Name: dual bioluminescence imaging of tumor progression and angiogenesis
Uploaded: 2019-08-01T13:00:00
Description: Watch this Scientific Journal Video about Dual Bioluminescence Imaging of Tumor Progression and Angiogenesis at JoVE.com

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...

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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>CO2 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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