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.
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.
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 process3,4. Additionally, these in vivo models are essential for developing breast cancer therapeutic agents5,6. 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.
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.
2. Virus preparation
3. Establishing cells stably expressing GFP and luciferase ("GFP + Luc+ cells")
4. Validating in vitro luciferase activity
5. Injecting mice with GFP+ Luc+ cells
6. Measuring the luciferase levels in GFP+ Luc+ mice
7. Acquiring ex vivo image using bioluminescence and fluorescence
8. Bioluminescence data analysis
9. Measuring the total flux
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.
Animal-based experiments are obligatory for cancer research7,8,9, and indeed many protocols have been developed3,6,10,11,12,13,14. 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.
The authors have nothing to disclose.
We thank the members of the Y.D.S. laboratory. We would like to thank The Wohl Institute for Translational Medicine at the Hadassah Medical Center, Jerusalem, for providing the small animal imaging facility. This study was supported by Research Career Development Award from the Israel Cancer Research Fund.
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 (90×15) | 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 |