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...

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis:+therapeutic+implications.">Tumor angiogenesis: therapeutic implications.</a> The New England Journal of Medicine. 285, 1182-1186 (1971).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis.">Tumor angiogenesis.</a> The New England Journal of Medicine. 358, 2039-2049 (2008).</li><li>Hosseinkhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+enigma+of+multicolor+bioluminescence+of+firefly+luciferase.">Molecular enigma of multicolor bioluminescence of firefly luciferase.</a> Cellular and Molecular Life Sciences. 68, 1167-1182 (2011).</li><li>Nakatsu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+spectral+difference+in+luciferase+bioluminescence.">Structural basis for the spectral difference in luciferase bioluminescence.</a> Nature. 440, 372-376 (2006).</li><li>McMillin, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-specific+bioluminescence+platform+to+identify+stroma-induced+changes+to+anticancer+drug+activity.">Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity.</a> Nature Medicine. 16, 483-489 (2010).</li><li>Madero-Visbal, R. A., Hernandez, I. C., Myers, J. N., Baker, C. H., Shellenberger, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+bioluminescent+imaging+of+xenograft+progression+in+an+orthotopic+mouse+model+of+HNSCC.">In situ bioluminescent imaging of xenograft progression in an orthotopic mouse model of HNSCC.</a> Journal of Clinical Oncology. 26, 17006 (2008).</li><li>Wang, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Tumor+Angiogenesis+and+Therapeutic+Effects+with+Dual+Bioluminescence.">Molecular Imaging of Tumor Angiogenesis and Therapeutic Effects with Dual Bioluminescence.</a> Current Pharmaceutical Biotechnology. 18, 422-428 (2017).</li><li>Rivera, L. B., Cancer Bergers, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+angiogenesis,+from+foe+to+friend.">Tumor angiogenesis, from foe to friend.</a> Science. 349, 694-695 (2015).</li><li>Zhang, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+therapeutic+effects+of+mesenchymal+stem+cell-derived+exosomes+with+an+injectable+hydrogel+for+hindlimb+ischemia+treatment.">Enhanced therapeutic effects of mesenchymal stem cell-derived exosomes with an injectable hydrogel for hindlimb ischemia treatment.</a> ACS Applied Materials &amp; Interfaces. 10, 30081-30091 (2018).</li><li>Du, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+proangiogenic+potential+of+mesenchymal+stem+cell-derived+exosomes+stimulated+by+a+nitric+oxide+releasing+polymer.">Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer.</a> Biomaterials. , 70-81 (2017).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+VEGF+signaling+is+required+for+vascular+homeostasis.">Autocrine VEGF signaling is required for vascular homeostasis.</a> Cell. 130, 691-703 (2007).</li><li>Dewhirst, M. W., Cao, Y., Moeller, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cycling+hypoxia+and+free+radicals+regulate+angiogenesis+and+radiotherapy+response.">Cycling hypoxia and free radicals regulate angiogenesis and radiotherapy response.</a> Nature Reviews. Cancer. 8, 425-437 (2008).</li><li>Wigerup, C., Pahlman, S., Bexell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+targeting+of+hypoxia+and+hypoxia-inducible+factors+in+cancer.">Therapeutic targeting of hypoxia and hypoxia-inducible factors in cancer.</a> Pharmacology &amp; Therapeutics. 164, 152-169 (2016).</li><li>Wong, P. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual-action+combination+therapy+enhances+angiogenesis+while+reducing+tumor+growth+and+spread.">Dual-action combination therapy enhances angiogenesis while reducing tumor growth and spread.</a> Cancer Cell. 27, 123-137 (2015).</li><li>Mezzanotte, L., van &#39;t Root, M., Karatas, H., Goun, E. A., Lowik, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+Molecular+Bioluminescence+Imaging:+New+Tools+and+Applications.">In vivo Molecular Bioluminescence Imaging: New Tools and Applications.</a> Trends in Biotechnology. 35, 640-652 (2017).</li><li>Du, W., Tao, H., Zhao, S., He, Z. X., Li, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+applications+of+molecular+imaging+in+cardiovascular+disease+and+stem+cell+therapy.">Translational applications of molecular imaging in cardiovascular disease and stem cell therapy.</a> Biochimie. 116, 43-51 (2015).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis,+biodistribution,+and+imaging+of+PEGylated-acetylated+polyamidoamine+dendrimers.">Synthesis, biodistribution, and imaging of PEGylated-acetylated polyamidoamine dendrimers.</a> Journal of Nanoscience and Nanotechnology. 14, 3305-3312 (2014).</li><li>Branchini, B. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red-emitting+chimeric+firefly+luciferase+for+in+vivo+imaging+in+low+ATP+cellular+environments.">Red-emitting chimeric firefly luciferase for in vivo imaging in low ATP cellular environments.</a> Analytical Biochemistry. 534, 36-39 (2017).</li><li>McLatchie, A. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+in+vivo+imaging+of+Trypanosoma+brucei+expressing+&amp;#34;red-shifted&amp;#34;+luciferase.">Highly sensitive in vivo imaging of Trypanosoma brucei expressing &amp;#34;red-shifted&amp;#34; luciferase.</a> PLoS Neglected Tropical Diseases. 7, e2571 (2013).</li></ol>

