This research was supported by the Deutsche Krebshilfe (Max Eder Group to AN: 110972), a DGVS doctoral thesis scholarship (to SMB), and an Else-Kr&#246;ner-Fresenius-Foundation scholarship (to RGG) at the University Medical Center Goettingen. We thank Jutta Blumberg and Ulrike Wegner for expert technical assistance. We also thank all animal technicians at the animal facility of the University Medical Center Goettingen for mouse keeping. All experiments were performed according to German animal welfare regulations.

This protocol is in accordance with the animal care guidelines at the University Medical Centre Goettingen, Germany (33.9-42502-04-15/2056). Depending on specific requirements of individual animal review boards, some of the protocol steps could be modified accordingly.
1. Abdominal Palpation of KPC Mice
<ol>
	<li>To avoid unnecessary ultrasound scans, palpate the mouse abdomen to identify mice which might possibly bear an intraabdominal lesion and should subsequently undergo abdominal ultrasound.
	<ol>
		<li>Perform abdominal palpation weekly starting from 8 weeks of age in the KPC mouse model. High resolution ultrasound can also be applied in transplanted tumor models (e.g., orthotopics) or other genetically engineered mouse models. 
		NOTE: Depending on tumor kinetics and study goal (prevention study, therapeutic intervention study), detection of very small tumors (1-2 mm) is feasible to monitor tumor onset before tumors can be palpated.</li>
	</ol>
	</li>
	<li>Place the mouse on the cage grid and gently lift the mouse by the tail and palpate the abdomen by gently going up and down with index and thumb of the non-fixing hand (Figure 1).</li>
	<li>If the mouse tolerates soft palpation, proceed by slowly increasing the pressure. A larger resistance can easily be detected in the upper and lower abdomen. Sometimes, hard feces can be mistakenly interpreted as a tumor mass.</li>
</ol>
2. Preparation of Work Space
<ol>
	<li>Provide space for at least three different work units: pre-scan area, scanning stage and recovery cage. Required material are depicted in Figure 2.</li>
	<li>Clean the main working plate (for example with 70% Ethanol) and switch on the heating platform prior to scanning (Figure 3). Make sure that the target temperature of 38-40 &#176;C is reached before the first mouse is transferred.</li>
	<li>Switch on the ultrasound machine and create a new folder for the acquired data (Figure 4). The B-mode setting is applied for abdominal imaging. Further, ensure the abdominal transducer is plugged in the active port of the machine. Fix the transducer head at the top of the working stage by using the transducer mounting system.</li>
	<li>Prepare a recovery cage on a heating plate or thermal mat (or use heating lamp) to ensure post-procedural thermoregulation and speedy recovery of scanned mice. Put a thin layer of tissue as cushion at the bottom of this cage to avoid aspiration of bedding with the risk of suffocation (Figure 5).</li>
</ol>
3. Inhalation Anesthesia
<ol>
	<li>Proceed by putting a mouse into the induction chamber. Open the valve for gas carrier flow (1 L/min oxygen or air) and set the isoflurane concentration to 4% allowing a slowly increasing saturation of the induction chamber. Make sure that proper isoflurane scavenging (passive or active filtering) is in place. After about 2-4 min the mouse will usually be under deep sedation.
	<ol>
		<li>Maintain sedation by administering 2% isoflurane through the nose cone on the working stage.</li>
	</ol>
	</li>
	<li>Carefully transfer the animal from the induction chamber to the working stage. Check the nose cone for a constant gas flow. Put the mouse snout in the nose cone of the anesthesia line outlet. The regular position for the initial scanning is supine position. The mouse can also be placed on its right or left side to improve visibility of pancreatic head or tail tumors (Figure 6).</li>
	<li>Although abdominal ultrasound is not painful per se, perform the foot pinch test as an indicator for a pain free condition and deep anesthetic state. If the mouse is still active or actively moving around, retransfer the animal back in the induction chamber for repeated and prolonged induction.</li>
	<li>Use eye protection ointment to prevent corneal injuries due to fluid loss.</li>
	<li>During anesthesia, use a monitoring system. However, ECG and rectal temperature is not regularly used for abdominal ultrasound scanning. Importantly, mice lose the ability to thermoregulate under anesthesia. Therefore, closely observe clinical signs for hypothermia or respiratory distress such as gasping or apnea. If any symptom is obvious stop isoflurane and place the mouse in the recovery cage immediately.</li>
</ol>
4. Ultrasound&#160;Settings
<ol>
	<li>Switch on the ultrasound system as described by manufacturer`s instructions. Make sure the primary setting is put on general imaging with the abdominal package activated. Usually B-Mode-imaging is used for data acquisition as a standard.</li>
