Method Article

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

DOI:

10.3791/60687

⸱

December 19th, 2019

 ,  , 
1Department of Molecular and Cellular Physiology, Albany Medical College, 2Department of Neuroscience and Experimental Therapeutics, Albany Medical College

In This Article

Summary

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

Introduction

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Cancer remains the second leading cause of death worldwide1 and metastasis is responsible for the majority of these deaths2,3. However, a limited understanding of the molecular mechanisms that govern metastatic colonization and subsequent growth has hindered the development of effective treatments for metastatic disease. The identification of novel therapeutic targets requires an assay to test how perturbed expression or function of a candidate gene influences metastasis formation and growth. While autochthonous mouse models have their advantages, they are time-consuming and expensive to generate, making them more suited for target validation rather than target discovery. Transplant model systems in which the candidate gene is perturbed in cancer cells in vitro and then effects on metastatic potential are assessed in vivo, are less expensive and higher throughput than autochthonous models. In addition, viral vectors for stable delivery of RNAi, CRISPR/CAS9, and transgenes are widely available, making it relatively easy to perturb virtually any gene or genes of interest in a cancer cell lines. This approach can also be used to assay the role of candidate genes in metastatic colonization and growth in human cancer cell lines by transplanting the cells into immunocompromised or humanized mice.

The two types of assays used to test metastasis formation by transplanted cancer cells in vivo are spontaneous metastasis assays and experimental metastasis assays. In spontaneous metastasis assays4,5, cancer cells are injected into mice, allowed to form a primary tumor, and then spontaneous metastasis formation and subsequent growth are assayed. The strength of this model is that the cells must complete all steps of the metastatic process in order to form metastatic tumors. However, many cancer cell lines do not metastasize efficiently in spontaneous metastasis models, and any manipulation of the cells that impacts primary tumor growth can confound the results of the metastasis assay. Experimental metastasis assays, in which cancer cells are injected directly into circulation, are used to avoid these pitfalls. Common experimental metastasis assays include the tail vein injection6,7,8 (and demonstrated here), intracardiac injection9, and portal vein injection10.

The purpose of the protocol presented here is to provide an in vivo experimental metastasis assay that allows a researcher to monitor metastasis formation and growth in real time, as well as to quantify end point metastasis number and size in the lungs of the same mouse. To accomplish this, traditional experimental tail vein metastasis assays6,7,8 are combined with live animal imaging, using an in vivo imaging device9,11,12,13,14. Tumor cells stably expressing both luciferase and a fluorescent protein are injected into mice via the lateral tail vein and then the in vivo imaging device is used to measure changes in metastatic burden in the lungs over time (Figure 1). However, the in vivo live animal imaging device cannot distinguish or measure the size of individual metastases. Thus, at the end of the experiment, a fluorescent stereomicroscope is used to count the number and measure the size of the fluorescent metastases in the lungs without the need for sectioning and histology or immunohistochemistry (Figure 1). This protocol can be used to test how altering the expression or function of a candidate gene influences metastasis formation and growth. Potential therapeutic compounds such as small molecules or function blocking antibodies can also be tested.

To demonstrate this approach, we first performed a proof of concept experiment in which the essential replication factor, replication protein A3 (RPA3) is knocked down in metastatic mouse breast cancer cells. We show that mice injected with RPA3 knockdown cells have significantly less metastatic burden at every time point compared to mice injected with control cells. Analysis of the metastasis-containing lungs shows that this reduced metastatic burden is the result of significantly reduced metastatic colonization and impaired growth of the metastases that form. To further demonstrate this technique, we tested whether simultaneous knock down of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) impairs metastatic colonization or subsequent growth. YAP and TAZ are two related transcriptional co-activators that are the critical downstream effectors of the Hippo Pathway. We15,16 and others have implicated YAP and TAZ in metastasis (reviewed in17,18,19), suggesting that these proteins are good therapeutic targets. Consistently, we found that mice injected with YAP/TAZ knockdown cells had significantly reduced metastatic burden. Analysis of the lungs showed that the YAP/TAZ knockdown cells formed many fewer metastases and that the metastases that did form were smaller. These experiments demonstrate how experimental metastasis assays allow a researcher to quickly and inexpensively test the role of a candidate gene in metastasis formation and growth. They further show how the combined use of live animal imaging and fluorescent quantification of metastases in whole lungs allows the researcher to better understand the steps during metastatic colonization.

Access restricted. Please log in or start a trial to view this content.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

This protocol involves the use of mice and biohazardous materials and requires approval from the appropriate institutional safety committees. All of the described in vivo work here is approved by the Albany Medical College institutional animal care and use committee (IACUC).

NOTE: For a protocol overview, see schematic in Figure 1.

