Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Summary

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Abstract

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology.

Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Introduction

Breast cancers are frequent types of cancer worldwide, with approximately 250,000 new cases diagnosed each year in the United States¹. Despite its high incidence, a new set of anticancer drugs has significantly improved breast cancer patient outcomes². However, these treatments are still inadequate, as many patients experience disease relapse and metastatic spread to vital organs², which is the primary cause of patient morbidity and mortality. Therefore, one of the main challenges in breast cancer research is identifying the molecular mechanisms regulating the formation of distal metastases to develop new means to inhibit their development.

Cancer metastasis is a dynamic process in which cells detach from the primary tumor and invade neighboring tissues through the blood circulation. Thus, animal models in which the cells undergo a similar metastatic cascade can facilitate the identification of the mechanisms that govern this process^3,4. Additionally, these in vivo models are essential for developing breast cancer therapeutic agents^5,6. However, these orthotopic models cannot indicate the actual tumor growth kinetics as the effect is only determined upon termination. Therefore, we established a luciferase-based tool to detect tumor development and metastatic colonization in real time. Additionally, these cells express GFP to detect the metastatic colonies. This approach is relatively simple and does not involve any invasive procedures³. Thus, combining luciferase and fluorescence detection is a helpful strategy to advance the preclinical studies of breast cancer therapeutics and disease management.

Protocol

All mouse experiments were carried out under the Hebrew University Institutional Animal Care and Use Committee-approved protocol MD-21-16429-5. In addition, the Hebrew University is certified by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

1. Cell line maintenance

NOTE: The human breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231) were used in this protocol.

1. Culture all the breast cancer cell lines in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in a humidified carbon dioxide (5% CO₂) incubator.
   ​NOTE: Check the cell density regularly; split and expand for future usage when they reach 70% confluency.

2. Virus preparation

1. Treat the HEK293T cells with 1 mL of trypsin until they detach.
2. Add 10 mL of DMEM (10% FBS) to neutralize the trypsin activity, and transfer the cell suspension to a new 15 mL centrifuge tube.
3. Centrifuge at 150 × g for 5 min to sediment the cells. Discard the supernatant after the centrifugation and add 1 mL of fresh DMEM medium.
4. Determine the cell concentration and seed 1.2 × 10⁶ cells/well in a six-well plate.
5. On the following day, prewarm all the necessary reagents, including transfection reagent, serum-free DMEM, envelope plasmid (VSV-G), lentivirus packaging plasmid (ΔVPR), and pLX304 Luciferase-V5 blast plasmid or FUW GFP plasmid.
6. In a 1.5 mL autoclaved centrifuge tube, mix 50 µL of serum-free DMEM with 0.3 µg of VSV-G plasmid, 1 µg of ΔVPR, and 1.2 µg of pLX304 Luciferase-V5 blast plasmid or FUW GFP plasmid.
7. After thoroughly mixing these plasmids with the medium, add 5 µL of the transfection reagent, mix gently, and incubate the mixture at room temperature for 15 min.
8. Following incubation, add the mixture dropwise to the HEK293T cells.
9. After 24 h, replace the growth medium with 2 mL of (30% FBS) DMEM. On the following day (48 h post-transfection), harvest the medium containing the viruses ("pLX304 Luciferase-V5 or the FUW GFP viruses").
   NOTE: The use of 30% FBS enhances the virus production efficiency.
10. To avoid any HEK293T cell residues, pass the virus-containing medium through a 0.45 µm syringe filter or centrifuge at 150 × g for 5 min, and collect the supernatant.
    NOTE: For long-term storage, keep the working aliquots of the virus at -80 °C.

3. Establishing cells stably expressing GFP and luciferase ("GFP  ⁺ Luc⁺ cells")

1. The day before infecting the cells, seed 8 × 10⁵ cells per well in a six-well plate.
2. After overnight incubation, replace the growth medium with fresh medium containing 8 µg/mL of polybrene. Add 200 µL of FUW GFP viruses dropwise to the cells.
3. Optional: To enhance the virus efficiency, centrifuge the plate at 560 × g for 30 min (37 °C) (spin infection).
4. Incubate the cells for 48 h, and verify the GFP expression by fluorescence microscopy.
5. Sort the GFP-expressing cells by fluorescence-activated cell sorting (FACS) (GFP⁺) (Figure 1A).
6. Infect the GFP-sorted cells with the pLX304 Luciferase-V5 blast viruses as described in step 3.2.
7. Treat the cells with blasticidin (10 µg/mL) 30 h post-infection to enrich for pLX304 Luciferase-V5 blast-expressing cells (GFP⁺, Luc⁺ cells). Then, every two days, replace with fresh medium containing blasticidin. Additionally, as a control, treat naïve cells with the same blasticidin-containing medium.
   NOTE: A clear difference between the infected and control cells should be observed after a few days of blasticidin treatment. This effect is cell line-dependent and usually takes ~8-10 days. A poor survival rate will indicate a low viral production yield. If so, produce a new batch of viruses as low efficiency may affect future experiments.

