Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Summary

This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Abstract

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable model for tracing tumor progression and angiogenesis simultaneously in a visual and sensitive manner. Bioluminescence imaging displays its unique superiority in living imaging due to its advantages of high sensitivity, strong specificity, and accurate measurement. Presented here is a protocol to establish a tumor-bearing mouse model by injecting a Renilla luciferase-labeled murine breast cancer cell line 4T1 into the transgenic mouse with angiogenesis-induced Firefly luciferase expression. This mouse model provides a valuable tool to simultaneously monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging in a single mouse. This model may be widely applied in anti-tumor drug screening and oncology research.

Introduction

Angiogenesis is an essential process in the progression of cancer from small, localized neoplasms to larger, potentially metastatic tumors^1,2. The correlation between tumor growth and angiogenesis becomes one of the points of emphasis in the field of oncology research. However, traditional methods of measuring morphologic changes fail to monitor tumor progression and angiogenesis simultaneously in living animals using a visualized approach.

Bioluminescence imaging (BLI) of tumor cells is a particularly appropriate experimental method to monitor tumor growth because of its non-invasiveness, sensitivity, and specificity^3,4,5,6. BLI technology is based on the principle that the luciferase can catalyze oxidation of a specific substrate while emitting bioluminescence. The luciferase expressed in  implanted tumor cells reacts with the injected substrate, which can be detected by a living imaging system, and signals indirectly reflect the changes in cell number or cell localization in vivo^6,7.

Except for tumor growth, tumor angiogenesis (the critical step in cancer progression) can also be visualized through BLI technology using Vegfr2-Fluc-KI transgenic mice^8,9,10. The vascular endothelial growth factor (Vegf) receptor 2 (Vegfr2), one type of Vegf receptor, is mostly expressed in the vascular endothelial cells of adult mice¹¹. In Vegfr2-Fluc-KI transgenic mice, the DNA sequence of Firefly luciferase (Fluc) is knocked into the first exon of the endogenous Vegfr2 sequence. As a result, the Fluc is expressed (which appears as BLI signals) in a manner that is identical to the level of angiogenesis in mice. To grow beyond a few millimeters in size, the tumor recruits new vasculatures from existing blood vessels, which highly express the Vegfr2 triggered by growth factors from tumor cells¹. This opens the possibility of using Vegfr2-Fluc-KI transgenic mice to non-invasively monitor tumor angiogenesis by BLI.

In this protocol, a tumor-bearing mouse model is established to monitor tumor progression and angiogenesis in a single mouse through Firefly luciferase (Fluc) and Renilla luciferase (Rluc) imaging, respectively (Figure 1). A 4T1 cell line (4T1-RR) is created that stably expresses Rluc and red fluorescent protein (RFP) to trace cell growth by Rluc imaging. To further investigate the dynamic changes of angiogenesis in the progression and regression of the tumor, another 4T1 cell line (4T1-RRT) is created that expresses suicide gene herpes simplex virus truncated thymidine kinase (HSV-ttk), Rluc, and RFP. By administration of ganciclovir (GCV), the HSV-ttk expressing cells are selectively ablated. Based on these cell lines, a tumor-bearing model in Vegfr2-Fluc-KI mice is built that serves as an experimental model bridging tumor progression and tumor angiogenesis in vivo.

Protocol

Experiments must comply with national and institutional regulations concerning the use of animals for research purposes. Permissions to carry out experiments must be obtained. The treatment of animals and experimental procedures of the study adhere to the Nankai University Animal Care and Use Committee Guidelines that conform to the Guidelines for Animal Care approved by the National Institutes of Health (NIH).

1. LV-Rluc-RFP (RR) and LV-Rluc-RFP-HSV-ttk (RRT) lentiviral packaging and production

NOTE: The pLV-RR carries the gene sequences of Renilla luciferase (Rluc) and red fluorescent protein (RFP) under the promoter EF1α, whereas the pLV-RRT carries the gene sequences coding Rluc, RFP, and herpes simplex virus truncated thymidine kinase (HSV-ttk) (Figure 2).