1. Packaging all required retroviruses and lentiviruses

NOTE: The described protocol uses lentiviral or retroviral vectors to stably express a luciferase enzyme and fluorescent protein as well as to manipulate the expression of a candidate gene. These viruses are packaged in HEK-293FT cells as described below.

  1. Day 1: Plate HEK-293FT cells on 6-well plates in 2 mL of complete growth media (10% FBS in DMEM with 1% penicillin/streptomycin and 2 mM L-Glutamine) so that they are 40-60% confluent on day 2. Incubate at 37 °C, 5% CO2 overnight.
  2. Day 2: For each viral vector to be packaged, transfect a 40-60% confluent well of HEK-293FT cells with the retroviral or lentiviral vector and the appropriate vectors encoding the viral coat and packaging proteins.
    NOTE: This protocol uses VSVG as the coat protein, psPAX2 for lentiviral packaging, and gag-pol for retroviral packaging (see Supplemental Table 1 for vector list).
  3. Make a co-transfection mixture for each well as follows:
    1. Combine 4 μL of lipid solution for transfections (see Table of Materials) and 96 μL of transfection buffer and incubate for 5 minutes.
    2. Add 1 μg of viral vector, 0.5 μg of coat protein vector (VSVG), and 0.5 μg of packaging protein vector (psPAX2 or gag-pol) and incubate for 20 minutes.
    3. Gently add the co-transfection mixture from step 1.3.2 to the HEK-293FT cells plated in step 1.1 and incubate at 37 °C, 5% CO2 overnight.
  4. Day 3: Remove the transfection-containing media from each well and gently add 2 mL of complete growth media to each well. Incubate at 37 °C, 5% CO2 for approximately 24 hours.
  5. Day 4: Collect the viral supernatant from each well using a 3 mL syringe and filter through a 0.45 μm filter into a 2 mL microcentrifuge tube.
  6. Optional: If collection of a second batch of virus is desired, gently add 2 mL of complete growth media to each well and incubate at 37 °C, 5% CO2 for approximately 24 hours.
  7. Day 5 (Optional): Collect a second round of viral supernatant as in step 1.5.
    NOTE: The protocol may be paused by freezing the viral supernatant.

2. Generation of cancer cells stably expressing luciferase and a fluorescent protein

NOTE: The following protocol describes how to first to stably label 4T1 cells with firefly luciferase and a fluorescent protein (ZsGreen) using two vectors with unique selection genes. Then a 3rd viral vector is used to manipulate the expression of a candidate gene. However, viral vectors that simultaneously deliver a fluorescent protein and a genetic manipulation can also be used as an alternative (as in the representative experiments below). Other cancer cells can be used, but the cell numbers should be optimized for steps 2.1 and 2.7.1.

  1. Plate 4T1 cells at 1.5 x 105 cells/well in a 12-well plate in complete growth media (10% FBS in DMEM with 1% penicillin/streptomycin and 2 mM L-Glutamine) and incubate at 37 °C, 5% CO2 overnight.
  2. Infect the 4T1 cells with the viral supernatant generated in step 1 as follows.
    1. Aspirate the growth media from the cells and add 500 μL of luciferase viral supernatant and 500 μL of fluorescent protein viral supernatant to simultaneously infect the cells with both the luciferase and fluorescent protein viral supernatants.
    2. Add 1 μL of 8 mg/mL hexadimethrine bromide (see Table of Materials).
    3. Incubate the cells at 37 °C, 5% until 75-90% confluent (typically 24-48 hours).
  3. Trypsinize the 4T1 cells with 500 μL of trypsin for 2-5 minutes (cells should freely rinse off the bottom of the well). Transfer all the cells to a 6 cm dish in 4 mL of selection media (complete growth media containing the appropriate antibiotics) thereby quenching the trypsin. Plate a 6 cm dish of non-infected control cells in the same selection media.
    NOTE: Appropriate antibiotic concentration should be determined ahead of time by testing several doses. Additionally, fluorescence-activated cell sorting can also be used to select the fluorescently labeled cells in place of drug selection.
  4. Incubate the cells at 37 °C, 5% CO2 in selection media and split the cells when necessary until all non-infected control cells are dead (dependent upon the selection gene and cell line).
  5. Confirm that the infected 4T1 cells are expressing the ZsGreen fluorescent protein using a green excitation (450-490 nm)/emission (500-550 nm) filter set on an inverted fluorescent microscope at 50-100x magnification.
  6. Confirm that the infected 4T1 cells are expressing luciferase using a commercially available luciferase activity kit as follows.
    1. Use a minimal volume of 1x passive lysis buffer to lyse luciferase-expressing 4T1 cells and control 4T1 cells that do not express luciferase, gently shaking for 30 minutes.
    2. Add 20 µL of cell lysate to a white-bottom 96-well plate and then 50 µL of the luciferase assay reagent from the luciferase activity kit.
    3. Use a plate reader to measure the luminescence from the cells at all wavelengths in the spectrum by using the luminescence setting.
      NOTE: The protocol may be paused by freezing down the stably-transduced cells.
  7. Manipulate the expression of the candidate gene as follows:
    1. Plate 6 x 105 labeled 4T1 cells from step 2.6 on a 60 mm dish in 4 mL of complete growth media and incubate at 37 °C, 5% CO2 overnight.
    2. Aspirate the growth media from each well and add 2 mL of complete growth media containing 4 μL of 8 mg/mL hexadimethrine bromide.
    3. Add 2 mL of viral supernatant from step 1 to the cells plated from step 2.7.2 and incubate at 37 °C, 5% CO2 overnight.
    4. Trypsinize the 4T1 cells with 500 μL of trypsin for 2-5 minutes (cells should freely rinse off the bottom of the well). Transfer all of the cells to a 10 cm dish in 10 mL of selection media (complete growth media containing the appropriate antibiotics) thereby quenching the trypsin. Plate some non-infected control labeled 4T1 cells in the same selection media.
    5. Incubate the cells at 37 °C, 5% CO2 in selection media and split the cells when necessary until all non-infected cells are dead.
    6. Confirm that the expression of the candidate gene is altered using a standard approach such as western blot20 or qPCR21.
    7. Assay luminescence and fluorescence as in steps 2.5-2.6 to determine how similar they are in control and knockdown cells, as this can change (see Discussion).
      NOTE: The protocol may be paused by freezing down the stably-transduced cells.

