Name: Multianimal Magnetic Resonance Imaging for Tumor Measurements in Pancreatic Cancer Mouse Models (Video) | JoVE Journal
Uploaded: 2026-02-03T14:20:00.000+00:00
Duration: 9 min 18 s
Description: Learn multianimal MRI for efficient pancreatic tumor detection in KRAS-driven mouse models. This protocol enables simultaneous imaging of up to four mice, improving throughput for preclinical therapeutic evaluation.

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5 minutes

This work was supported, in part, by the National Cancer Institute R01 CA258314 grant and the MSU Tetrad Initiative for Interdisciplinary Research grant 2024 edition to LFS. We are thankful to MSU Precision Health Program Tissue Analysis Core, Institute for MSU Quantitative Health Science and Engineering/Precision Health Program Advanced Molecular Imaging Facility, MSU Campus Animal Resources, MSU Veterinary Diagnostic Laboratory, and MSU Quantitative Bio Element Analysis and Mapping (QBEAM) Center for their technical assistance.

All experiments described were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee at Michigan State University. All procedures involving live animals comply with established guidelines to ensure the welfare and humane treatment of animals. This protocol describes the use and treatment of the KPC mouse model of PDAC: LSL-K-Ras G12D (Krastm4Tyj); p53lox/lox (Trp53tm1Brn); Pdx1-Cre (Pdx1-cre6Tuv)9.
1. Preimaging animal preparation
<ol>
	<li>Ensure proper genotyping verification of KPC mice before use. Transport experimental mice from the animal facility to the imaging facility.</li>
	<li>Anesthetize up to four mice in a prewarmed sealed induction chamber with 1.5-2% isoflurane and 0.8-2 L/min oxygen. Confirm anesthesia by applying a noxious stimulus (toe-pinching).</li>
	<li>Apply ophthalmic lubricant to both eyes of each mouse to prevent corneal desiccation.</li>
</ol>
2. Multianimal MRI bed setup
<ol>
	<li>Place two anesthetized mice in a ventral position on the bottom part (left and right) of the 3D printed bed insert, which is attached to the vendor-provided standard single-rat PET/MRI scan holder. Ensure the animals are positioned with their spines and limbs straight. 
	NOTE: A 3D printer was used to print the bed inserts (Supplemental File 1 and Supplemental File 2).</li>
	<li>Connect the air input in the vendor bed to the nose cones using tubing (inner diameter 1/16&#34;, outer diameter 1/8&#34;), and luer fittings (e.g., 7 x 1/16&#34;-male, 2 x 1/16&#34; female, 2 female-female connectors, 2 T socket to socket connectors).</li>
	<li>Secure the mouse head in a nose cone to maintain anesthesia (1.5-2% isoflurane, 0.8-2 L/min oxygen). Cover the bottom with the top of the 3D printed bed inserts (Figure 2B).</li>
	<li>Place the other two anesthetized mice in a ventral position on the top part (left and right) of the 3D printed bed insert.</li>
	<li>Secure the mouse head in a nose cone to maintain anesthesia (1.5-2% isoflurane, 0.8-2 L/min oxygen).</li>
	<li>Tape the respiratory monitoring pad to the top left mouse. Ensure that the monitoring pad is at the level of the abdomen, not the chest, to measure respiration without a signal from the heartbeat.</li>
	<li>Set the isoflurane level to maintain a respiration rate of 30-40 bpm throughout the scan, typically 1.5-2%. If the breathing trace is not visible or the amplitude is too low, make sure the pad is securely held in place by the tape and that all connections in the tubing are tight.</li>
	<li>Cover the bed to complete the multianimal MRI chamber (Figure 2B). 
	NOTE: The vendor-provided single-rat PET/MRI bed is equipped with circulating warm air to maintain the body temperature of the animals (~35-37 &#730;C). Up to four mice can be scanned simultaneously with 3D-printed inserts. The exact settings of the warm water temperature, as well as air intake and air exhaust rates, will depend on the setup at each site, including distance from the control room to the MRI. These can be set up at each site using a series of pilot scans where temperature is monitored throughout and then standardized for each study.</li>
</ol>
3. Prescan setup and imaging calibration
<ol>
	<li>Ensure that the mice are in the center of the MRI coil lengthwise by aligning the red laser with the center of the multi-chamber bed before sending the bed holder into the bore.</li>
	<li>Assign a label to the scan using the mouse ID and scan date in the following format: Strain, Mouse ID, Scan Date.</li>
	<li>Set up a T1-weighted FLASH scan with one axial slice, two coronal slices, and two sagittal slices to visualize the mice in the four-chamber bed and define the imaging area using the following settings: TR/TE 50/2.4 ms, 2 averages, flip angle 20&#176;, resolution 0.313 mm, 1 mm slice thickness, scan time 13 s.</li>
	<li>Verify that the whole mouse body is in the field of view and that the mice are straight. If not, repeat the mouse positioning steps and/or move the mouse bed into or out of the bore. 
	NOTE: These parameters were based on the specifications of the MRI system used. These parameters can be adjusted as needed to optimize image acquisition according to experimental requirements and/or the MRI system&#39;s capabilities.</li>
</ol>
4. Multianimal magnetic resonance imaging
<ol>
