Name: dynamic contrast enhanced magnetic resonance imaging of an orthotopic pancreatic cancer mouse model
Uploaded: 2015-04-18T15:00:00.000+00:00
Duration: 6 min 24 s
Description: Watch this Scientific Journal Video about Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model at JoVE.com

Authors thank Jeffrey Sellers to assist orthotopic pancreatic cancer mouse modeling. This work was supported by Research Initiative Pilot Awards from the Department of Radiology at UAB and NIH grants 2P30CA013148 and P50CA101955.

All procedures were approved by the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham.
1. Orthotopic Pancreatic Tumor Mouse Modeling
<ol>
	<li>Culture standard human pancreatic-cancer cell lines in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum. Maintain all cultures at 37 &#176;C in humidified atmosphere with 5% CO2.</li>
	<li>Use 8-10 week-old female severe combined immunodeficient&#160;mice. Place animal cages at 12 hr light and 12 hr darkness cycle at RT (21 &#177; 2 &#176;C) and 60% humidity.</li>
	<li>Anesthetize all animals using ventilation with 2% of isoflurane mixed with oxygen (2 L/min) throughout surgery. Confirm the depth of anesthesia by toe pinch reflex. Place animals on a heating pad (37 &#176;C) to maintain the body temperature.&#160;Apply veterinary ophthalmic ointment on eyes to prevent dryness while under anesthesia. &#160;</li>
	<li>Remove the hair in the left upper quadrant of the abdomen of each mouse, and give an analgesic drug (carpofen, 5 mg/kg of body weight subcutaneously) into the area. Apply a betadine solution to the exposed skin. Prepare autoclaved surgical instruments.</li>
	<li>Make a 1 cm incision in the skin and peritoneum using iris straight scissors. Gently remove the pancreas from the abdomen using surgical tweezers.</li>
	<li>Insert 28 G needle of a 0.5 ml insulin syringe into the tail of pancreas and then slowly infuse a solution of 2.5 million human pancreatic cancer cells in 30 &#181;l of DMEM. Confirm that a small bleb is created in the head of pancreas by the solution.</li>
	<li>Gently place the pancreas back into the abdomen using surgical tweezers. Close the peritoneum and the skin in 1 layer with 2 interrupted 5-0 Prolene sutures, and then terminate the anesthesia. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.&#160;Remove sutures at 7~10 days post surgery.</li>
	<li>Give another dose of the analgesic drug (carpofen, 5 mg/kg of body weight subcutaneously) at 24 hr after surgery.</li>
	<li>Check tumor size by palpating the surgery area using two fingers. Tumors typically feel denser and bumpier than the surrounding tissues and organs. Usually it takes ~1 - 2 weeks to start feeling a tumor.</li>
	<li>Monitor animals daily for signs of illness. When animals appear ill (lack normal grooming and avoidance behaviors), we terminated them using cervical dislocation while under anesthesia.</li>
</ol>
2. Magnetic Resonance Imaging
<ol>
	<li>Apply MRI when tumor size is about 5 - 7 mm in diameter at usually 2~4 weeks after cell implantation. Use an MR scanner dedicated to small animal imaging or a clinical MR scanner equipped with a specialized coil for small animal imaging. 
	NOTE: We used a 9.4T small animal MR scanner with a combination of a 1H volume resonator/transmitter and a surface coil receiver (30 mm in diameter)(Bruker BioSpin Corp., Billerica, MA). A surface coil provides better signal-to-noise ratio (SNR)14.</li>
	<li>Prepare a gadolinium-based MRI contrast agent to inject ~0.1 - 0.2 mmol/kg to each animal in ~0.1 - 0.2 ml PBS (phosphate buffered saline). 
	NOTE: We used gadoteridol, and injected 0.2 mmol/kg in 0.15 ml PBS over a period of 15 sec (0.1 ml/sec).</li>