3. Optimization of the in vivo experimental design

  1. Optimize appropriate cell number and duration of experiment for the desired metastatic burden as follows.
    1. Expand the fluorescent and bioluminescent 4T1 cells from step 2.6 in complete growth media so that excess cells are available on the desired day of injection.
    2. Prepare the cells for tail vein injections as described in step 4.2. To determine the optimal cell number for injections, resuspend the cells at several different concentrations in 100 μL of 1x PBS.
      NOTE: We recommend testing a range of cell numbers/mouse from 25,000 up to 500,000.
    3. Keep the cell suspensions on ice until injection.
    4. Inject each dilution of 4T1 cells from step 3.1.2 into 3-4 mice via the lateral tail vein described in step 4.3 (see below).
    5. Return the mouse to its cage and monitor for 15 minutes to ensure a full recovery. Mice should be checked for signs of pain or distress 3x weekly.
    6. Monitor mice for metastasis formation and growth for 3-6 weeks (cell line and mouse strain dependent) using an in vivo live animal imaging device (see steps 5 and 6).
      1. Euthanize mice 3-6 weeks after the tail vein injection according to standard institutional guidelines.
      2. Prepare the lungs and assess metastasis size and number described in step 7.
      3. Choose the appropriate length of time for the metastases to grow for the desired metastatic burden (see discussion).

4. Tail vein injection of labeled cancer cells

NOTE: Step 4.2.4 has been optimized for 4T1 cells growing in syngeneic BALB/c mice. If other cancer cell lines and mouse strains are used, the number of cells injected, and the length of the assay should first be optimized.

  1. Expand the 4T1 cell lines generated in step 2 on two 15 cm dishes in complete growth media so that excess cells are available on the day of injection.
  2. Prepare the cells for tail vein injection as follows:
    1. Aspirate the media and rinse the cell plates with 1x PBS.
    2. Trypsinize the cells with 5 mL of trypsin per 15 cm plate for 2-5 minutes (cells should freely rinse off the bottom of the well). Transfer all of the cells to a conical tube. Wash remaining cells from the tissue culture dish with enough complete growth media to quench the trypsin and add the wash to the same conical tube.
    3. Count the cells using an automated cell counter to determine the total cell number.
    4. Centrifuge the cells at 122 x g for 3 minutes, aspirate the supernatant, and resuspend the cells in 1x PBS at the desired concentration. Here, 2.5 x 104 cells are injected into each mouse in 100 μL of PBS, so the resuspend cells at 2.5 x 105 cells/mL. Keep the cell suspensions on ice until injection.
      NOTE: it is important to limit the amount of time between trypsinization of the cells and the tail vein injection to roughly 1 hour.
  3. Inject 4T1 cells from step 4.2.5 into mice via the lateral tail vein as follows:
    1. Working in a hood at the animal facility, gently but thoroughly mix the cells by inverting the tube or using a 1 mL syringe to ensure that they are uniformly resuspended. Always ensure the cells are resuspended prior to loading the syringe.
    2. Load a 1 mL Luer-lock syringe with cell suspension and expel excess air bubbles. Place a ½ inch, 30-gauge needle on the syringe with the bevel up and expel air bubbles.
    3. Gently place the mouse in a rodent restrainer.
    4. The lateral tail veins should be visible and dilated. If not, gently pinch the base of the tail and dip the tail in warm tap water to dilate the veins.
      NOTE: Dilation of the veins may also be achieved by placing the mouse cage under a heating lamp and/or on top of a heating pad.
    5. Use an alcohol wipe to clean the tail. Insert the needle into the tail vein, bevel side up, and inject 100 μL of cell suspension.
      NOTE: If the needle is inserted properly into the vein, it should easily slide slightly forward and back, and there should not be resistance when the plunger is pushed. Successful injections should also result in a "flush" in which the blue color of the vein turns white for a few seconds following the injection.
  4. Slowly remove the needle and using a sterile gauze, apply pressure to the injection site to stop any bleeding.
  5. Return the mouse to its cage and monitor for 15 minutes to ensure full recovery. Mice should be checked for signs of pain or distress 3x weekly.
  6. Monitor mice for metastasis formation and growth for 3-6 weeks (cell line and mouse strain dependent) using an in vivo live animal imaging device (see steps 5 and 6).