4. Validating in vitro luciferase activity

1. Grow the MCF-7, MDA-MB-468, and MDA-MB-231 GFP⁺ Luc⁺ cells in a 15 cm plate to 80% confluency. Harvest the cells by trypsinization, as described in steps 2.1-2.2.
2. Seed an increasing number of cells in each well (0.1, 0.5, 1, 2, 3, 4 × 10⁴) into a black 96-well plate.
   NOTE: Black 96-well plates are more suited for measuring luciferase levels as white or transparent plates will produce autoluminescence signals. As a control, use phosphate-buffered saline (PBS) alone in one well to ensure there is no autoluminescence from PBS.
3. Fill all the wells with 100 µL of DMEM and incubate for 16-24 h.
4. Prepare luciferin solution in PBS at a 1.5 mg/mL concentration from the stock of 30 mg/mL. Aliquot the stock of luciferin solution and store at -20 °C.
5. Wash the cells once with PBS gently, add 100 µL of luciferin solution into each well, and wait for 2 min. Finally, measure the luciferase activity in all the breast cancer cells using bioluminescence.
   NOTE: Examining in vitro GFP and luciferase expression prior to animal experiments is crucial. Additionally, blank wells are used to subtract the background.

5. Injecting mice with GFP⁺ Luc⁺ cells

1. Transfer 5 × 10⁶ (MCF-7 and MDA-MB-468) or 2 × 10⁶ (MDA-MB-231) GFP⁺ Luc⁺ cells into 200 µL or 100 µL PBS, respectively.
2. Before injection, clean the sterile biological hood with 5% disinfectant solution (see the Table of Materials). Then, anesthetize the mice with filtered (0.2 µm) air containing 4% isoflurane at an airflow rate of 1 L/min for 2−3 min.
   NOTE: It is essential to confirm proper anesthetization; pinch the mouse's toe and look for any response.
3. Place a cone over the anesthetized mouse head in a supine position. Apply vet ointment to its eyes to prevent dryness while under anesthesia.
4. Wipe the abdominal area of the mouse, above the mammary gland, with ethanol using a cotton swab and lift the 4^th mammary gland with forceps.
5. Insert the needle 27 G x 3/4 (0.4 x 19 mm) under the fat pad and slowly inject 100 µL of the cell suspension.
   NOTE: A round bulge will appear under the skin. In addition, an improper injection may lead to deviation in the tumor growth rate or absence of tumor in the same experimental group.
6. After the injection procedure, take the mice out of the hood and transfer them to a new cage. Monitor the mice until they return to consciousness.
   ​NOTE: Ensure that all the used needles and syringes are discarded in the sharps box.

6. Measuring the luciferase levels in GFP⁺ Luc⁺ mice

1. Before the bioluminescence detection, restrain the conscious mouse by holding its neck with the left hand. Then, tilt the hand to the left, resulting in the mouse face upward with the lower body in a supine position.
2. Inject 100 µL of luciferin (30 mg/mL) intraperitoneally (i.p.) into the abdominal surface of the mouse in the lower-left abdominal quadrant using a 1.0 mL syringe with needle size 27 G x 3/4 (0.4 x 19 mm).
   NOTE: The tip of the needle should not be inserted more than 3-5 mm from the abdominal wall, as it might penetrate visceral organs. Additionally, it is recommended to perform i.p. injection without anesthesia as luciferin distribution in the body of anesthetized mice is slower than in conscious mice. Thus, monitoring luciferase levels in conscious mice is a faster procedure.
3. Keep the mouse for 7 min without anesthesia followed by 3 min within the anesthesia chamber before measuring the tumor kinetics.
   NOTE: The incubation time varies between the different experiments, cell lines, and species to species. Thus, it is recommended to calibrate the incubation time before starting the experiments.
4. Anesthetize the mice as described in steps 5.2-5.3.
5. Open the software (Table of Materials) during the incubation, initialize the imaging system, and click the Initialize button.
   NOTE: Be aware that the camera will take ~10 min to cool down and reach -90 °C. Additionally, as background control, measure the luciferase levels in naïve mice (i.e., GFP⁺ mice injected with cells that do not express the luciferase gene).
6. Keep the setup in auto exposure using the following settings: exposure time auto, 60 s; Binning Medium, F/Stop 1; Excitation filter blocked; Emission filter open. When Initialization ends, select Imaging Wizard | Bioluminescence and then click Next | Open Filter | select Mouse in the image subject.
7. In Field View, select stage C (10 cm) and Subject Height: 1.50 cm.
8. Click Image Setup Stage to C, and before clicking the Acquire button, ensure that the mouse is placed on the stage in the proper supine position.
9. Close the door, click the Acquire button, and wait for an image to appear on the screen.
   NOTE: With the Auto-Exposure option, a strong signal takes 5-20 s depending on the signal; a weak signal will take around 60 s.
10. Repeat this step for the other mice.
11. To detect lung metastasis, cover the strong signal of the primary tumor using thick black cardboard paper and expose only the ventral side of the lungs towards the camera. Capture the image using the same parameters described above.