	<li>Prepare a micro-polyethelene tube (length: 7.62 mm, inner diameter: 0.28 mm, outer diameter: 0.64 mm). Insert a 30 G needle (12.7 mm length) into one end of the tube, and a 30 G blunt tip needle (9.5 mm length) into the other end. Connect a 1 ml syringe containing MR contrast agent to the blunt tip needle, and slowly push the syringe to fill up the entire tube with the MR contrast agent.</li>
	<li>Anesthetize animals using ventilation with ~1 - 2% of isoflurane mixed with oxygen (2 L/min) throughout preparation and imaging. Confirm the depth of anesthesia by toe pinch reflex. Apply veterinary ophthalmic ointment on eyes to prevent dryness while under anesthesia. Dilate the tail vein using a heat lamp before needle insertion. Grab the middle of the 30 G needle using Kelly forceps, and insert it into the tail vein. Tape both the tail and tube onto a piece of plastic or cardboard paper (10 mm width x 100 mm length) to keep the tail straight.</li>
	<li>Place the animal in supine position in an animal bed equipped with circulating warm water (or warm air) to regulate body&#160;temperature during imaging. Set the temperature on the bed to 37 &#176;C. Insert a rectal temperature probe to monitor the body temperature during imaging.</li>
	<li>Apply an orthogonally bent plastic board into the abdominal area. Make sure the tumor is located behind the upper end of the board, and then pull down the board slightly (~2 mm) to ensure the tumor is caught by the board. Tape the board to animal bed firmly.</li>
	<li>Tape a respiration pad transducer (SA Instrument, Inc., Stony Brook, NY)&#160;&#160;on the chest area to monitor animal respiration during imaging. Place a surface coil on the top of the tumor region, and tape it to animal bed firmly. Push the animal bed into the MR scanner to place the tumor region at the center of the volume coil (inner diameter: 72 mm).</li>
	<li>Perform matching and tuning for both the receiver and transmitter, followed by shimming.</li>
	<li>Begin with an anatomical MR sequence to locate the tumor. Use a T2-weighted (T2W) turbo spin-echo sequence to obtain axial images with the following acquisition parameters. Repetition time (TR)/echo time (TE) = 3,000/34 msec, 128 x 128 matrix, 30 x 30 mm field of view, number of averages = 1, echo train length = 4, and 20 contiguous 1 mm thick slices in an interlaced mode to cover the entire tumor region (total scanning time: 1.6 min). 
	NOTE: Since orthotopic pancreatic tumors are more difficult to be located than subcutaneous ones, conventional localizer images having lower resolution may not be useful.</li>
	<li>Acquire T1-weighted (T1W) images with various flip angles to retrieve T1 map. For this purpose, use a gradient echo multiflip angle approach with the following parameters: repetition time (TR)/echo time (TE) = 115/3 msec, 128 x 128 matrix,&#160;a 30 x 30 mm field of view, number of averages = 4, ~5 - 7 contiguous 1 mm thick slices in an interlaced mode to cover the tumor region, and seven flip angles of 10, 20, 30, 40, 50, 60, and 70 (total scanning time per flip angle: 1 min). 
	NOTE: However, multiflip angle approach is efficient only when B1 field homogeneity is high. If not, T1 maps can be obtained with multiple TR approach instead15.</li>
	<li>Acquire T1W images before and after gadolinium based MR contrast injection. Use the same acquisition parameters and geometry for T1 mapping but with the fixed flip angle of 30. Use linear encoding to ensure steady state when the center of k-space is obtained, especially when a short TR and a low degree flip angle are used. Acquire 5 baseline images before contrast injection. Then acquire 40 images after contrast injection (total scanning time: 45 min). Use a syringe pump to inject contrast agent at a constant rate (0.01 ml/sec).</li>