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 ...

Angiogenesis play important role in tumor metastasis and progression. In this protocol, we will use dual bioluminescence imaging method to monitor the dynamic tumor progression and angiogenesis in a mouse model of breast cancer. In this model, we can track the tumor growth and angiogenesis simultaneously in the single mouse through Firefly luciferase and Renilla luciferase imaging, respectively.This model may be widely applied in antitumor drug screening and oncology research. This dual bioluminescence model can be used to track tumor progression and regression and to detect tumor-related molecular processes in response to therapeutic strategies. Angiogenesis is a crucial process of tumor progression.Therefore, monitoring tumor progression and angiogenesis in a visual and sensitive manner is necessary for developing effective tumor treatments. The procedures will be demonstrated by Kaiyue Zhang, Chen Wang, and Shang Chen, students from our lab. For lentivirus packaging and production seed one times 10 to the sixth of 293T cells in two milliliters of DMEM supplemented with 10%fetal bovine serum per well into a six-well plate for overnight culture in a humidified incubator at 37 degrees Celsius and 5%carbon dioxide.The next morning mix 7.5 microliters of liposome with 250 microliters of Minimal Essential Medium in two separate 1.5-milliliter tubes for a five minute incubation at room temperature. To prepare the DNA solutions add the plasmid lentivirus RR vector and helper plasmids to 250 microliters of Minimal Essential Medium in individual 1.5-milliliter tubes as outlined in the table. At the end of the incubation gently add the DNA RR solutions to the liposome suspensions in a drop-wise manner and allow the DNA to bond to the lipid membranes for 20 minutes at room temperature.While the mixture is incubating, replace the supernatant of the 293T cell culture with one milliliter of fresh culture medium, and gently add 0.5 milliliters of the appropriate liposome DNA mixture to each well. Return the cells to the cell culture incubator for 12 to 16 hours before replacing the supernatant in each well with one milliliter of fresh culture medium supplemented with antibiotics. After an additional 36 hours of incubation, pool the supernatants into one conical tube per lentivirus strain to sediment the 293T cells by centrifugation.Then transfer the lentivirus RR-containing supernatants into individual 1.5-milliliter sterile polypropylene storage tubes for storage at minus 80 degrees Celsius. For lentiviral transduction for gene expression in 4T1 mammary tumor cells, replace the supernatant in each well of a six-well 4T1 cell culture plate with one milliliter of fresh RPMI-based cell culture medium supplemented with fetal bovine serum and one milliliter of lentiviral stock. Then add eight micrograms per milliliter of Polybrene to each well, and gently pipette a few times to mix.Centrifuge the plate to help increase the transduction efficiency, and return the cells to the cell culture incubator for four to 12 hours. At the end of the incubation, replace the supernatant in each well with fresh culture medium supplemented with serum and antibiotics to remove any lentiviral particles and Polybrene. To select for the lentivirus-transduced cells, passage the transduced 4T1 cell cultures at a one-to-three to one-to-four ratio with selection medium, changing the medium every two to three days.After seven days of selection medium culture, view the transduced cell cultures under a fluorescence inverted phase contrast microscope and count the number of red fluorescent protein-positive or RFP-4T1 cells and all of the 4T1 cells in three fields of vision to estimate the RFP-positive ratio. To set up a tumor-bearing mouse model, wash 80%confluent transduced cell cultures with PBS before detaching the cells with two milliliters of trypsin-EDTA per 60-millimeter culture plate. When the cells have lifted from the plate bottoms stop the reaction with five to 10 milliliters of serum supplemented medium per plate.And transfer the cultures into individual 15-milliliter centrifuge tubes for counting. Dilute the cells to a one times 