	<li>Set up the T2-weighted fast spin echo sequence with the following parameters: Repetition time (TR): 3,200 ms, Echo time: 46 ms, Number of averages: 10, In-plane resolution: 300 x 300 &#181;m, Number of coronal slices: 14, Intraslice gap: 0.3 - 0.7 mm, Fat suppression: Enabled, Rare factor: 8, slice packages: 2, Total image acquisition time: 14 min.</li>
	<li>Click on the center (translation) and edge (rotation) of the yellow and green boxes on the localizer image to adjust the slice package positioning as needed to optimize coverage of the pancreas region and other abdominal organs for the top and bottom set of animals.</li>
	<li>Confirm that all four mice are correctly aligned in the multianimal bed setup before proceeding with the scanning.</li>
	<li>Monitor respiration throughout the imaging sequences and adjust isoflurane level if necessary.</li>
	<li>After the MRI scan is completed, check that the image quality is adequate. Look for motion artifact, which shows as blurring, if a mouse moved during the scan. Check for full coverage of the abdomen. If needed, reposition the mice and redo the scan.</li>
</ol>
5. Mouse recovery
<ol>
	<li>When the scan is completed and verified, remove the bed from the MRI system.</li>
	<li>Open the top of the multichamber bed. Remove the respiratory monitor from the top left mouse and transfer the animals from the top and bottom of the 3D printed inserts into the recovery cage.</li>
	<li>Place animals into a recovery cage over a 37 &#176;C-heating pad for postanesthesia recovery.</li>
	<li>Monitor respiratory rate and movement to ensure full recovery from anesthesia before returning mice to home cages. Signs of recovery include normal breathing and movement (walking).</li>
</ol>
6. Magnetic resonance imaging file processing and analysis
<ol>
	<li>Export the DICOM images from MRI software to an image analysis program to crop the images from one image with four mice to one image for each individual mouse.</li>
	<li>In the software, open the image for the top set of animals in the View module. The automatic window/level settings are sufficient, but adjust in the general image display control tab if needed, so that the mice are visible. In the VOI tab, manually draw a generous box around the top right mouse using a cube analytic object; then crop the image around that box from the Masking and contouring operations tab. When saving the new file, edit the DICOM metadata and file name to the correct mouse ID; then, repeat for the top left mouse using the same image, open the image for the bottom set of mice, and repeat the ROI cropping and saving. 
	NOTE: The VOI does not have to be precise.</li>
	<li>Use MRI viewer software to analyze images stored in DICOM files.</li>
	<li>Analyze imaging slices from dorsal to ventral or ventral to dorsal. Locate the spleen and stomach to establish a proper context for the identification of pancreas and/or pancreatic tumors.</li>
	<li>Define &#34;Region of Interest&#34; (ROI) by drawing a line on the tumor boundary and perform semiautomated segmentation to calculate tumor volume. Take note of fluid cysts and other hyperchromatic findings&#160;(Figure 3 and Figure 4). 
	NOTE: An MRI viewer should be used to analyze DICOM files.</li>
</ol>
7. Gemcitabine treatment
<ol>
	<li>Perform MRI scans of KPC animals weekly until the tumor starts forming and can be detected (volume of &#8805;20&#160;mm3). Before scanning, ensure that mice are in good health (no excessive weight loss or signs of distress, difficulty breathing, and/or gasping).</li>
	<li>Set up a treatment regimen once tumor criteria have been met. 
	NOTE: For the treated group, gemcitabine was injected intraperitoneally (i.p.) at a dose of 100 mg/kg twice a week. 
	CAUTION: Gemcitabine is a cytotoxic chemotherapeutic agent. Handle with appropriate personal protective equipment (PPE), including gloves and a lab coat. Dispose of contaminated syringes and materials in accordance with institutional hazardous waste protocols.</li>
	<li>Perform MRI scans (following sections 1-6) once every week to monitor tumor response to gemcitabine.</li>
	<li>Sacrifice animals when the tumor meets the euthanasia criteria based on volume measurements from MRI analysis (Section 6) and/or other symptoms.</li>
	<li>When MRI scanning treated mice, place a protective barrier to prevent the potential transfer of the chemotherapeutic agent&#160;through excretions. During and after working with gemcitabine, follow good personal hygiene practices. Dispose of used empty syringes in a chemotherapy sharps container.</li>
	<li>After completing the work, spray all the anesthesia equipment, MRI beds, and all working surfaces with 70% ethanol. Place chemically contaminated protective barriers and gloves used during imaging into a plastic bag, tie off the bag, and place it in a fiber barrel for incineration.</li>
	<li>Schedule imaging sessions before the next round of treatment to minimize exposure to the chemotherapeutic agent. 
	NOTE: A frequency of MRI scans of every other week may be considered depending on the tumor growth rate and treatment regimen. Responses to treatment by tumor kinetics can be assessed through longitudinal imaging (Figure 5).</li>
</ol>

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</ol>

The authors have no conflicts of interest to disclose.