	<li>Monitor animal breathing continuously, and adjust isoflurane concentration to keep the respiratory rate to 50 - 100 breaths per min. Monitor animal body temperature throughout imaging.</li>
	<li>After completing DCE-MRI, take off the needle and other probes, and place the animal in an empty cage bedded with paper towels.&#160; Massage softly the lower abdominal area.&#160; The cage should be placed half under a heat lamp to allow the animal to move in and out of the heat gradient as it recovers. &#160;Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.</li>
</ol>
3. Image Processing and Analysis
<ol>
	<li>Segment tumor region in T2W images. In T2W images, the signal intensity in tumor region is brighter than that of surrounding tissues, so the tumor boundary can be manually delineated. 
	NOTE: Semi-automatic segmentation techniques such as global thresholding or active contouring can be used16,17, but uneven background intensity should be corrected especially when a surface coil is used.</li>
	<li>Create T1 and proton density maps. In T1W images acquired with a gradient echo sequence, assuming that echo time (TE) is much less than T2* value, the pixel value is determined by 
	<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq1.jpg" /> 
	where S0 is proton density, T1 is T1 relaxation time constant, TR is repetition time, and &#952; is a flip angle. Equation (1) can be rewritten to 
	<img alt="Equation 2" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq2.jpg" /> 
	when S(&#952;)/sin&#952; is replaced with Y, and S(&#952;)/tan&#952; is replaced with X. Equation (2) is a linear equation, and its slope and intercept can be used to retrieve T1 and S0 values, respectively.</li>
	<li>Calculate MR contrast concentration in DCE-MR images. When gadolinium based MR contrast agent is injected, T1 relaxation time constant is changed over time. So, equation (1) can be rewritten to 
	<img alt="Equation 3" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq3.jpg" /> 
	T1(t) is related with MR contrast concentration, C(t), as follows, 
	<img alt="Equation 4" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq4.jpg" /> 
	where r1 is longitudinal relaxivity of MR contrast agent. So, by combining equations (3) and (4), MR contrast concentration is determined by 
	<img alt="Equation 5" fo:content-width="3in" src="/files/ftp_upload/52641/52641eq5.jpg" /></li>
	<li>Quantitate the pharmacokinetic parameters of MR contrast agent. Cp(t) presents MR contrast concentration in blood plasma at time t after initiating contrast injection. Cp(t) is called arterial input function (AIF). If AIF is available, the pharmacokinetic parameters of MR contrast agent can be calculated by 
	<img alt="Equation 6" fo:content-width="4in" src="/files/ftp_upload/52641/52641eq6.jpg" /> 
	where Ct(t) is MR contrast concentration in a target tissue, vp is fractional blood plasma volume, ve is fractional extravascular extracellular volume, and Ktrans is volume transfer constant. Flux rate constant, kep, is equal to Ktrans divided by ve. If AIF is not available, then the reference region model can be used instead18,19. The reference region model is based on the flow-limited Kety model20 and uses contrast concentration in a reference region to remove the need for AIF as follows, 
	<img alt="Equation 7" fo:content-width="5in" src="/files/ftp_upload/52641/52641eq7.jpg" /> 
	where Ct,ROI(t), Ktrans,ROI, and ve,ROI are contrast concentration, volume transfer constant, and fractional extravascular-extracellular volume, respectively, in the region of interest (ROI), while Ct,RR(t), Ktrans,RR, and ve,RR are those in the reference region. Paravertebral muscle is often selected as the reference region, and ve,RR in murine paravertebral muscle is assumed to be constant at 0.08 21. We used the reference region model.</li>
</ol>

