ImageJ software15.

5. In Vitro Luciferase Assay

  1. Prepare a cell suspension of 3 - 5 x 104 MDA-MB-231 cells in 1 mL of DMEM media containing 5% FBS.
  2. Infect cell suspension with 2.5 μL of Ad-CAGA12-Luc (titer = 5 x 1010 PFU/mL, MOI = 2,500).
  3. Seed the infected cells into a 96 well plate at 3,000 cells/100 µL/well.
  4. Place the plate in the incubator at 37 °C with 10% CO2 for 24 h.
  5. Remove old media from wells and replace with 100 µL of fresh 5% FBS media with or without 0.1 μL of TGF-β per well (1 mg/mL in stock, 1 ng/μL as the final concentration) and in the presence or absence of 0.17 μL of TGF-β inhibitor per well (7 mg/mL in stock, 12 ng/μL as final concentration) (a novel TGF-β ligand trap protein, Chen et al., unpublished work).
  6. Place the plate in the incubator at 37 °C with 10% CO2 for 24 h.
  7. Thaw out the luciferase assay system substrate and prepare 1x cell culture lysis reagent in double distilled water (DDW).
  8. Remove media from the 96 wells and lyse wells with 50 μL of 1x cell culture lysis buffer on ice using an orbital shaker with gentle agitation for 30 min.
  9. Transfer 30 μL of lysate from each well into an opaque luciferase plate and allow the plate to warm to room temperature.
  10. On the luminometer software, create a new protocol. Select for 1 injector with measurements before and after injection. Set measurements before injection at 1 s for both Delay in Measurement and Integration Time. For after injection measurement, set the Injection Volume to 40 µL with a Delay in Measurement of 1 s after injection and 5 s Integration Time. Confirm settings by selecting OK.
  11. Place the injector tube into DDW and click the Injector 1 button under the Prime settings to clean the injector tube before use. Next remove the injector tube from the DDW and place in air followed by two further Primes of injector 1 to flush all solution from the tube. Place the injector tube into Firefly substrate and again Prime twice to prepare the substrate.
  12. Place the opaque luciferase plate with the samples into the luminometer and select Options on the software. Highlight the wells of interest and click Apply. Press Start to measure luciferase signal.
  13. Once measurements are complete, remove and rinse the plate with water. Recover any remaining substrate into the injector tube by placing the tube into air and Prime injector 1 back into firefly substrate tube. Place the injector tube into DDW and perform two further Primes to clean the injector tube of any remaining substrate.

6. Cell Preparation for Live Signaling Imaging In Vivo

  1. 48 h before tumor implantation, seed 2 - 3x106 MDA-MB-231 cells into 175 cm2 flasks (10 flasks in total). Culture cells in 20 mL of DMEM media containing 5% FBS at 37 °C, 10% CO2.
  2. 24 h later, add 300 μL of Ad-CAGA12-Luc (titer = 5 x 1010 PFU/mL, MOI = 2,500) into each flask. Keep cells in culture for another 24 h.
  3. On the day of implantation, remove the old media and wash the adenovirus infected cells once with 20 mL of PBS, pH 7.4. Add 2 mL of 0.05% trypsin-EDTA into the flask and shake the flask slightly to allow trypsin to cover all the flask surface. Place the flask in to the incubator 37 °C, 10% CO2 for 3 min.
  4. Quench the trypsin by adding 8 mL of FBS-containing DMEM media into flasks. Transfer all cell suspensions into a glass reagent bottle.
  5. Count the cell number using a hemocytometer and transfer 4.8 x 107 cells in two 50ml centrifuge tubes.
  6. Centrifuge the cells at 400 x g for 5 min at RT to remove trypsin and resuspend cells in 720 mL of fresh FBS-DMEM media.
  7. Transfer the cell suspensions into a sterile 5 mL tube and add 160 μL Matrigel (10% total matrigel suspension).
  8. Keep cells on ice until implantation.

7. Orthotopic Implantation

  1. Weigh 12 SCID mice and randomly allocate mice into control and treatment groups (6 mice/group).
  2. Perform the implantation in a sterile bio-cabinet to maintain a sterile environment. Anaesthetize the mouse using 2.5 - 4.5% isoflurane with an oxygen flow rate of 1 L/min. Place the mouse on a heat pad and dispense a drop of lubricating eye ointment onto both eyes to avoid corneal damage. Fix the mouse to the heat pad in a supine position while maintaining anaesthesia by placing the snout into a nose cone connected to the isoflurane.
  3. Confirm the success of anaesthesia by the lack of reaction to toe pinch. Reduce isoflurane to a maintenance dose of 1.5 - 2.5% with 1 L/min oxygen for the reminder of implantation.
  4. Clean the skin from the fourth nipple on both sides to the midline by using the cotton swab dipped into 80% ethanol.
  5. Find the 4th mammary fat pad through palpation and squeeze the fat pad to further expose the tissue.
  6. Mix the cell suspension well by pipetting up and down. Gently aspirate 50 µL of cell mixture into a 27G insulin syringe and inject into the mammary fat pad on both sides of the mouse.
  7. Confirm a successful injection by feeling for swelling of the fat pad. Release the fat pad gently and place the mouse back into the cage.
  8. Leave the cages on the heat pad for at least 30 min to allow the mice to gain consciousness and then place the cages back in the holding room for 3 days.