	<li>Achieve optimized image quality with following parameters: frequency 40 MHz, 2D-gain 25-30 dB, image depth 10 mm, image width 10 mm, 3 focal zones with center between 3-6 mm.</li>
</ol>
5. Mouse Preparation
<ol>
	<li>Fix the upper and lower extremity using adhesive tape. Be as careful as possible to avoid any injury to the animal due to fixation (Figure 7).</li>
	<li>Remove all abdominal fur using electronic pet clippers. Due to the fur texture, start from the lower abdomen up to the ribcage. Take care at the processus xiphoideus to avoid skin injuries in this area. 
	NOTE: Mouse fur reflects ultrasound waves and decreases visibility.</li>
	<li>Use depilatory cream to perform an optimized skin preparation and complete removal of fur. Administer a thin layer of cream onto the shaved abdomen using cotton tips (Figure 8). Remove the cream (after 30 s or later depending on the cream used) with dry tissues followed by plenty of water to completely remove all remnants (Figure 9, Figure 10, Figure 11). Cream remnants could cause skin and mucosa irritations after the ultrasound and may thus affect the health.</li>
	<li>To ensure adequate scanning conditions apply plenty of ultrasound gel onto the abdomen (Figure 12). Usually, warm ultrasound gel is used in order to minimize hypothermia for the mouse.</li>
	<li>Manually apply a thin layer of ultrasound gel on the transducer head. Perform a test by touching on the transducer end with the notch. At the screen a signal at the left upper side can be seen if the transducer is correctly positioned.</li>
</ol>
6. Abdominal Ultrasound
<ol>
	<li>Adjust the transducer onto the abdomen using firm pressure. Heavy compression of the mouse abdomen may lead to respiratory or circulatory impairment and should be avoided.</li>
	<li>Scan up and down. Adjust the frame of the working platform and the wheels for x-axis and y-axis.</li>
	<li>As the first step identify both kidneys. Usually they appear as hyperechogenic homogenous structures in the lower abdomen. The connected vena renalis can be easily found which further leads to the vena cava inferior (Figure 13).</li>
	<li>Use the vena lienalis serves as landmark vessel for the pancreas. Attempt to distinguish between vena renalis and vena lienalis. Upon detection of the vena lienalis the pancreas can be exactly localized. The normal pancreas is rather a membrane spread through the abdominal cavity than a solid structure (Figure 14 and Figure 15).</li>
</ol>
7. Pancreatic Tumor Detection and Volume Evaluation
<ol>
	<li>Identify pancreatic lesions from all surrounding structures. They generally appear as hypoechoic inhomogeneous round or elongated tissue masses (Figure 16). The tumor is usually firm and can hardly be compressed by the scan head. Note, from time to time it might be tricky to clearly distinguish between a tumor and a small bowel loop. Pay attention to the bowel peristalsis that should not be observed in pancreatic tumors.</li>
	<li>Upon tumor detection screen up and down to get an impression of the total size and measure the largest diameter by using the ultrasound caliper. Once a suitable image is found, freeze the screen and use the measure tool to precisely determine the tumor size. Note, for later use it will be important to differentiate whether a tumor is localized in the tail, body or head of the pancreas.</li>
	<li>Before changing back to live mode, make sure to save pictures to record the work. Note, while freezing the image it might happen that the adjustment wheel is released, and the ultrasound probe is even more pressed to the mouse abdomen. Therefore, fix the adjustment of the compression before changing the mode to freeze.</li>
	<li>After finishing the measurement of the first plane either rotate the scan head by 90&#176; in longitudinal position (Figure 17), or turn the mouse to its side, to detect the tumors&#39; longitudinal expansion. Some tumors may require changing the position of the mouse for adequate visibility. For pancreatic tail tumors, the mouse can be placed on its right side (Figure 10 and Figure 19), for pancreatic head tumors on its left side (Figure 11 and Figure 20).</li>
</ol>
8. Quantification of Tumor Volume
Note: One major aim of all efforts is the correct determination of tumor volume. Although there are several techniques available a calculation method including the formula of an ellipsoid is preferred at the University Medical Center Goettingen.
<ol>
	<li>Equipped with all three previously obtained diameters, calculate the approximate volume of the corresponding tumor (Figure 21).</li>
	<li>Alternatively, use a motor system of the scan head to perform an automated scan of the tumor region with subsequent volume calculation and 3-D-reconstruction.</li>
</ol>
9. Recovery
<ol>
	<li>After the scanning, carefully remove all adhesive tape and carefully remove all ultrasound gel with dry tissue.</li>
	<li>Carefully transfer the mouse into the recovery cage ( Figure 21). Avoid any covering tissue at the mouse&#180;s face that might cause respiratory impairment. A side position for recovery should be preferred.</li>
</ol>