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Authors do not have any conflicts of interest to declare.

We have introduced the detailed methods of orthotopic pancreatic tumor modeling using immunodeficient mice, DCE-MRI of abdominal tumors in mice, and quantification of its kinetic parameters. In orthotopic pancreatic tumor modeling, care must be taken when inserting a needle into the tail of pancreas. If successful, cells will be transferred to the head of pancreas creating a small bleb. When applying an orthogonally bent plastic board, it is critical to confirm that the tumor is located below the upper end of the board. Since the pancreatic tumor is near to the diaphragm, the board may be not able to hold it firmly, especially when the tumor size is less than 5 mm in diameter. After imaging, the abdominal area should be massaged gently until the animal is conscious. It is recommended not to exceed the total imaging and preparation time for each animal longer than 90 min in order to prevent abdominal pain.
The dimension of the plastic board may need to be readjusted according to the size of tumors or animals. If the tumor size is smaller than 5 mm, the tumors may not be firmly caught by the orthogonally bent board having 4 mm depth and thereby motion artifact may occur in MR images. Also if the animal size is larger than 20 g, the 4 mm depth may not be enough to hold the tumor. However, too high pressure into abdomen can damage the abdominal nervous system paralyzing the lower body of the animal.&#160;
Orthotopic pancreatic tumor modeling in mice using standard human pancreatic cancer cell lines has been used to study the therapeutic response of pancreatic tumors. However, tumor take rate (percentage of that an animal will have a palpable tumor after implantation) and growth rate were highly dependent upon tumor cell characteristics, so it is recommended to start with two-fold more animals and select half of them bearing similar size tumors. Tumors with irregular shape should be excluded, since they might respond to a therapy differently; the shape irregularity can be detected by routine palpation, but ultrasound imaging will provide better assessment9. Some cell lines can develop metastasis usually in the liver. Liver metastases (METs) can be detected commonly using bioluminescent imaging when luciferase-positive cells were implanted or by liver dissection after animals died. Liver METs shorten the life span of the animals, so the study plan should be established accordingly.
Quantitative DCE-MRI has been successfully implemented for an orthotopic pancreatic tumor mouse model. To quantitate pharmacokinetic parameters of MR contrast agent based on the flow-limited Kety model20, the arterial input function (AIF) must be available. In this application, the abdominal aorta is within the field of view, so AIF can be obtained by directly measuring the change of the MR contrast concentration in the abdominal aorta. However, the inner diameter of the mouse abdominal aorta is less than 1 mm (0.5 - 0.7 mm)22, and empirically the pixel size should be at least four times smaller than the diameter (0.125 mm) to prevent severe partial volume effect. Furthermore, the ideal sampling rate of AIF is 1 - 2 sec23. In MRI, spatial and temporal resolutions compromise with SNR. Therefore it is challenging to obtain a reliable AIF in DCE-MRI of mice. Alternatively, the reference region (RR) model can be used. RR model employs the data of contrast enhancement in a reference region (typically muscle tissue) to remove the need of AIF in the pharmacokinetic-parameter quantification. However, the fractional extravascular extracellular volume in the reference region must be assumed, which caused about 35% error in one study (n = 9)21. Nonetheless, the change of tumor microvasculature following therapy can be accurately measured, if the same reference region is selected at each time.
In conclusion, we have demonstrated an inexpensive and simple method to suppress motion artifact in abdominal area of mice during DCE-MRI. This technique can be easily adapted for other orthotopic mouse models of gastrointestinal cancers such as gastric and colorectal cancers24,25.

The overall goal of this method is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to assess microvasculature in a target tissue by monitoring the change of MR contrast over a certain period of time after injection. DCE-MRI has been utilized to diagnose malignant tumors and to assess tumor response to various therapies1-4. Quantitative DCE-MRI has presented high reproducibility5. To quantitate pharmacokinetic parameters of an MR contrast agent in a target tissue, all DCE-MR images acquired at different time points and T1 map obtained before contrast injection must be coregistered6. However, due to respiratory and peristaltic motions in the abdominal area, quantitative DCE-MRI has had limited application for gastrointestinal tumors.
Orthotopic pancreatic tumor models have been utilized to assess pancreatic-tumor response following biological therapies and chemotherapies7,8. Orthotopic tumor models are considered superior to conventional subcutaneous models, since the microenvironment in the original tumor site is reflected and thereby human tumor response to therapy can be more accurately predicted. However, the mouse pancreas is located in the left upper quadrant of the abdomen, so quantitative DCE-MRI of orthotopic pancreatic tumor xenografts in mice has not been readily implemented.
We have established a protocol of DCE-MRI of abdominal tumors in mice by fixing the tumors using an orthogonally bent plastic board to prevent motion transfer from the chest region9. The pressure applied by this board was localized on the abdominal area, and has not resulted in respiratory difficulty. An automated image coregistration technique has been validated for DCE-MRI of abdominal organs in a free-breathing mode, but it performs effectively only when the target regions move slowly and regularly10. Respiratory rate of animals is variable during imaging, so physical restraint in the abdominal area will be necessary to retrieve reliable pharmacokinetic parameters in orthotopic pancreatic tumor mouse models. We have successfully quantitated the pharmacokinetic parameters of an MR contrast agent in orthotopic pancreatic tumor xenografts using the orthogonally bent plastic board in DCE-MRI11-13. Here we present the detailed procedure of orthotopic pancreatic tumor modeling, DCE-MRI of the tumor xenografts in mice, and quantification of pharmacokinetic parameters.

