This research was supported by National Key R&#38;D Program of China (2017YFA0103200), National Natural Science Foundation of China (81671734), and Key Projects of Tianjin Science and Technology Support Program (18YFZCSY00010), Fundamental Research Funds for the Central Universities (63191155). We acknowledge the Gloria Nance&#8217;s revisions, which were valuable in improving the quality of our manuscript.

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 EF1&#945;, whereas the pLV-RRT carries the gene sequences coding Rluc, RFP, and herpes simplex virus truncated thymidine kinase (HSV-ttk) (Figure 2).
<ol>
	<li>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 &#176;C with Dulbecco&#8217;s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS).</li>
	<li>Prepare the liposome suspension: mix 7.5 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>After incubating in a humidified incubator with 5% CO2 at 37 &#176;C for 12&#8211;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&#8722;streptomycin.</li>
	<li>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 &#176;C. 
	NOTE: A Biosafety Level 2 (BSL-2) facility is required in order to work with recombinant lentivirus.</li>
	<li>Repeat steps 1.1&#8211;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 &#176;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.</li>
</ol>
2. LV-RR and LV-RRT lentiviral transduction for gene expression in 4T1 cells
<ol>
	<li>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 &#176;C.</li>
	<li>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 &#181;g/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.</li>
	<li>Spin transduction solution in a centrifuge at 1,000 &#215; g for 60 min at RT to help increase transduction efficiency. After centrifugation, culture 4T1 cells for 4&#8211;12 h and maintain in a humidified incubator with 5% CO2 at 37 &#176;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.</li>
	<li>Refresh the medium of transduced 4T1 cells with 2 mL of RPMI 1640 medium containing 10% FBS and 100 U/mL penicillin&#8722;streptomycin to remove lentiviral particles and polybrene.</li>
</ol>
3. Drug screening and identification of LV-RR and LV-RRT transduced 4T1 cells
<ol>
	<li>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.</li>
	<li>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&#8722;streptomycin, and 5 &#181;g/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.</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>Culture 4T1-RR cells and 4T1-RRT cells in 60 mm Petri dishes in a humidified incubator with 5% CO2 at 37 &#176;C, respectively. When the cells are at 80% confluence, remove the medium and rinse with phosphate buffered saline (PBS).</li>
	<li>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 &#176;C) until the cells detach.</li>
	<li>Add 5&#8211;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 &#181;L in RPMI 1640 medium.</li>
	<li>Anesthetize the Vegfr2-Fluc-KI mice with 1%&#8211;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.</li>
	<li>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.</li>
	<li>Subcutaneously inject 4T1-RR cells (1 x 106 cells at a 100 &#181;L total volume) and 4T1-RRT cells (1 x 106 cells at a 100 &#181;L 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.</li>
	<li>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).</li>
	<li>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).</li>
</ol>
5. Dual bioluminescence imaging of tumor (Rluc) and angiogenesis (Fluc)
<ol>
	<li>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 &#176;C before able to start imaging. The temperature will turn green when the CCD camera is cooled.</li>
	<li>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.</li>
	<li>Weigh and record the mice and calculate the volume of coelenterazine (CTZ; 2.5 mg/kg) and D-luciferin (150 mg/kg) needed.</li>
	<li>Anesthetize tumor-bearing mouse by 1%&#8211;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.</li>
	<li>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 &#956;L to deliver 50 &#956;g of CTZ) by using an insulin syringe needle.</li>
	<li>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.</li>
	<li>Intraperitoneally inject 150 mg/kg D-luciferin (30 mg/mL) using an insulin syringe needle (e.g., for a 20 g mouse, inject 100 &#956;L to deliver 3 mg of D-luciferin). Keep the mouse at RT for 10 min before Fluc imaging.</li>
	<li>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.</li>
	<li>Repeat the procedures steps 5.4&#8211;5.8 for each mouse.</li>
	<li>After imaging, maintain the mice in a warm environment until animals wake up.</li>
	<li>At the desired time point (day 3, 7, 14, and 21), repeat above procedures (step 5.3&#8211;5.10) to detect the tumor progression and tumor angiogenesis over time.</li>
	<li>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.</li>
	<li>Analyze the Rluc and Fluc signals of ROI by using graphics software (Figure 4).</li>
</ol>

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The authors have nothing to disclose.

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 ...