<ol>
	<li>Kersten, K., de Visser, K. E., van Miltenburg, M. H., Jonkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+in+oncology+research+and+cancer+medicine.">Genetically engineered mouse models in oncology research and cancer medicine.</a> EMBO Molecular Medicine. 9 (2), 137-153 (2017).</li><li>Olive, K. P., Politi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Translational therapeutics in genetically engineered mouse models of cancer. (2), 131-143 (2014).</li><li>Westphalen, C. B., Olive, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+engineered+mouse+models+of+pancreatic+cancer.">Genetically engineered mouse models of pancreatic cancer.</a> The Cancer Journal. 18 (6), 502-510 (2012).</li><li>Hingorani, S. 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=Trp53R172H+and+KrasG12D+cooperate+to+promote+chromosomal+instability+and+widely+metastatic+pancreatic+ductal+adenocarcinoma+in+mice.">Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice.</a> Cancer Cell. 7 (5), 469-483 (2005).</li><li>Frese, K. 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=nab-Paclitaxel+potentiates+gemcitabine+activity+by+reducing+cytidine+deaminase+levels+in+a+mouse+model+of+pancreatic+cancer.">nab-Paclitaxel potentiates gemcitabine activity by reducing cytidine deaminase levels in a mouse model of pancreatic cancer.</a> Cancer Discovery. 2 (3), 260-269 (2012).</li><li>Paredes, J. L., 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=A+non-invasive+method+of+quantifying+pancreatic+volume+in+mice+using+micro-MRI.">A non-invasive method of quantifying pancreatic volume in mice using micro-MRI.</a> PLoS One. 9 (3), e92263 (2014).</li><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Aung, 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=Immunotargeting+of+Integrin+alpha6beta4+for+Single-Photon+Emission+Computed+Tomography+and+Near-Infrared+Fluorescence+Imaging+in+a+Pancreatic+Cancer+Model.">Immunotargeting of Integrin alpha6beta4 for Single-Photon Emission Computed Tomography and Near-Infrared Fluorescence Imaging in a Pancreatic Cancer Model.</a> Molecular Imaging. 15, (2016).</li><li>Akladios, C. Y., 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=Contribution+of+microCT+structural+imaging+to+preclinical+evaluation+of+hepatocellular+carcinoma+chemotherapeutics+on+orthotopic+graft+in+ACI+rats.">Contribution of microCT structural imaging to preclinical evaluation of hepatocellular carcinoma chemotherapeutics on orthotopic graft in ACI rats.</a> Bulletin du Cancer. 98 (2), 120-132 (2011).</li><li>Neesse, A., 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=CTGF+antagonism+with+mAb+FG-3019+enhances+chemotherapy+response+without+increasing+drug+delivery+in+murine+ductal+pancreas+cancer.">CTGF antagonism with mAb FG-3019 enhances chemotherapy response without increasing drug delivery in murine ductal pancreas cancer.</a> Proceedings of the National Academy of Science USA. 110 (30), 12325-12330 (2013).</li><li>Sastra, S. A., Olive, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+murine+pancreatic+tumors+by+high-resolution+ultrasound.">Quantification of murine pancreatic tumors by high-resolution ultrasound.</a> Methods in Molecular Biology. 980, 249-266 (2013).</li></ol>

The authors have nothing to disclose.