10 to the sixth cells per 100 microliters of culture medium without serum concentration. And subcutaneously inject 100 microliters of the 4T1-RR cell line into the left shoulder of an anesthetized tumor-bearing model mouse, and 100 microliters of the 4T1-RRT cell line into the right shoulder of the same mouse.After the injections, place each mouse in an appropriate recovery area with a thermal support until fully recovered. Palpate the tumor masses every day for seven days to confirm that the mice are tumor-bearing. At day seven post-implantation, intraperitoneally inject 50 micrograms per kilogram of Ganciclovir into each tumor-bearing mouse two times per day until the end of the experiment.For tumor growth, open the Living Imaging System. Initialize the Living Imaging software and initialize the system. While the system is initializing, use an insulin syringe to inject each mouse to be imaged with the appropriate volume of coelenterazine into the retrobulbar.Place the injected mice into the camera chamber and immediately obtain several pictures of the mouse dorsally to acquire the Renilla luciferase signals from 4T1 cells until the bioluminescent signals fade away. 10 minutes after the Renilla luciferase imaging, use an insulin syringe to intraperitoneally inject the appropriate volume of D-luciferin into each animal. 10 minutes after the D-luciferin injection, place the mice into the camera chamber and obtain several images of the mouse dorsal to acquire the Firefly luciferase signals from angiogenesis.After lentivirus transduction, more than 99%of the cells are RFP-positive with no differences in the cell culture morphology observed between the two transduced cell lines. Subsequently, bioluminescence imaging of the transduced cells reveals that both cell lines emit strong bioluminescent signals of a similar strength. After subcutaneous injection of the transduced cell lines into a transgenic tumor-bearing mouse model, angiogenesis-induced tumor growth can be evaluated by Firefly luciferase signals in the presence of D-luciferin in the same animal.At seven day post-implantation, Ganciclovir administration induces death in the 4T1-RRT cells resulting in a dramatic reduction in the Renilla luciferase signal in the tumors established by these cells. Firefly luciferase signals also increase in conjunction with an increase in Renilla luciferase expression and decrease following luciferase decline. Taken together, these results suggest that there is a direct correlation between tumor angiogenesis and tumor growth.Indeed, Ganciclovir-induced tumor cell death may lead to an inhibition of tumor angiogenesis. Further, immunostaining for anti-vascular endothelial growth factor receptor II, as a marker of angiogenesis, reveals a more significantly established microvascular structure in 4T1-RR tumor tissue sections than in sections from 4T1-RRT tumors, consistent with the decline in Firefly luciferase signal observed after Ganciclovir administration. The most critical step of the protocol is using two kinds of luciferase, such as FLuc and RLuc for simultaneous tracking of the cells and the angiogenesis.This technology could be used to treat cancer cells within different locations, and it can be used widely in virus cancer models. This mouse model allowing the tumor growth and the anti-tumor effects of the HSV-ttk/GCV tracking system to be visualized dynamically in a living animal. A biosafety level II facility is required to prevent a lentivirus transfection, as the media contains lentivirus particles that could be harmful to humans.

dual-bioluminescence-imaging-of-tumor-progression-and-angiogenesis

Research

JoVE Journal

Medicine

8.0K visualizações.  Nankai University School of Medicine. A angiogênese desempenha um papel importante na metástase e progressão tumoral. Neste protocolo, usaremos o método de imagem de bioluminescência dupla para monitorar a progressão dinâmica do tumor e a angiogênese em um modelo de camundongos de câncer de mama. Neste modelo, podemos acompanhar o crescimento do tumor e a angiogênese simultaneamente no camundongo único através da luciferase Firefly e da imagem luciferase de Renilla, respectivamente.Este modelo pode ser amplamen...