Angiogenesis is an essential process in the progression of cancer from small, localized neoplasms to larger, potentially metastatic tumors1,2. 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 specificity3,4,5,6. BLI technology is based on the principle that the luciferase can catalyze oxidation of a specific substrate while emitting bioluminescence. The luciferase expressed in &#160;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 vivo6,7.
Except for tumor growth, tumor angiogenesis (the critical step in cancer progression) can also be visualized through BLI technology using Vegfr2-Fluc-KI transgenic mice8,9,10. 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.

<table><tbody><tr><td>0.25% Trypsin-0.53 mM EDTA</td><td>Gibco</td><td>25200072</td><td></td></tr><tr><td>1.5 mL Tubes</td><td>Axygen Scientific</td><td>MCT-105-C-S</td><td></td></tr><tr><td>15 mL Tubes</td><td>Corning Glass Works</td><td>601052-50</td><td></td></tr><tr><td>293T</td><td>ATCC</td><td>CRL-3216</td><td></td></tr><tr><td>4T1</td><td>ATCC</td><td>CRL-2539</td><td></td></tr><tr><td>60 mm Dish</td><td>Corning Glass Works</td><td>430166</td><td></td></tr><tr><td>6-well Plate</td><td>Corning Glass Works</td><td>3516</td><td></td></tr><tr><td>Biosafety Cabinet</td><td>Shanghai Lishen Scientific</td><td>Hfsafe-900LC</td><td></td></tr><tr><td>Blasticidine S Hydrochloride (BSD)</td><td>Sigma-Aldrich</td><td>15205</td><td></td></tr><tr><td>Cell Counting Kit-8</td><td>MedChem Express</td><td>HY-K0301</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Tegulated Incubator</td><td>Thermo Fisher Scientific</td><td>4111</td><td></td></tr><tr><td>Coelenterazine (CTZ)</td><td>NanoLight Technology</td><td>479474</td><td></td></tr><tr><td>D-luciferin Potassium Salt</td><td>Caliper Life Sciences</td><td>119222</td><td></td></tr><tr><td>DMEM Medium</td><td>Gibco</td><td>C11995500BT</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>BIOIND</td><td>04-001-1A</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>Ti-E/U/S</td><td></td></tr><tr><td>Ganciclovir (GCV)</td><td>Sigma-Aldrich</td><td>Y0001129</td><td></td></tr><tr><td>Graphics Software</td><td>GraphPad Software</td><td>Graphpad Prism 6</td><td></td></tr><tr><td>Insulin Syringe Needles</td><td>Becton Dickinson</td><td>328421</td><td></td></tr><tr><td>Isoflurane</td><td>Baxter</td><td>691477H</td><td></td></tr><tr><td>Lentiviral Packaging System</td><td>Biosettia</td><td>cDNA-pLV03</td><td></td></tr><tr><td>Liposome</td><td>Invitrogen</td><td>11668019</td><td></td></tr><tr><td>Living Imaging Software</td><td>Caliper Life Sciences</td><td>Living Imaging Software 4.2</td><td></td></tr><tr><td>Living Imaging System</td><td>Caliper Life Sciences</td><td>IVIS Lumina II</td><td></td></tr><tr><td>MEM Medium</td><td>Invitrogen</td><td>31985-070</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Invitrogen</td><td>15140122</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Corning Glass Works</td><td>R21031399</td><td></td></tr><tr><td>Polybrene</td><td>Sigma-Aldrich</td><td>H9268-1G</td><td></td></tr><tr><td>RPMI1640 Medium</td><td>Gibco</td><td>C11875500BT</td><td></td></tr><tr><td>SORVALL ST 16R Centrifuge</td><td>Thermo Fisher Scientific</td><td>Thermo Sorvall ST 16 ST16R</td><td></td></tr><tr><td>Ultra-low Temperature Refrigerator</td><td>Haier</td><td>DW-86L338</td><td></td></tr><tr><td>XGI-8 Gas Anesthesia System</td><td>XENOGEN Corporation</td><td>7293</td><td></td></tr></tbody></table>

dual bioluminescence imaging of tumor progression and angiogenesis

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.

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...

Watch this Scientific Journal Video about Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis at JoVE.com

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

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

Angiogenesis, as a crucial process of tumor progression, has become a research hotspot and target of anti-tumor therapy. However, there is no reliable ...

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Research

JoVE Journal

Medicine

8.1K Views.  Nankai University School of Medicine. This protocol describes the establishment of a tumor-bearing mouse model to monitor tumor progression and angiogenesis in real-time by dual bioluminescence imaging.

Video: Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Neuroscience

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Immunology and Infection

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Biology

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

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.

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 ...

Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis

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Chapters in this video

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

In vivo Dual Substrate Bioluminescent Imaging

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

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