With this protocol, a detailed description for quantifying pancreatic tumors using high-resolution abdominal ultrasound imaging in genetically engineered mouse models is provided. Recently, Sastra et al. published a detailed description how to quantify pancreatic tumors in mouse models, but no visualized instructions about the preparation and handling as prerequisite for all further steps were shown11. The overall goal of this manuscript is to provide a comprehensive visual guide for high-resolution ultrasound in mice.
Although we focus our description on endogenous pancreatic tumors in genetically engineered mice, this method can also be used for orthotopic transplantation models. For orthotopic models, even very small tumors up to 1-2 mm can be detected abdominally to investigate the take rate and tumor onset. As abdominal palpation can be subjective and difficult to reproduce, ultrasound screening can also be applied without prior palpation in the KPC model to detect the onset of very small pancreatic tumors. Furthermore, abdominal structures such as small bowel loops filled with air may be superimposed. In this regard, it might be helpful to change the position of the animal on the working stage. Most tumors can be visualized in a supine position; however, some tumors may only be detected when the mouse is placed on its left side (pancreatic head tumors) or right side (pancreatic tail tumors). Thus, an adequate positioning of the mouse is critical to increase efficient visibility.
Insufficient visibility of abdominal organs or anatomical variability might occur. To increase visibility and resolution of the ultrasound images, brightness and contrast settings as well as the focus should be altered accordingly. Image artefacts might be due to defective/wrong probe, incomplete removal of mouse fur, insufficient amounts of ultrasound gel or insufficient contact pressure of the probe with the mouse abdomen. Furthermore, slightly tilting the probe may also considerably improve visibility.
Furthermore, tumor growth kinetics can be monitored longitudinally by small animal ultrasound. Major advantages of the technique include low costs (given the availability of an ultrasound machine), no radiation exposure, short scanning time, and thus little strain for mice. Especially in light of longitudinal cancer studies, repeated scanning is easy to perform and puts little strain on the animals without the need of lengthy periods of sedation or anesthesia that is necessary for small animal CT or MRI scans.
Since technical advances with more powerful ultrasound systems are currently being developed with improved resolution and increasingly automated image optimization tools, small animal, high resolution ultrasound will be used more often in various preclinical disciplines in the future. Critical steps within the protocol include correct mouse preparation and positioning, as well as correct identification of abdominal organs and vessels.
High resolution ultrasound is a valuable tool for the detection and quantification of pancreatic tumors in genetically engineered and orthotopic mouse models. Compared to other established imagining techniques this ultrasound-based method combines fast session times and cost efficiency. Since the correct acquisition and interpretation of ultrasound images requires proper hands-on training and experience this video protocol serves as a detailed guideline for all aspects in pancreatic cancer models.