5. Monitoring the metastatic burden by fluorescence with an in vivo live animal imaging device

NOTE: Do not image animal for fluorescence with active luminescent signal.

  1. If only imaging bioluminescence, skip to step 6.
  2. Turn on the in vivo live animal imaging device.
  3. Set up the in vivo live animal imaging anesthesia system according to manufacturer's guidelines to deliver between 1.5% and 2% isoflurane to the anesthesia chamber and the imaging chamber.
  4. Anesthetize mice with fluorescently labeled metastases from step 4 by placing them in an anesthesia chamber and delivering 1.5-2.5% isoflurane.
  5. Open the image software (see Table of Materials) and login.
  6. Click the Initialize button and wait for the machine to initialize.
  7. Change the Field of View to D.
    NOTE: Field of View C can also be used if a closer view of a mouse is required, but this limits the number of mice that can be imaged simultaneously.
  8. Once the software has indicated that the camera has reached the appropriate temperature, click the Imaging Wizard button, choose Fluorescence and then choose the appropriate filter pair from the drop-down menu.
    NOTE: If the fluorophore being used is not an option, choose Input EX/EM and type the excitation and emission required.
  9. Place the mouse in the chamber as described in step 3.2.4.
  10. Click Acquire Sequence.
  11. After imaging, return the mouse to its cage and monitor for 15 minutes to ensure full recovery.
  12. Repeat step 5.2 and steps 5.7-5.9 with at least one mouse that does not contain metastases. NOTE: This mouse will be used to quantify and subtract background signal during analysis (step 8).
  13. Image 2-3 times weekly for the duration of the experiment.

6. Monitoring the metastatic burden by bioluminescence with an in vivo live animal imaging device

  1. Turn on the in vivo live animal imaging device and setup the program as follows.
    1. Open the image software (see Table of Materials) and login. Click the Initialize button and wait for the machine to initialize. Change the Field of View to D.
      NOTE: Field of View C can be used for a closer view to image the full mouse body; however, this limits the number of mice that can be imaged at once.
    2. For first time use, edit exposure settings as follows: click Edit | Preferences | Acquisition | Auto Exposure and change the maximum exposure time from the default 60 seconds to 300 seconds and click OK.
      NOTE: Do not change any other parameters in the auto exposure tab.
  2. Set-up the anesthesia system according to manufacturer's guidelines to deliver between 1.5% and 2% isoflurane to the anesthesia chamber and the imaging chamber.
  3. Ensure the camera has reached the appropriate temperature before proceeding to step 6.4.
  4. Anesthetize metastasis-containing mice from step 4 by placing them in an anesthesia chamber and delivering 1.5-2.5% isoflurane.
  5. Prepare mice for bioluminescence imaging as follows.
    1. Load a 1 mL syringe with D-luciferin (30 mg/mL in D-PBS) and then add a ½ inch 30-gauge needle to the syringe and expel air bubbles.
    2. Measure and record the mass of the anesthetized mouse.
    3. Restrain the mouse by pinching the scruff of their neck using the thumb and pointer fingers and grasping the tail between the pinkie finger and the base of the hand. Invert the mouse at a 45-degree angle, with its head pointed downward.
    4. Insert the needle, bevel side up, into the mouse's left side intraperitoneal (IP) space. Confirm entry into the IP space by drawing back a small volume. There should not be color at the base of the needle when drawing back in the IP space.
    5. Inject the appropriate volume of D-luciferin for a dose of 150 mg/kg.
    6. Immediately after D-luciferin administration, start a timer and place the mouse flat on its back in the imaging device with its nose in the nose cone and ensure that 1.5-2.5% isoflurane is being administered. Place dividers between each mouse if imaging multiple mice. Ensure mice are positioned as flat as possible (i.e. not leaning to one side).
    7. Click the Luminescent and Photograph boxes and then click Acquire in the Acquisition Control Panel.
  6. Continuously acquire bioluminescent images until the peak signal is achieved and use the image with the peak bioluminescent signal for analysis.
  7. After imaging, return the mouse to its cage and monitor for 15 minutes to ensure full recovery.
  8. Image 2-3 times weekly for the duration of the experiment.