<table><tbody><tr><td>DMEM</td><td>Invitrogen</td><td>11965-118</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Harlan Laboratories</td><td>BT-9501</td><td></td></tr><tr><td>Betadine</td><td>Purdue products</td><td>67618-153-01</td><td></td></tr><tr><td>5-0 Prolene sutures</td><td>Ethicon</td><td>8720H</td><td></td></tr><tr><td>9.4T MR scanner</td><td>Bruker Biospin Corporation</td><td>BioSpec 94/20 USR</td><td></td></tr><tr><td>Gadoteridol</td><td>Bracco Diagnostics Inc</td><td>NDC 0270-1111-03</td><td></td></tr><tr><td>Micro-polyethelene tube</td><td>Strategic Applications, Inc</td><td>#PE-10-25</td><td></td></tr><tr><td>30 G blunt tip needle</td><td>Strategic Applications, Inc</td><td>89134-194</td><td></td></tr><tr><td>Monitoring and gating system</td><td>SA instruments, Inc</td><td>Model 1030</td><td>This is an MR compatiable system to measure resiratory rating and body temperature of small animals at the same time.</td></tr><tr><td>Syringe pump</td><td>New Era Pump Systems, Inc.</td><td>NE-1600</td><td></td></tr></tbody></table>

dynamic contrast enhanced magnetic resonance imaging of an orthotopic pancreatic cancer mouse model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

Human pancreatic tumor cells grow successfully in mouse pancreas creating a solid tumor. Figure 1 shows photographs of (A) a normal pancreas where tumor cell solution is injected, and (B) a representative mouse bearing an orthotopic pancreatic tumor xenograft (MIA PaCa-2). Tumor is located in the left upper quadrant of abdomen, next to the spleen. It usually takes 2 - 4 weeks for the tumors to grow up to 5 - 7 mm in diameter after cell implantation.
Motion of orthotopic pancreatic tumor xenografts was substantially suppressed, although motion artifact was present in MR images to a certain magnitude. In T2W MR images, the standard deviation of MR signal in the air above the tumor region was about 2.5 fold larger when pancreatic tumor cells were orthotopically implanted than that when a subcutaneous tumor model was employed. Figure 2A shows the schematic of a setup for DCE-MRI of an orthotopic pancreatic tumor xenograft. The orange circle represents the tumor, caught by the upper end of an orthogonally bent plastic board. Figure 2B shows the schematic of the plastic board with dimensions. The board width (30 mm) was designed to be the same with the width of the animal bed for mice. The board length was designed to be long enough to apply tape firmly. The depth of the board tip (4 mm) was designed for 20 g mice. Figure 2C shows the T2W MR images (sagittal view) of a representative mouse bearing an orthotopic pancreatic tumor xenograft (MIA PaCa-2). Tumor is indicated in a dotted red circle, and the indentation induced by the plastic board is indicated with a white arrow.
Quantitative DCE-MRI is successfully applied for orthotopic pancreatic tumor xenografts using the reference region (RR) model, as described in equation (7). Figure 3A shows contrast maps in tumor region (color scale) at 1 minute before MR contrast (gadoteridol) injection and at 5 and 40 min after injection, respectively, overlapped with T2W MR images (gray scale). The mouse was bearing a MIA PaCa-2 tumor xenograft orthotopically. Figure 3B shows contrast enhancement curves averaged in the tumor region (3 x 3 window) and paravertebral muscle region (9 x 9 window) indicated with two white squares, respectively, in Figure 3A. Figures 3C and 3D show Ktrans and kep maps, respectively.
<img alt="Figure 1" src="/files/ftp_upload/52641/52641fig1highres.jpg" width="600" /> 
Figure 1. Photographs of (A) a normal pancreas injected with tumor cell solution and (B) an orthotopic pancreatic tumor xenograft in an immunodeficient mouse. Tumor is located in the left upper quadrant of abdomen, indicated with a white dotted circle. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52641/52641fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52641/52641fig2highres.jpg" width="600" /> 
Figure 2. Schematics in DCE-MRI of an orthotopic pancreatic tumor xenograft. (A) Schematic of a setup for DCE-MRI of an orthotopic pancreatic tumor xenograft (orange circular region). A catheter is inserted into the tail vein to infuse MR contrast agent. (B) Schematic of an orthogonally bent plastic board with dimensions. (C) T2W MR images (sagittal view) of a mouse bearing an orthothopic pancreatic tumor xenograft indicated with a dotted red circle, when an orthogonally bent plastic board was applied (indicated with a white arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52641/52641fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52641/52641fig3highres.jpg" width="700" /> 
Figure 3. DCE-MR images and paramatric maps of an orthotopic pancreatic tumor xenograft. (A) Contrast maps at 1 min before or at 5 and 40 min after MR contrast (gadoteridol) injection. (B) Contrast enhancement curves averaged in the tumor region (3 x 3 window) and paravertebral muscle region (9 x 9 window) indicated with 2 white squares, respectively, in Figure 3A. (C) Ktrans map. (D) kep map. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52641/52641fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