Genetically engineered mouse models have gained an increasing importance in cancer research due to their ability to closely recapitulate the complex nature of human carcinogenesis1,2,3. One of the most frequently used models to study pancreatic cancer development, progression and therapeutic response is characterized by an activating mutation in the Kras oncogene combined with an inactivation of the tumor suppressor p534. This LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model mimics the step-wise progression from pre-invasive pancreatic intraepithelial neoplasia (PanIN) lesions to invasive carcinoma. Phenotypically, nearly all mice develop PDAC within the first six months after birth. However, compared to transplanted models, the KPC model reveals a highly variable tumor onset from 8 weeks to several months4. Once pancreatic tumors reach a certain size (5-9 mm in diameter), tumor growth accelerates rapidly and mice will have to be enrolled in preclinical trials5. Therefore, the exact detection of tumor onset and tumor size is an essential prerequisite for preclinical study logistics and therapy monitoring. In general, several approaches like magnetic resonance imaging (MRI)6, computed tomography scanning7,8,9 or high resolution ultrasound can be employed to conduct tumor screening and therapy10. Each technique has its advantages and drawbacks. Although MRI-or computed tomography (CT) -imaging allows high resolution data acquisition as well as accurate volume calculation, prolonged examination time under general sedation, and very expensive equipment is required, and does not permit frequent scanning over a long period of time. In contrast, small animal ultrasonography is an established method that can be employed to screen for abdominal pathologies in mice11. Advantages of this imaging method are short scanning times, high resolution, and the possibility to use doppler ultrasound or contrast enhanced ultrasound (CEUS) to visualize perfusion of organs in parallel. However, anatomical knowledge, 3D imagination and thorough practical training are required for correct image interpretation.
In the following article, a detailed protocol for utilizing high resolution ultrasound in the KPC model is provided. Furthermore, standard ultrasound images are depicted and labelled with organ structures to facilitate orientation for the investigator.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Visual Sonics Vevo2100 High Resolution Ultrasound System, including imaging stage and anesthesia line</td>
			<td>FUJIFILM VisualSonics Inc, Canada</td>
			<td>VS-11945</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo 2100 MicroScan Transducer MS-550-D (22-55MHz)</td>
			<td>FUJIFILM VisualSonics Inc, Canada</td>
			<td>VS-11874</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Anesthesia System (anesthesia induction chamber with fresh and waste gas inlet)</td>
			<td>FUJIFILM VisualSonics Inc, Canada</td>
			<td>SA-12055</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo Imaging Station (working stage with nose cone for anesthesia supply)</td>
			<td>FUJIFILM VisualSonics Inc, Canada</td>
			<td>SA-11982</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;electronic pet clippers</td>
			<td>Panasonic Marketing Europe, Germany</td>
			<td>5025232484324</td>
			<td>Panasonic ER-PA10-s</td>
		</tr>
		<tr>
			<td>Labotect Hot plate</td>
			<td>Labor tech G&#246;ttingen, Germany</td>
			<td>13854</td>
			<td></td>
		</tr>
		<tr>
			<td>eye cream (ophthalmic ointment)</td>
			<td>Sch&#252;lke&#38;Mayr, Germany</td>
			<td>9080249</td>
			<td></td>
		</tr>
		<tr>
			<td>veterinary isoflurane</td>
			<td>Abbvie, Germany</td>
			<td>4831867</td>
			<td></td>
		</tr>
		<tr>
			<td>depilatory cream</td>
			<td>RB healthcare UK, United Kingdom</td>
			<td>8218535</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol (v/v) in distilled water</td>
			<td>TH. Geyer, Germany</td>
			<td>22941000</td>
			<td></td>
		</tr>
		<tr>
			<td>ultrasound gel</td>
			<td>Asmuth, Germany</td>
			<td>13477</td>
			<td></td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>Kimberly-Clark Germany, Germany</td>
			<td>7558</td>
			<td></td>
		</tr>
		<tr>
			<td>cotton tips</td>
			<td>Meditrade, Germany</td>
			<td>75481116</td>
			<td></td>
		</tr>
		<tr>
			<td>glass bowl for ultrasound gel</td>
			<td>ARC France, France</td>
			<td>H1149</td>
			<td></td>
		</tr>
		<tr>
			<td>water bowl</td>
			<td>W &#38; P Trading Co., USA</td>
			<td>B00K2P6PLQ</td>
			<td></td>
		</tr>
		<tr>
			<td>gauze sponges</td>
			<td>Fuhrmann, Germany</td>
			<td>960504</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilizing high resolution ultrasound to monitor tumor onset and growth in genetically engineered pancreatic cancer models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