7. Quantification of the number and size of metastases

NOTE: The length of time the metastases are allowed to grow should be determined for each cell line and mouse strain, and will be influenced by the number of cells injected.

  1. Euthanize the mice according to standard institutional guidelines.
  2. Isolate and remove the lungs from each mouse and rinse in 1x PBS to remove excess blood.
  3. Gently separate the lungs into lobes.
  4. Acquire images of ZsGreen metastases in the lobes at 10x in bright field and fluorescence using a fluorescent stereoscope with a GFP wideband filter (excitation 470/40x).
    NOTE: Maintain the same magnification and brightness for all samples. The magnification used may vary depending on the size, number, and brightness of metastases as well as the field of view for the microscope used.
  5. Use image analysis software to quantify the size and number of metastases from the images.
    NOTE: The protocol for image analysis is software dependent and could be optimized with tumors from step3. Alternatively, count the number of metastases in each lung manually using the fluorescent stereoscope. Protocol can be paused here and step 8 can be done at any point after all the in vivo images have been collected.

8. Processing and analysis of the data from the images acquired with the in vivo live animal imaging device

  1. Open all image files for each mouse in the image software.
    NOTE: Use the image with the peak bioluminescent signal for analysis
  2. Ensure the units are in Radiance for bioluminescent data and Efficiency for fluorescent data by clicking the arrow at the top left of the image window and changing it to the appropriate unit.
  3. Use the image from the last timepoint to create a region of interest (ROI) as follows.
    1. Click ROI Tools in the Tool Palette window. Insert one ROI by clicking the arrow and selecting 1.
    2. Click the border of the ROI and move it over the chest of the mouse. Adjust the size of the ROI so it covers the chest of the mouse and does not exclude signal.
  4. Click measure ROIs and copy or type the raw number into an excel sheet.
    NOTE: For bioluminescent data select the total flux (photons/second), which is the sum of all the radiance in the ROI. Since metastases do not necessarily grow uniformly, the total flux is preferred over average radiance because it measures the sum of the metastatic burden. Similarly, for fluorescent data, the total efficiency % (emission light (photons/second)/excitation light (photons/second)) should be used instead of average efficiency.
  5. Right click in the image file to copy the ROI used in step 8.3 and paste it into every image file.
    NOTE: When quantifying fluorescent images, quantify the same region on a mouse that was imaged with no metastasis. Use this signal as the background signal and subtract it from each fluorescent metastasis-containing mouse image acquired.
  6. Move pasted ROIs over the same region selected in step 8.3.4 for each image and repeat step 8.4.
  7. Plot and analyze the raw data as follows (see Supplemental Table 2).
    1. Do a log10 transformation of the raw data for each mouse using the indicated formula (Supplemental Table 2) and plot as in Figure 2D and Figure 4F. The log10 transformation linearizes the growth curve which tends to be geometric and minimizes heteroscedasticity.
    2. Using linear regression, calculate the slope of the fitted line to the log10 transformed data for each mouse plotted in step 8.7.1 (Supplemental Table 2). Refer to the formula in Supplemental Table 2 to fit the line and calculate the slope in one step.
    3. Plot the numerical values of the slopes as in Figure 2E and Figure 4G. Use a Student's t-test or one-way ANOVA (for more than 2 groups) on the slopes to determine statistical significance.

Access restricted. Please log in or start a trial to view this content.

Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

To demonstrate the above approach, we performed a proof of concept experiment in which a critical replication factor, RPA3 was knocked down in a metastatic mouse mammary carcinoma cell line (4T122). While the protocol describes labeling the cells with both luciferase and fluorescent proteins prior to genetic manipulation, we used a modified approach because THE RNAi vectors also deliver ZsGreen (Figure 2A). First, 4T1 cells were stably transduced with...

Access restricted. Please log in or start a trial to view this content.

Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Critical steps of the method
It is critical to optimize the number of cells injected (step 3) for a given cell line and mouse strain as this can greatly influence the number of metastases that form and the length of the experiment. If too many cells are injected or the metastases grow for too long, the metastases may be difficult to count making the effects of the genetic manipulation difficult to assess. However, if too few cells are injected, few or no metastases may form. Thus, a preliminary exp...