7. Acquiring ex vivo image using bioluminescence and fluorescence

1. Euthanize the mice using carbon dioxide (CO₂) inhalation in the desiccator and dissect the mice using autoclaved scissors and forceps.
2. Perfuse the mice using 0.9% saline, harvest the organs, and rinse with 1x PBS to eliminate bloodstains from the organ.
3. Transfer the rinsed organ to a Petri dish and place it into the stage of the bioluminescence machine. Apply the same bioluminescence setting as described in steps 6.2-6.6.
   NOTE: Due to the decrease in luciferase signal, this step is time-limiting. Thus, after euthanizing mice, immediately visualize the organ by bioluminescence.
4. For GFP images, apply the same setting as described in step 6.3, using Fluorescence GFP filter instead of Bioluminescence.
5. Take lung images using a stereo microscope to examine the presence of GFP⁺ colonies.

8. Bioluminescence data analysis

1. Double-click the software and select Open from the File menu.
2. Open all the files and minimize them. Ensure all the units are Radiance (Photons). Additionally, click Apply to All to keep the same parameters for all the images.
3. From the View menu, select the Tool Palette, which will open a new window.
4. From the Tool Palette window, select the ROI Tools tab and in Type, choose the Average Bkg ROI.
5. From the ROI tools, select Circle, and draw a small circle on the thoracic area of the mouse.
6. Double-click on the circle, select the Use as BKG for future ROIs option from the Background ROI tab, and click Done.

9. Measuring the total flux

1. From the Tool Palette window, select the ROI Tools tab and in Type, choose the Measurement of ROI.
2. From the ROI tools, select Circle, and draw a big circle on the primary tumor of the mouse.
3. Right-click Copy ROI and right-click Paste ROI into every individual file of each mouse.
4. Click Measure ROIs from the ROI Tools tab, which will open a new window named ROI Measurements. From this tab, keep the Measurements Types as Radiance (Photons) and Image Attributes as All Populated Values.
5. Export the file as Measurements File (*.txt) or Csv (*.csv) format.
6. Open the exported data in a spreadsheet and take the Total Flux (p/s) and the values for the weeks.
   NOTE: This step can be used to measure different groups. For example, mice treated with a vehicle vs. those treated with a drug.
7. Generate an XY plot, where the Time is presented along the X-axis and Total Flux (p/s) along the Y-axis. For each week, take the Total Flux (p/s) parameter for each sample group and determine any significant differences using the non-parametric Student's t-test.

Representative Results

We generated breast cancer cell lines (MDA-MB-231, MCF-7, and MDA-MB-468) expressing GFP and luciferase vectors. Specifically, this was achieved by a sequential infection. First, the breast cancer cell lines were infected with a lentivirus vector expressing fluorescent GFP. The GFP-positive cells (GFP⁺) were sorted 2 days post-infection (Figure 1A,B) and infected with the pLX304 Luciferase-V5 vector. Then, blasticidin was used to select for luciferase to generate the indicated (GFP⁺, Luc⁺) cells. To validate the in vitro luciferase activity, we demonstrated a cell number-dependent increase in the luciferase activity levels (Figure 1C). In addition, a linear correlation was found between the luciferase activity and the cell number (Figure 1D).

To confirm luciferase detection in the mice, all three GFP⁺, Luc⁺ breast cancer cell lines were injected into the mammary fat pad of female NOD/SCID mice. Then, the mice were subjected to bioluminescence reading every two weeks to determine the tumor growth kinetics. We found that tumor growth kinetics varies between the cell lines; it is faster in the more aggressive MDA-MB-231 and slower in the less aggressive cell lines MCF-7 and MDA-MB-468 (Figure 2).