1. Seed 1 x 10⁶ of 293T cells per well into a 6 well plate and culture overnight in a humidified incubator with 5% CO₂ at 37 °C with Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS).
2. Prepare the liposome suspension: mix 7.5 µL of liposome and 0.25 mL of minimal essential medium (MEM) into a 1.5 mL tube following incubation for 5 min at room temperature (RT) to disperse liposomes equally.
3. Prepare the DNAs solution (DNAs-RR): separately, add the pLV-RR vector and helper plasmids to 0.25 mL of MEM in a 1.5 mL tube as described in Table 1.
4. Obtain the liposome/DNAs-RR compound: gently add the DNAs-RR solution into prepared liposome suspension drop by drop and incubate for 20 min at RT so the DNA bonds to the lipid membrane.
5. Replace the medium of the 293T cells with 1 mL of DMEM containing 10% FBS and add the liposome/DNAs-RR compound to the medium of the 293T cells gently.
6. After incubating in a humidified incubator with 5% CO₂ at 37 °C for 12–16 h, replace the liposome/DNAs-RR compound containing medium of the 293T cells with 1 mL of DMEM containing 10% FBS and 100 U/mL penicillin−streptomycin.
7. Continue culturing the 293T cells in the humidified incubator for 48 h after transfection. Then, collect the supernatant of the 293T cells and centrifuge the medium at 300 x g for 5 min to pellet the 293T cells. Transfer the lentivirus-RR (LV-RR)-containing supernatant into 1.5 mL sterile polypropylene storage tubes and store at -80 °C.
   NOTE: A Biosafety Level 2 (BSL-2) facility is required in order to work with recombinant lentivirus.
8. Repeat steps 1.1–1.7 and use pLV-RRT vector instead of pLV-RR vector in step 1.3 to obtain the lentivirus-RRT (LV-RRT). Store the LV-RRT at -80 °C.
   NOTE: The non-purified lentiviral stock may inhibit cell growth in some cases. Lentiviral stock may need to be purified. The lentiviral stocks containing LV-RR or LV-RRT particles should be divided into 1.5 mL tubes (1 mL per tube) for storage to avoid multiple free-thaw cycles.

2. LV-RR and LV-RRT lentiviral transduction for gene expression in 4T1 cells

1. Seed 4T1 cells into a 6-well plate (5 x 10⁵ cells/well) and culture with Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS overnight in a humidified incubator with 5% CO₂ at 37 °C.
2. Remove the medium from the culture plate and replace it with 1 mL fresh RPMI 1640 medium as well as 1 mL of lentiviral stock (LV-RR or LV-RRT) to each well. Add 8 µg/mL polybrene and gently blend the medium containing lentiviral particles by pipetting up and down.
   NOTE: Please be aware that the medium contains lentiviral particles, which could transduce human cells.
3. Spin transduction solution in a centrifuge at 1,000 × g for 60 min at RT to help increase transduction efficiency. After centrifugation, culture 4T1 cells for 4–12 h and maintain in a humidified incubator with 5% CO₂ at 37 °C.
   NOTE: For some cell lines, polybrene may be toxic for long-term culture. Therefore, the incubation time for transducing different cells may be changeable. Check the cell status multiple times to find appropriate incubation time.
4. Refresh the medium of transduced 4T1 cells with 2 mL of RPMI 1640 medium containing 10% FBS and 100 U/mL penicillin−streptomycin to remove lentiviral particles and polybrene.

3. Drug screening and identification of LV-RR and LV-RRT transduced 4T1 cells

1. Select transduced cells with medium containing blasticidin (BSD) according to the BSD-resistance gene carried by LV-RR or LV-RRT as the following steps described.
   NOTE: Alternatively, the transduced cells which are RFP-positive could be selected by flow cytometry according to the RFP gene carried by LV-RR or LV-RRT.
2. 48 h after transduction, passage 4T1 cells at the ratio of 1:3 to 1:4 with selection medium (RPMI 1640 medium containing 10% FBS, 100 U/mL penicillin−streptomycin, and 5 µg/mL BSD). Change medium every 2 or 3 days.
   NOTE: The optimal BSD concentration may vary from cell line to cell line. Therefore, a pilot experiment of kill curve should be performed to determine the optimal concentration of BSD before initial experiment.
3. 7 days post-drug screening, observe the LV-RR transduced 4T1 cells (4T1-RR) and LV-RRT transduced 4T1 cells (4T1-RRT) under the fluorescence inverted phase-contrast microscope. Count the number of RFP⁺ 4T1 cells and all 4T1 cells in three fields of vision to estimate the RFP-positive ratio, respectively (Figure 2).
   NOTE: Alternatively, the RFP-positive ratio of transduced 4T1 cells could be identified by flow cytometry.
4. Measure the renilla signals of 4T1-RR cells and 4T1-RRT cells by using a living imaging system to detect the linear relationship between cell numbers and renilla signals (Figure 3).
5. Expand BSD-screened 4T1-RR and 4T1-RRT cells with selection medium at split ratios between 1:3 and 1:4 and store the cell line stocks in liquid nitrogen.