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

We thank Emily Norton for assisting with viral infections and critical reading of the manuscript. We also thank Ryan Kanai for help with acquiring images of the lungs and Kate E. Tubbesing for help with image analysis of the green metastases in the lungs. We thank the animal research facility staff for their support and for assistance in the preparation of this video. This work was supported by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

Access restricted. Please log in or start a trial to view this content.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
10% SDS-PAGE GelFor western blot
2.5% TrypsinGibco15090-046Trypsin for tissue culture
96 well flat bottom white assay plateCorning3922For measuring luciferase and renilla signal in cultured cells
Alcohol wipesFor sterolizing the injection site before tail vein injecitons
BALB/C mice (female, 6 weeks)TaconicBALB-FFor tail vein metastatic colonization and burden assays
BSA regularVWR Ameresco97061-416For western blot
Cell lysis bufferCell SignalingFor collecting protien samples
Celltreat Syringe Filters, PES 30mm, 0.45 μmCelltreat40-229749-CSFor filtering viral supernatant
CO2 and euthanasia chamberFor euthanasing the mice
Dual-luciferase reporter assay kitPromegaE1960For measuring luciferase and renilla signal in cultured cells
Dulbecco&39;s phosphate buffered salineHimediaTS1006For PBS
EDTAVWR97061-406Used to dilute trypsin for tissue culture
FBS 100% US originVWR97068-085Component of complete growth media
Fujifilm LAS-3000 gel imagerFujifilmFor western blot
GAPDH(14C10) Rabibit mAbCell Signaling2118For western blot
Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugateThermo Scientific31460For western blot
Human embryonic kidney cells, HEK-293FTInvitrogenR70007Cell line used for packging virus
HyClone DMEM/High clucoseGE Healthcare life sciencesSH30243.01Component of complete growth media
Hygromycin B, Ultra Pure GradeVWR Ameresco97064-810For antibiotic selection of infected cells
I3-P/i3 Multi-Mode Microplate/EAMolecular devicesFor measuring luciferase and renilla signal in cultured cells
ImagejUsed for image analysis of lung metastases: threshold set to 25 & 100
Immuno-Blot PVDF MembraneBiorad1620177For western blot
IsofluraneFor mouse anesthesia
IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device)PerkinElmerCLS136340For in vivo imaging of metastatic burden
Leica M205 FA & Lecia DCF3000 G (GFP and bright field filters)Leica MicrosystemsMicroscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain
L-GlutamineGibco25030-081Component of complete growth media
Lipofectamine 3000Life technologiesL3000008For YAP/TAZ-TEAD reporter transfection
Living Image 3.2 (image software program)PerkinElmerSoftware for IVIS
Mouse breast cancer cells, 4T1Karmanos Cancer InstituteAslakson, CJ et al.,1992Mouse metastatic breast cance cell line
Multi-Gauge version 3.0FujifilmSoftware for quantifying western blot band intensity
Opti-MEM (transfection buffer)Gibco31985-062For packaging virus and transfection
Penicillin StreptomycinGibco15140-122Component of complete growth media
Pierce BCA protein assay kitThermo Scientific23225For quantifying protein concentration
Pierce Phosphatase Inhibitor Mini TabletsThermo ScientificA32957Added to cell lysis buffer
Pierce Protease Inhibitor Mini TabletsThermo ScientificA32953Added to cell lysis buffer
Polybrene (hexadimethrine bromide)Sigma-Aldrich45-H9268For infection
PuromycinSigma-Aldrich45-P7255For antibiotic selection of infected cells
Rodent restrainerFor restraining mice during tail vein injeciton
SDS-PAGE running bufferFor western blot
TAZ (V3886) AntibodyCell Signaling4883For western blot
TBST bufferFor western blot
TC20 automated cell counterBio-RadFor counting cells
VectorsSee Table 1 for complete list of vectors
VWR Inverted Fluorescence MicroscopeVWR89404-464For visualizing fluorescence in ZSGreen labeled cells
Western transfer bufferFor western blot
XenoLight D-Luciferin K+ SaltPerkinElmer122799Substrate injected into mice for in vivo bioluminescent IVIS images
X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection)Roche6365787001For packaging virus
YAP (D8H1X) XP Rabbit mAbCell Signaling14074For western blot