Next, the fluorescence readings of the isolated tumors generated by the MDA-MB-231 cell line were obtained. Specifically, 6 weeks post injection, the tumors were harvested from the mice to confirm the GFP fluorescence; the tumors were found to maintain their GFP expression (Figure 3A). The next goal was to determine whether metastatic colony formation could be assessed in real time in the lung of a living mouse using the bioluminescence machine; positive bioluminescence readings were obtained from the lung of the whole mouse (Figure 3B). To verify that these were positive metastatic colonies, the lung was harvested, and the metastatic colonies were observed for GFP and bioluminescence (Figure 3C).

Figure 1: Validation of GFP expression and luciferase activity in cells. (A) The GFP cells were sorted by FACS. Representative images of MDA-MB-231 non-GFP and GFP⁺ cells. (B) MDA-MB-231, MCF-7, and MDA-MB-468 cells were infected with GFP-expressing virus, followed by FACS sorting. An image of each cell line is represented in brightfield (left) and GFP(right). The cells were captured under a Nikon Eclipse 80i microscope at 10x magnification. Scale bars = 100 µm. (C) The bioluminescence due to luciferase activity in each cell was determined by a luminometer. An increasing number of cells (as in A) were seeded in a black 96-well plate. The color bar represents the intensity of luminescence. (D) An XY plot demonstrating the luciferase activity of MDA-MB-231 cells (as measured in C). Abbreviations: GFP = green fluorescent protein; SSC-A = area of side-scattered peak; GFP⁺ = GFP-positive; FACS = fluorescence-activated cell sorting. Please click here to view a larger version of this figure.

Figure 2: Kinetics of tumor growth was determined by the bioluminescence machine. In vivo tumor growth kinetics were determined weekly in NOD-SCID mice, and the representative images were captured using bioluminescence for (A) MDA-MB-231, (B) MCF-7, (C) MDA-MB-468. The color bar represents the intensity of luminescence. (D) Quantification of the luminescence activity is presented as total flux. (E) MDA-MB-231 mice; individual reading represented as a plot. Please click here to view a larger version of this figure.

Figure 3: Different approaches to validate tumor formation and lung metastasis. (A) The mice were perfused, and the tumors generated from MDA-MB-231 cells were harvested. The GFP levels in the tumors were measured by the bioluminescence machine. (B) Lung metastasis in the whole mice, as shown by the bioluminescence. (C) To confirm the presence of metastases, the lungs were harvested and observed under SMZ18 Nikon Stereomicroscope (brightfield and GFP). Bioluminescent-Luc-samples were taken immediately after euthanizing the mice. The color bar represents the intensity of luminescence. Abbreviations: GFP = green fluorescent protein; Luc = luciferase; BLI = bioluminescence imaging. Please click here to view a larger version of this figure.

Discussion

Animal-based experiments are obligatory for cancer research^7,8,9, and indeed many protocols have been developed^{3,6,10,11,12,13,14}. However, most of these studies determined the biological effect only at the end of the experiments, and thus the impact on tumor growth kinetics or metastasis colonization remains undetermined. Here, we provide a noninvasive dual bioluminescence approach by inoculating cells expressing GFP and luciferase into the mammary fat pad. Using this powerful tool, tumor development and metastasis can be monitored in mice in real time¹⁴. However, this technique contains a few critical steps, which demand extra caution. For example, one of the critical steps for the success of this experiment is to verify the efficiency of infection by monitoring the luciferase and GFP expression levels in the cells before mouse injection. Thus, the blasticidin dosages¹⁵ and the lentiviral production¹⁶ protocol should be optimized for each cell line to increase the experimental efficiency.

A few technical issues may affect the bioluminescence signal in the in vivo experiment. These issues include the mouse's movement during the bioluminescence reading, which may interfere with the image quality and thus affect the tumor kinetic curves. Thus, the animals must be fully anesthetized after the substrate injection and during the entire procedure. Additionally, placing multiple animals in the machine simultaneously may lead to inconsistency in luminescence reading as mice with a high signal can mask those of less intensity. Therefore, the luminescence readings must be taken individually for each mouse.

When conducting the in vitro bioluminescence reading, it is vital to replace the culture medium with PBS, as the medium contains serum and other supplements that may interfere with the signal. Additionally, it is necessary to eliminate the background reading by measuring the luminescence signal of a sample that only contains PBS (no cells).