8. IVIS Imaging

  1. Open the Living Image software.
  2. Initialize the IVIS system by clicking the Initialize IVIS System button on the control panel. Wait until the temperature sign turns green.
  3. Select the Luminescent Imaging Mode on the control panel.
  4. Turn on isoflurane anaesthesia to the chamber and IVIS system.
  5. Anaesthetize the mice by using 2.5 - 4.5% isoflurane with 1 L/min oxygen. Once anaesthetized, set isoflurane at 1.5 - 2.5% for maintenance. Inject 150 mg/kg body weight of D-luciferin into mice by intraperitoneal (i.p.) injection.
  6. Immediately transfer mice into the IVIS system, place them onto the temperature-controlled imaging platform and place their nose into the nose cone.
  7. Click on the Sequence Setup button on the control panel and select Medium Binning.
  8. Set up the imaging exposure time as follows: 10 s, 20 s, 30 s, 60 s, and 120 s. Start imaging approximately 3 min after the luciferin injection and continue to image until the signal starts to drop (normally this will take around 20 min).
  9. Remove the mice from IVIS while maintaining isoflurane anaesthetic through a nose cone and treat the mice with 50 µL PBS or 50 µL TGF-β signaling inhibitor (50 μg/tumor) via intra-tumor injection.
  10. Put the mice back in the cage and leave them on the heat pad for at least 30 min. Then place the cage back in the holding room.
  11. Repeat IVIS imaging procedure as described above the day following treatment.

9. Data Analysis

  1. Open saved files in Living Image software.
  2. Find the image information using View | Image Information.
  3. Select photos taken at a similar time point with the same exposure time. Load photos as a group.
  4. Uncheck the Individual box in Image Adjust to normalize the intensity.
  5. Select Photon mode to analyze the intensity. Quantify the intensity of the signal by selecting the Region of Interest (ROI) button in ROI Tools.
  6. Drag the ROI circle to the region containing the bioluminescent signal and click the Measure button to acquire the signal intensity.

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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...

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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...

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Disclosures

The authors report no conflicts of interest.

Acknowledgements

This work was supported by grants from the National Health and Medical Research Council (NHMRC) to H-JZ. TMBW is a recipient of an Australia Postgraduate Award from the Australian Government and the Ann Henderson Top-Up Scholarship from Australian Rotary Health in partner with Rotary of Templestowe and Dine for a Cure.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
DMEMThermoFisher1881024Warm in 37 °C waterbath before use
Foetal Bovine SerumScientifix LifeFBS500-SHeat inactivated before use
Recombinant Human TGF-β1PEPROTECH100-21Aliquot in DDW to make final concentration at 10 mg/mL
Hoechst-33258Tocris Bioscience5117Dilute in to PBS to make final concentration at 1 μL/mL
Luciferase Reporter Assay KitPromega197897Dilute 5x in PBS before use
LuminometerPromega9100-002
Phase contrast fluorescence microscopyOLYMPUSIX50
Centrifugeeppendorf5810 R
VivoGl LuciferinPromegaP1041
IVIS Lumina III In Vivo Imaging SystemPerkinElmerCLS136334
0.5% Trypsin-EDTA (10x)ThermoFisher15400-054Diltue to 0.05% (1x) in PBS
Cell Culture Lysis 5x ReagentPromegaE153ADilute to 1x in DDW
10% FormalinSigma-AldrichF5554-4L
HEK 293AThermoFisherR70507
MDA-MB-231ATCCCRM-HTB-26
PRKDC-SCIDAnimal Resources CentreSCIDF6
MatrigelCorning354234
IsofluraneZoetis26675-46-7
EthanolChem-supplyEA043-10L-P
Refresh Night TimeAllergan1750DLubricating Eye Ointment
SolutionComposition
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

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TGF beta Smad3 SignalingIn Vitro ImagingIn Vivo ImagingMDA MB 231 CellsCAGA12 Td Tomato ReporterCAGA12 Luciferase ReporterFluorescence MicroscopyIVIS Imaging
Live Cell Imaging of the TGF- β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System
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