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Ferlay, J. S. I., et al. GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11. , Available from: http://globocan.iarc.fr (2013).
  2. Gupta, G. P., Massague, J. Cancer metastasis: building a framework. Cell. 127 (4), 679-695 (2006).
  3. Chaffer, C. L., Weinberg, R. A. A perspective on cancer cell metastasis. Science. 331 (6024), 1559-1564 (2011).
  4. Wendt, M. K., Molter, J., Flask, C. A., Schiemann, W. P. In vivo dual substrate bioluminescent imaging. Journal of Visualized Experiments. (56), (2011).
  5. Moret, R., et al. Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis. Journal of Visualized Experiments. (147), (2019).
  6. Lizardo, M. M., Sorensen, P. H. Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay. Journal of Visualized Experiments. (133), (2018).
  7. Mohanty, S., Xu, L. Experimental metastasis assay. Journal of Visualized Experiments. (42), (2010).
  8. Welch, D. R. Technical considerations for studying cancer metastasis in vivo. Clinical and Experimental Metastasis. 15 (3), 272-306 (1997).
  9. Lim, E., et al. Monitoring tumor metastases and osteolytic lesions with bioluminescence and micro CT imaging. Journal of Visualized Experiments. (50), (2011).
  10. Goddard, E. T., Fischer, J., Schedin, P. A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer. Journal of Visualized Experiments. (118), (2016).
  11. Cordero, A. B., Kwon, Y., Hua, X., Godwin, A. K. In vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer. Journal of Visualized Experiments. (42), (2010).
  12. Ozawa, T., James, C. D. Establishing intracranial brain tumor xenografts with subsequent analysis of tumor growth and response to therapy using bioluminescence imaging. Journal of Visualized Experiments. (41), (2010).
  13. Lim, E., Modi, K. D., Kim, J. In vivo bioluminescent imaging of mammary tumors using IVIS spectrum. Journal of Visualized Experiments. (26), (2009).
  14. Oshima, G., et al. Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones. Journal of Visualized Experiments. (117), (2016).
  15. Lamar, J. M., et al. SRC tyrosine kinase activates the YAP/TAZ axis and thereby drives tumor growth and metastasis. Journal of Biological Chemistry. 294 (7), 2302-2317 (2019).
  16. Lamar, J. M., et al. The Hippo pathway target, YAP, promotes metastasis through its TEAD-interaction domain. Proceedings of the National Academy of Sciences U S A. 109 (37), 2441-2450 (2012).
  17. Warren, J. S. A., Xiao, Y., Lamar, J. M. YAP/TAZ Activation as a Target for Treating Metastatic Cancer. Cancers (Basel). 10 (4), (2018).
  18. Janse van Rensburg, H. J., Yang, X. The roles of the Hippo pathway in cancer metastasis. Cell Signal. 28 (11), 1761-1772 (2016).
  19. Harvey, K. F., Pfleger, C. M., Hariharan, I. K. The Drosophila Mst ortholog, hippo, restricts growth and cell proliferation and promotes apoptosis. Cell. 114 (4), 457-467 (2003).
  20. Journal of Visualized Experiments Science Education Database. Basic Methods in Cellular and Molecular Biology. The Western Blot. Journal of Visualized Experiments. , (2019).
  21. Wong, W., Farr, R., Joglekar, M., Januszewski, A., Hardikar, A. Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs. Journal of Visualized Experiments. (98), (2015).
  22. Aslakson, C. J., Miller, F. R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Ressearch. 52 (6), 1399-1405 (1992).
  23. Fellmann, C., et al. Functional identification of optimized RNAi triggers using a massively parallel sensor assay. Molecular Cell. 41 (6), 733-746 (2011).
  24. Reticker-Flynn, N. E., et al. A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis. Nature Communications. 3, 1122(2012).
  25. Sun, Z., et al. Prognostic Value of Yes-Associated Protein 1 (YAP1) in Various Cancers: A Meta-Analysis. Public Library of Science One. 10 (8), 0135119(2015).
  26. Feng, J., Ren, P., Gou, J., Li, Z. Prognostic significance of TAZ expression in various cancers: a meta-analysis. Onco Targets and Therapy. 9, 5235-5244 (2016).
  27. Ge, L., et al. Yes-associated protein expression in head and neck squamous cell carcinoma nodal metastasis. Public Library of Science One. 6 (11), 27529(2011).
  28. Zhang, X., et al. The Hippo pathway transcriptional co-activator, YAP, is an ovarian cancer oncogene. Oncogene. 30 (25), 2810-2822 (2011).
  29. Vlug, E. J., et al. Nuclear localization of the transcriptional coactivator YAP is associated with invasive lobular breast cancer. Cellular Oncology (Dordrecht). 36 (5), 375-384 (2013).
  30. Kim, T., et al. MRTF potentiates TEAD-YAP transcriptional activity causing metastasis. European Molecular Biology Organization Journal. 36 (4), 520-535 (2017).
  31. Nallet-Staub, F., et al. Pro-invasive activity of the Hippo pathway effectors YAP and TAZ in cutaneous melanoma. Journal of Investigative Dermatology. 134 (1), 123-132 (2014).