This protocol describes a noninvasive technique to measure breast cancer cell growth and metastases. Specifically, this paper describes the injection of breast cancer cell lines, expressing both GFP and luciferase into the mouse mammary fat pad. This combination provides a quick and reliable method to measure metastatic colonization in vivo and ex vivo.

Despite the clear advantages of this method, it has some limitations. The primary constraint is the need for a bioluminescence machine, as this is a relatively expensive machine and therefore not always available. In addition, each read is time-consuming, and thus the machine can be overbooked and unavailable. Another limitation refers to the protocol itself. To detect the bioluminescence signal in the ex vivo samples, it is recommended to euthanize the mice and examine the sample immediately. This step is a time-limiting stage and is not feasible for a large set of experiments.

In conclusion, this noninvasive bioluminescence tool is highly sensitive to detecting tumor development and metastasis in mice. This protocol is not restricted to breast cancer and could be applied to other carcinomas such as lung and pancreatic cancer. Furthermore, because it is noninvasive, it can be applied to measure the efficacy of anticancer drugs¹² and their effects on tumor growth kinetics in real time.

Materials

Name	Company	Catalog Number	Comments
1.7 mL eppendorf tubes	Lifegene	LMCT1.7B-500
10 µL tips	Lifegene	LRT10
1000 µL tips	Lifegene	LRT1000
15 mL tubes	Lifegene	LTB15-500
200 µL tips	Lifegene	LRT200
6 well cell culture plate	COSTAR	3516
96 well Plates BLACK flat bottom	Bar Naor	BN30496
Automated Cell Counters	Thermofisher	A50298
BD FACSAria III sorter	BD
BD Microlance 3 Needles 27 G (3/4'')	BD	302200
BD Plastipak Syringes 1 mL x 120	BD	303172
Corning 100 mm x 20 mm Style Dish	CORNING	430167
Corning 150 mm x 20 mm Style Dish	CORNING	430599
Countess cell counting chamber slides	Thermofisher	C10228
Dulbecco's modified Eagle's medium (DMEM), high glucose, no glutamine	Biological Industries	01-055-1A
Eclipse 80i microscope	Nikon
eppendorf Centrifuge 5810 R	Sigma Aldrich	EP5820740000
Fetal Bovine Serum (FBS)	Biological Industries	04-127-1A
FUW GFP			Gifted from Dr. Yossi Buganim's lab (Hebrew University of Jerusalem)
HEK293T			Gifted from Dr. Lior Nissim's lab (Hebrew University of Jerusalem)
Isoflurane, USP Terrell	Piramal	NDC 66794-01-25
IVIS Spectrum In Vivo Imaging System	Perkin Elmer	124262
L-Glutamine Solution	Biological industries	03-020-1A
Living Image Software	PerkinElmer		bioluminescence measurement
MCF-7	ATCC	ATCC HTB-22
MDA-MB-231	ATCC	ATCC HTB-26
MDA-MB-468	ATCC	ATCC HTB-132
Pasteur pipettes	NORMAX	2430-475
PBS	Hylabs	BP655/500D
pCMV-dR8.2-dvpr	Addgene	#8455	Provided by David M. Sabatini’s lab (Whitehead institute, Boston, USA)
pCMV-VSV-G	Addgene	#8454	Provided by David M. Sabatini’s lab (Whitehead institute, Boston, USA)
Penicillin-Streptomycin Solution	Biological Industries	03-031-1B
Petri dish 90 mm (90x15)	MINI PLAST	820-090-01-017
Pipettes 10ml	Lifegene	LG-GSP010010S
Pipettes 25ml	Lifegene	LG-GSP010050S
Pipettes 5ml	Lifegene	LG-GSP010005S
pLX304 Luciferase-V5 blast plasmid	Addgene	#98580
Polybrene	Sigma Aldrich	#107689
Prism 9	GraphPad
Reagent Reservoirs	Bar Naor	BN20621STR200TC
SMZ18 Stereo microscopes	Nikon
Sodium Chloride	Bio-Lab	190359400
Syringe filters	Lifegene	LG-FPV403030S
Trypan Blue 0.5% solution	Biological industries	03-102-1B
Trypsin EDTA Solution B (0.25%), EDTA (0.05%)	Biological Industries	03-052-1a
Vacuum driven Filters	SOFRA LIFE SCIENCE	SPE-22-500
Virusolve		disinfectant
VivoGlo Luciferin, In Vivo Grade	Promega	P1043
X-tremeGENE HP DNA Transfection Reagent	Sigma Aldrich	#6366236001