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15.1K Views.  University of Alabama at Birmingham. The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy. 

Video: Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Cancer Research

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.

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 ...

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 ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

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 ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, immune cells, vessels, nerves, and extracellular matrix components. Over the years, different mouse models of PDAC have been developed to address the challenges posed by its progression, metastatic potential, and phenotypic heterogeneity. Immunocompetent mouse orthotopic allografts of PDAC have shown good promise owing to their fast and reproducible tumor progression in comparison to genetically engineered mouse models. Moreover, combined with their ability to mimic the biological features observed in autochthonous PDAC, cell line-based orthotopic allograft mouse models enable large-scale in vivo experiments. Thus, these models are widely used in preclinical studies for rapid genotype-phenotype and drug-response analyses. The aim of this protocol is to provide a reproducible and robust approach to successfully inject primary mouse PDAC cell cultures into the pancreas of syngeneic recipient mice. In addition to the technical details, important information is given that must be considered before performing these experiments.

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

Pancreatic ductal adenocarcinoma (PDAC) is a very complex disease characterized by a heterogeneous tumor microenvironment made up of a diverse stroma, ...

DCE-MRI of Orthotopic Pancreatic Tumor Xenografts: A Method to Assess Microvasculature in a Target Tumor Tissue

Source: Kim, H. et al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/52641/" target="_blank">Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model</a>. J. Vis. Exp. (2015).
This video describes the technique of DCE-MRI for orthotropic pancreatic tumor xenografts in mice.&#160;DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy. This method will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

Source: Kim, H. et al.&#160;Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model. J. Vis. Exp. (2015).
This ...

JoVE Encyclopedia of Experiments

Pancreatic Cancer

Assays & techniques

Bioluminescent Orthotopic Pancreatic Cancer Mouse Model: A Non-invasive Technique to Monitor Cancer Progression in Mice

Source: Chai, M. G., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/50395/" target="_blank">Bioluminescent Orthotopic Model of Pancreatic Cancer Progression</a>.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and&#160;in vivo&#160;bioluminescence imaging for non-invasive monitoring of tumor progression. The orthotopic model is suited to both syngeneic and xenograft models and could 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.

Source: Chai, M. G., et al. Bioluminescent Orthotopic Model of Pancreatic Cancer Progression.&#160;J. Vis. Exp.&#160;(2013).
This video demonstrates an ...

Orthotopic Homograft Tumor Transplantation: A Technique to Generate Pancreatic Tumor Mouse Models

Source: An, X. et al.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57460/" target="_blank">Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry</a>. J. Vis. Exp. (2018)
The video explains a stepwise surgical method for orthotopic implantation of a tumor fragment from a cancerous donor to the pancreas of a non-cancerous recipient mice for development of pancreatic cancer. Orthotopically implanted PDAC could recapture the tissue microenvironment of human pancreatic cancers than conventional subcutaneous (SC) models more accurately.

Source: An, X. et al.&#160;Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry. ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

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