Ultrasound imaging is a versatile and non-invasive technique that is used to address several issues in murine models of human diseases. Compared to all other imaging approaches major advantages are high-throughput, cost efficiency, short acquisition time and real-time imaging. However, this tool needs expertise to generate accurate, high quality images. Particularly in the case of unwanted artefacts at least some experience with ultrasound imaging in general is very helpful. In relation to pancreatic cancer this tool allows to determine tumor onset, progression and response to therapy. Furthermore, by measurement of different diameters it allows an exact quantification of the tumor volume over time. This study demonstrates and describes in detail how to use high resolution ultrasound imaging as a screening tool for pancreatic cancer in genetically engineered mouse models.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56979/56979fig1.jpg" /> 
Figure 1: Abdominal palpation. Fixing hand gently pulls up tail while non-fixing hand starts procedure by gently moving up and down. <a href="/files/ftp_upload/56979/56979fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56979/56979fig2.jpg" /> 
Figure 2: Pre-scanning area with overview of all needed items. skin disinfection (1), gauze sponges (2), 70% isopropanol (3), ultrasound gel with glass bowel (4), ophthalmic ointment (5), tissue wipes (6), medical tape (7), water container (8), pet clippers (9) depilatory cream with small container (10), cotton tips to apply eye ointment or ultrasound gel (11). <a href="/files/ftp_upload/56979/56979fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56979/56979fig3.jpg" /> 
Figure 3: Scanning area with Isoflurane vaporizer. Working stage with adjustable probe holder (1), induction chamber (2) including isoflurane vaporizer (3). <a href="/files/ftp_upload/56979/56979fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56979/56979fig4.jpg" /> 
Figure 4: Ultrasound device. The ultrasound system is supplied with three ultrasound probes and corresponding ports. Keyboard and PC mouse are included in the system. <a href="/files/ftp_upload/56979/56979fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56979/56979fig5.jpg" /> 
Figure 5: Recovery cage. The bottom of the cage is prepared with thin tissue and pre-heated to allow for sufficient heating after the scanning procedure. The temperature is shown in Celsius (C&#176;). <a href="/files/ftp_upload/56979/56979fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56979/56979fig6.jpg" /> 
Figure 6: Fixed mouse on working stage. The mouse is placed in a supine position on the working stage. <a href="/files/ftp_upload/56979/56979fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56979/56979fig7.jpg" /> 
Figure 7: Shaved mouse after using pet clippers. The abdominal fur is cut, but smaller parts of hair persist which regularly cause artefacts. <a href="/files/ftp_upload/56979/56979fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/56979/56979fig8.jpg" /> 
Figure 8: Use of depilatory cream. Administer a thin layer of depilatory cream on the shaved region of the abdomen. <a href="/files/ftp_upload/56979/56979fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/56979/56979fig9.jpg" /> 
Figure 9: Completely shaved abdomen of a mouse. Plenty of water is used to rinse off all cream remnants, only a complete removal of fur allows an optimal scanning procedure. <a href="/files/ftp_upload/56979/56979fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/56979/56979fig10.jpg" /> 
Figure 10: Right side of mouse. Optimal orientation for scanning pancreatic tail tumors. <a href="/files/ftp_upload/56979/56979fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/56979/56979fig11.jpg" /> 
Figure 11: Left side of mouse. Optimal orientation for scanning pancreatic head tumors. <a href="/files/ftp_upload/56979/56979fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/56979/56979fig12.jpg" /> 
Figure 12: Ultrasound probe on mouse abdomen. To prevent any injury to the mouse do not use too much pressure. <a href="/files/ftp_upload/56979/56979fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/56979/56979fig13.jpg" /> 
Figure 13: Ultrasound image of the right abdominal part. Main vessel structures such as abdominal aorta and vena cava inferior are located in close proximity to the kidney and liver. <a href="/files/ftp_upload/56979/56979fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/56979/56979fig14.jpg" /> 
Figure 14: Ultrasound image of the left abdominal part. Between left kidney and spleen the membranous structure of the pancreas is located, as a landmark, the vena lienalis is running through the organ (yellow arrow) and can be used as a guiding structure. <a href="/files/ftp_upload/56979/56979fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/56979/56979fig15.jpg" /> 
Figure 15: Border of the pancreas. The stomach (with contents) marks the left border of the pancreas (yellow arrow). <a href="/files/ftp_upload/56979/56979fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/56979/56979fig16.jpg" /> 
Figure 16: Identifying pancreatic tumor. Supine position with a hypodense appearing round pancreatic tumor, arrow indicates the border of the surrounding normal tissue. <a href="/files/ftp_upload/56979/56979fig16large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/56979/56979fig17.jpg" /> 
Figure 17: Longitudinal position. Determining the length of the pancreatic tumor. <a href="/files/ftp_upload/56979/56979fig17large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 18" class="xfigimg" src="/files/ftp_upload/56979/56979fig18.jpg" /> 
Figure 18: Longitudinal scan of pancreatic tumor. Interference caused by air filled duodenum. <a href="/files/ftp_upload/56979/56979fig18large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 19" class="xfigimg" src="/files/ftp_upload/56979/56979fig19.jpg" /> 
Figure 19: Right side of the mouse: pancreatic tail tumor <a href="/files/ftp_upload/56979/56979fig19large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 20" class="xfigimg" src="/files/ftp_upload/56979/56979fig20.jpg" /> 
Figure 20: Left side of the mouse: pancreatic head tumor <a href="/files/ftp_upload/56979/56979fig20large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 21" class="xfigimg" src="/files/ftp_upload/56979/56979fig21.jpg" /> 
Figure 21: Tumor volume quantification. After acquisition of all three diameters in the previously indicated two scanning positions, a volume calculation is performed. Therefore, the formula of an ellipsoid is used: 4/3 &#960; a/2* b/2* c/2. <a href="/files/ftp_upload/56979/56979fig21large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 22" class="xfigimg" src="/files/ftp_upload/56979/56979fig22.jpg" /> 
Figure 22: Recovery. After scanning the animal is transferred to a heating plate until fully recovered, notice the white eye ointment at the mouse`s eye. <a href="/files/ftp_upload/56979/56979fig22large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models at JoVE.com