4. Vegfr2-Fluc-KI mice and tumor-bearing mouse model

NOTE: The transgenic Vegfr2-Fluc-KI mice, 6-8 weeks old and female, are used in this experiment to non-invasively monitor angiogenesis in vivo by BLI.

1. Culture 4T1-RR cells and 4T1-RRT cells in 60 mm Petri dishes in a humidified incubator with 5% CO₂ at 37 °C, respectively. When the cells are at 80% confluence, remove the medium and rinse with phosphate buffered saline (PBS).
2. Remove the PBS and add an additional 2 mL of 0.25% trypsin-0.53 mM EDTA solution respectively. Keep the dish at RT (or at 37 °C) until the cells detach.
3. Add 5–10 mL of fresh medium containing 10% FBS, then aspirate and dispense cells to resuspend 4T1-RR and 4T1-RRT cells into 15 mL centrifuge tubes, respectively. Count two types of 4T1 cells using a counting chamber and prepare the cell suspensions at a concentration of 1 x 10⁶ per 100 µL in RPMI 1640 medium.
4. Anesthetize the Vegfr2-Fluc-KI mice with 1%–3% isoflurane in 100% oxygen at anesthesia induction chamber with a flow rate of 1 L/min. Monitor the toe pinch response of the mouse to confirm the status of anesthesia. Then, apply ophthalmic ointment to the eyes of mouse to prevent dehydration.
5. Remove mouse from chamber and position in nosecone. Entirely remove the hair of the shoulder of mouse by using electric shaver and hair removal cream, which could provide a good view of surgical field and avoid blocking the BLI signals in following-up experiments.
6. Subcutaneously inject 4T1-RR cells (1 x 10⁶ cells at a 100 µL total volume) and 4T1-RRT cells (1 x 10⁶ cells at a 100 µL total volume) in left and right shoulders of each mouse, respectively (record as Day 0). Place mice in recovery area with thermal support until fully recovered.
7. After implantation of 4T1-RR and 4T1-RRT cells, touch the tumor masses to check that the mice are tumor-bearing every day (Figure S1). At day 7 post-implantation, intraperitoneally inject 50 mg/kg ganciclovir (GCV) to the tumor-bearing mice two times per day until the end of experiment.
   NOTE: Before this experiment, the cytotoxic of GCV on 4T1-RRT cells should be detected. The killing efficiency of GCV could be evaluated by cell counting assay with different concentration of GCV (Figure S2).
8. On the day 0, 3, 7, 14, and 21 after 4T1 implantation, monitor the tumor growth and angiogenesis of tumor-bearing mice and assess by both Rluc and Fluc imaging (Figure 4).

5. Dual bioluminescence imaging of tumor (Rluc) and angiogenesis (Fluc)

1. Open the living imaging system, initialize the living imaging software, and then initialize the system.
   NOTE: The system initialization will take few minutes to cool down the charge-coupled device (CCD) camera to -90 °C before able to start imaging. The temperature will turn green when the CCD camera is cooled.
2. Use the following camera settings:
   Check the Luminescence and Photograph.
   Check Overlay.
   Luminescence settings:
   Exposure Time sets AUTO in normal conditions.
   Binning sets to 8.
   F/Stop sets to 1.
   Emission Filter sets Open.
   Photograph settings:
   Binning sets to medium.
   F/Stop sets to 8.
   IVIS system settings:
   Field of view: C=1 mouse view, D=5 mice view.
   Subject height sets 1.5 cm.
3. Weigh and record the mice and calculate the volume of coelenterazine (CTZ; 2.5 mg/kg) and D-luciferin (150 mg/kg) needed.
4. Anesthetize tumor-bearing mouse by 1%–3% isoflurane in 100% oxygen at anesthesia induction chamber with a flow rate of 1 L/min. Monitor the toe pinch response of the mouse to confirm the status of anesthesia. Then, dispense a drop of lubricating eye ointment onto both eyes to avoid corneal damage.
5. Inject 2.5 mg CTZ (3.33 mg/mL) per kilogram body weight into the retrobulbar of the mouse (e.g., for a 20 g mouse, inject 15 μL to deliver 50 μg of CTZ) by using an insulin syringe needle.
6. Move the tumor-bearing mouse into the camera chamber with its nose in the anesthesia cone gently and acquire several pictures of the mouse dorsal immediately to get the Rluc signals from 4T1 cells until the BLI signals fade away.
   NOTE: The half-life of CTZ is very short and the signals of Rluc drop precipitously ~30 s. To ensure any residual Rluc signal has dissipated and the interval between Rluc and Fluc imaging should be more than 10 min.
7. Intraperitoneally inject 150 mg/kg D-luciferin (30 mg/mL) using an insulin syringe needle (e.g., for a 20 g mouse, inject 100 μL to deliver 3 mg of D-luciferin). Keep the mouse at RT for 10 min before Fluc imaging.
8. Move this mouse into camera chamber with its nose in the anesthesia cone again and acquire several pictures of the mouse dorsal to get the Fluc signals from angiogenesis.
   NOTE: The Fluc kinetic monitor should be performed for each mouse until the signals reach the maximum and then fade.
9. Repeat the procedures steps 5.4–5.8 for each mouse.
10. After imaging, maintain the mice in a warm environment until animals wake up.
11. At the desired time point (day 3, 7, 14, and 21), repeat above procedures (step 5.3–5.10) to detect the tumor progression and tumor angiogenesis over time.
12. Analyze the Rluc and Fluc signals data to investigate the relationship between the tumor growth and angiogenesis in tumor progression.
    NOTE: The regions of interest (ROI) which cover the BLI signal site are used to analyze the data. Measure the total radiance (Photons) of ROI in the unit of Photons/seconds/cm²/steradian (p/s/cm²/sr) for every timepoint.
13. Analyze the Rluc and Fluc signals of ROI by using graphics software (Figure 4).