  32. Hsu, Y. L., et al. Angiomotin decreases lung cancer progression by sequestering oncogenic YAP/TAZ and decreasing Cyr61 expression. Oncogene. 34 (31), 4056-4068 (2015).
  33. Lau, A. N., et al. Tumor-propagating cells and Yap/Taz activity contribute to lung tumor progression and metastasis. European Molecular Biology Organization Journal. 33 (5), 468-481 (2014).
  34. Gu, J. J., et al. Inactivation of ABL kinases suppresses non-small cell lung cancer metastasis. Journal of Clinical Investigation Insight. 1 (21), 89647(2016).
  35. Diepenbruck, M., et al. Tead2 expression levels control the subcellular distribution of Yap and Taz, zyxin expression and epithelial-mesenchymal transition. Journal of Cell Science. 127, Pt 7 1523-1536 (2014).
  36. Han, S., et al. Suppression of miR-16 promotes tumor growth and metastasis through reversely regulating YAP1 in human cholangiocarcinoma. Oncotarget. 8 (34), 56635-56650 (2017).
  37. Yin, K., et al. Netrin-1 promotes metastasis of gastric cancer by regulating YAP activity. Biochemical Biophysical Research Communications. 496 (1), 76-82 (2018).
  38. Guo, L., et al. Knockdown of TAZ modifies triple-negative breast cancer cell sensitivity to EGFR inhibitors by regulating YAP expression. Oncology Reports. 36 (2), 729-736 (2016).
  39. Liu, Y. N., et al. Loss of Androgen-Regulated MicroRNA 1 Activates SRC and Promotes Prostate Cancer Bone Metastasis. Molecular and Cellular Biology. 35 (11), 1940-1951 (2015).
  40. Bartucci, M., et al. TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells. Oncogene. 34 (6), 681-690 (2015).
  41. Wang, T., et al. YAP promotes breast cancer metastasis by repressing growth differentiation factor-15. Biochimica et Biophysica Acta Molecular Basis of Disease. 1864, 5 Pt A 1744-1753 (2018).
  42. Wang, J., Rouse, C., Jasper, J. S., Pendergast, A. M. ABL kinases promote breast cancer osteolytic metastasis by modulating tumor-bone interactions through TAZ and STAT5 signaling. Science Signaling. 9 (413), (2016).
  43. Sun, S., Irvine, K. D. Cellular Organization and Cytoskeletal Regulation of the Hippo Signaling Network. Trends in Cell Biology. 26 (9), 694-704 (2016).
  44. Sharif, G. M., et al. Cell growth density modulates cancer cell vascular invasion via Hippo pathway activity and CXCR2 signaling. Oncogene. 34 (48), 5879-5889 (2015).
  45. Li, C., et al. A ROR1-HER3-lncRNA signalling axis modulates the Hippo-YAP pathway to regulate bone metastasis. Nature Cell Biology. 19 (2), 106-119 (2017).
  46. Pei, T., et al. YAP is a critical oncogene in human cholangiocarcinoma. Oncotarget. 6 (19), 17206-17220 (2015).
  47. Qiao, Y., et al. YAP Regulates Actin Dynamics through ARHGAP29 and Promotes Metastasis. Cell Reports. 19 (8), 1495-1502 (2017).
  48. Zhou, W., Li, X., Premont, R. T. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes. Journal of Cell Science. 129 (10), 1963-1974 (2016).
  49. Haemmerle, M., et al. Platelets reduce anoikis and promote metastasis by activating YAP1 signaling. Nature Communcations. 8 (1), 310(2017).
  50. Liu, Y., et al. Increased TEAD4 expression and nuclear localization in colorectal cancer promote epithelial-mesenchymal transition and metastasis in a YAP-independent manner. Oncogene. 35 (21), 2789-2800 (2016).
  51. Hiemer, S. E., et al. A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma. Molecular Cancer Research. 13 (6), 957-968 (2015).
  52. Lee, H. J., et al. Fluid shear stress activates YAP1 to promote cancer cell motility. Nature Communications. 8, 14122(2017).
  53. Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. M. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. Journal of Virology. 70 (8), 5701-5705 (1996).
  54. Mahoney, W. M., Hong, J. H., Yaffe, M. B., Farrance, I. K. The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members. Biochemical Journal. 388, Pt 1 217-225 (2005).
  55. Zhao, H., et al. Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. Journal of Biomedical Optics. 10 (4), 41210(2005).
  56. Chen, M. B., Lamar, J. M., Li, R., Hynes, R. O., Kamm, R. D. Elucidation of the Roles of Tumor Integrin beta1 in the Extravasation Stage of the Metastasis Cascade. Cancer Resesearch. 76 (9), 2513-2524 (2016).
  57. Labelle, M., Begum, S., Hynes, R. O. Direct signaling between platelets and cancer cells induces an epithelial-mesenchymal-like transition and promotes metastasis. Cancer Cell. 20 (5), 576-590 (2011).

Access restricted. Please log in or start a trial to view this content.

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Breast Cancer MetastasisLung ColonizationBioluminescence ImagingFluorescence ImagingMetastatic BurdenYAP TAZ KnockdownMetastasis QuantificationIn Vivo Experimental Design

Related Articles