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

utilizing-high-resolution-ultrasound-to-monitor-tumor-onset-growth

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.7K Views.  University Medical Center Goettingen. This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

Video: Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables associated to specific pancreatic pathologies to help preoperative differential diagnosis and patient profiling. Pancreatic juice is a relatively unexplored body fluid, which, due to its close proximity to the tumor site, reflects changes in the surrounding tissue. Here we describe in detail the intraoperative collection procedure. Unfortunately, translating pancreatic juice collection to murine models of PDAC, to perform mechanistic studies, is technically very challenging. Tumor interstitial fluid (TIF) is the extracellular fluid, outside blood and plasma, which bathes tumor and stromal cells. Similarly to pancreatic juice, for its property to collect and concentrate molecules that are found diluted in plasma, TIF can be exploited as an indicator of microenvironmental alterations and as a valuable source of disease-associated biomarkers. Since TIF is not readily accessible, various techniques have been proposed for its isolation. We describe here two simple and technically undemanding methods for its isolation: tissue centrifugation and tissue elution.

Pancreatic juice is a precious source of biomarkers for human pancreatic cancer. We describe here a method for intraoperative collection procedure. To overcome the challenge of adopting this procedure in murine models, we suggest an alternative sample, tumor interstitial fluid, and describe here two protocols for its isolation.

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

Molecular and Immunologic Techniques in a Genetically Engineered Mouse Model of Gastrointestinal Stromal Tumor

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor types, numerous mouse models have been developed in order to investigate the next generation of cancer therapies. However, in GIST, most in vivo studies use xenograft mouse models which have inherent limitations. Here, we describe an immunocompetent, genetically engineered mouse model of gastrointestinal stromal tumor harboring a KitV558&#916;/+ mutation. In this model, mutant KIT, the oncogene responsible for most GISTs, is driven by its endogenous promoter leading to a GIST which mimics the histological appearance and immune infiltrate seen in human GISTs. Furthermore, this model has been used successfully to investigate both targeted molecular and immune therapies. Here, we describe the breeding and maintenance of a KitV558&#916;/+ mouse colony. Additionally, this paper details the treatment and procurement of GIST, draining mesenteric lymph node, and adjacent cecum in KitV558&#916;/+ mice, as well as sample preparation for molecular and immunologic analyses.

The goal of this manuscript is to describe the KitV558&#916;/+&#160;mouse model and techniques for successful dissection and processing of mouse specimens.

Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the KIT receptor. Across tumor ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features of a unique and convenient model system. As highly proliferative cells are more susceptible to radiation-induced DNA damage, zebrafish embryos are a front-line in vivo model in radiation research. In addition, this model projects the effect of radiation and different drugs within a short time, along with major biological events and associated responses. Several cancer studies have used zebrafish, and this protocol is based on the use of radiation modifiers in the context of radiotherapy and cancer. This method can be readily used to validate the effects of different drugs on irradiated and control (non-irradiated) embryos, thus identifying drugs as radio sensitizing or protective drugs. Although this methodology is used in most drug screening experiments, the details of the experiment and the toxicity assessment with the background of X-ray radiation exposure are limited or only briefly addressed, making it difficult to perform. This protocol addresses this issue and discusses the procedure and toxicity evaluation with a detailed illustration. The procedure describes a simple approach for using zebrafish embryos for radiation studies and radiation-based drug screening with much reliability and reproducibility.

The zebrafish has recently been exploited as a model to validate potential radiation modifiers. The present protocol describes the detailed steps to use zebrafish embryos for radiation-based screening experiments and some observational approaches to evaluate the effect of different treatments and radiation.

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...