Results

In this experiment, a breast cancer mouse model was established using 4T1 cells to investigate the relationship between tumor growth and tumor angiogenesis (Figure 1). Firstly, two lentivirus were packaged, which carried gene sequences expressing Rluc/RFP (LV-RR) and Rluc/RFP/HSV-ttk (LV-RRT), respectively, as previously reported⁷. Then, two different 4T1 cell lines, named 4T1-RR and 4T1-RRT, were created by transducing LV-RR and LV-RR...

Discussion

In this protocol, a non-invasive dual BLI approach is described for monitoring tumor development and angiogenesis. The BLI reporter system is first developed, containing the HSV-ttk/GCV suicide gene for tracking tumor progression and regression in vivo by Rluc imaging. Meanwhile, tumor angiogenesis is assessed using Vegfr2-Fluc-KI mice via Fluc imaging. This tumor-bearing mouse model is able to provide a practical platform for continuous and non-invasive tracking tumor development and tumor angiogenesis by dual BLI in a ...

Materials

Name	Company	Catalog Number	Comments
0.25% Trypsin-0.53 mM EDTA	Gibco	25200072
1.5 mL Tubes	Axygen Scientific	MCT-105-C-S
15 mL Tubes	Corning Glass Works	601052-50
293T	ATCC	CRL-3216
4T1	ATCC	CRL-2539
60 mm Dish	Corning Glass Works	430166
6-well Plate	Corning Glass Works	3516
Biosafety Cabinet	Shanghai Lishen Scientific	Hfsafe-900LC
Blasticidine S Hydrochloride (BSD)	Sigma-Aldrich	15205
Cell Counting Kit-8	MedChem Express	HY-K0301
CO2 Tegulated Incubator	Thermo Fisher Scientific	4111
Coelenterazine (CTZ)	NanoLight Technology	479474
D-luciferin Potassium Salt	Caliper Life Sciences	119222
DMEM Medium	Gibco	C11995500BT
Fetal Bovine Serum (FBS)	BIOIND	04-001-1A
Fluorescence Microscope	Nikon	Ti-E/U/S
Ganciclovir (GCV)	Sigma-Aldrich	Y0001129
Graphics Software	GraphPad Software	Graphpad Prism 6
Insulin Syringe Needles	Becton Dickinson	328421
Isoflurane	Baxter	691477H
Lentiviral Packaging System	Biosettia	cDNA-pLV03
Liposome	Invitrogen	11668019
Living Imaging Software	Caliper Life Sciences	Living Imaging Software 4.2
Living Imaging System	Caliper Life Sciences	IVIS Lumina II
MEM Medium	Invitrogen	31985-070
Penicillin-Streptomycin	Invitrogen	15140122
Phosphate Buffered Saline (PBS)	Corning Glass Works	R21031399
Polybrene	Sigma-Aldrich	H9268-1G
RPMI1640 Medium	Gibco	C11875500BT
SORVALL ST 16R Centrifuge	Thermo Fisher Scientific	Thermo Sorvall ST 16 ST16R
Ultra-low Temperature Refrigerator	Haier	DW-86L338
XGI-8 Gas Anesthesia System	XENOGEN Corporation	7293