This protocol utilizes a vendor-provided standard single-rat PET/MRI bed, modified with custom-made 3D-printed inserts, which allow for the simultaneous scanning of up to four mice (Figure 1). This multianimal configuration substantially reduces not only the expensive MRI scanning time but also the overall procedure duration. It allows for the parallel preparation (anesthesia, positioning in bed, and bed loading), scanning, and recovery of four animals in one session, compared to the additional time required for preparing, scanning, and recovering each animal individually in four separate sessions. The setup also includes individual nose cones for controlled anesthesia delivery to each animal, dedicated vital monitoring for one animal, and integrated heating capabilities to maintain body temperature for all scanned mice (Figure 2). The 3D printed inserts provide consistent animal alignment and improve segmentation consistency. Real-time respiratory monitoring enables continuous assessment and adjustment of anesthesia during scanning, thereby minimizing motion artifacts.
This protocol was used to detect and longitudinally monitor pancreatic tumor progression in the KPC model (Figure 5A-C) treated with the chemotherapeutic agent gemcitabine, serving as a proof-of-concept preclinical study. Gemcitabine, alone or in combination, is a first- or second-line treatment for patients with local or metastatic pancreatic cancer28,29,30. These results with gemcitabine-treated animals are consistent with previous reports, which have also shown a survival benefit31. More importantly, this protocol provides a suitable and robust platform to study combination therapies of new therapeutic agents and SOC chemotherapy in the KPC model or similar models of PDAC. These combination therapies may be small molecule inhibitors, immunotherapy, siRNAs, microRNA activity modulators, and other therapeutic oligonucleotides against key molecular targets20,21,22,23,24,25,26,27. These combination therapies may also include genetically modified targets to understand their interaction with SOC chemotherapy or other therapies at an early stage of development9,20,32.
While the focus of this protocol is the imaging of the pancreas and other abdominal cavity organs, this multianimal protocol and described MRI sequences can be applied for anatomical characterization of other mouse models of cancer, such as breast cancer and glioblastoma, or other diseases involving the kidney, and/or liver33. We are aware of other protocols describing multianimal MRI scanning18,34. For example, Dazai et al. described neuroanatomical MRI scanning of up to seven mice18. While this setup enables brain imaging of seven animals, it does not enable individual control of anesthetic and temperature for each mouse. Their housing chamber is designed for mice weighing less than approximately 32 g; in contrast, our multichamber bed supports a broader range of mouse body sizes. Depending on the intended organ, scanning time, and resolution, Dazai et al.&#39;s protocol may provide some advantages over the protocol described here.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal malignancies. Despite modest improvements in survival over recent years, the 5-year survival rate is still only 13%1. In most cases, PDAC is detected at an advanced stage, often involving local invasion or distant metastasis, which complicates treatment. The disease is biologically diverse and marked by highly invasive tumor cells, a dense desmoplastic stroma, and poor vascularization2,3,4. This complex tumor microenvironment continues to hinder efforts to fully characterize the cellular and non-cellular components that influence disease progression. To address this, effective preclinical models are needed that closely mimic human disease progression and desmoplasia.
A variety of mouse models can be used to study the biology of cancer progression and evaluate the efficacy of novel therapeutics5,6. KRas-driven genetically engineered mouse model (GEMM) spontaneously develops pancreatic tumors that recapitulate many pathophysiological and molecular features of human PDAC3,7,8,9,10,11. In particular, the Kras-driven, p53-deleted KPC model (LSL-KrasG12D; p53lox/+; Pdx1-Cre) is a well-characterized PDAC GEMM7,8,9. Tumor imaging is an important component of preclinical studies. Tumor detection and measurement of tumor size enable the selection of animals for preclinical studies, longitudinal monitoring of tumor growth and disease progression, and assessment of treatment response.
Advanced in vivo imaging modalities such as ultrasound imaging (US), bioluminescence (BLI), computer tomography (CT), fluorescence imaging (FLI), positron emission tomography (PET), and magnetic resonance imaging (MRI) can be used for tumor imaging in the KPC model and similar PDAC models12,13,14,15,16. Yet, tumor imaging in the KPC model can be challenging, and each of these imaging modalities offers unique strengths and limitations. Optical imaging technologies, such as bioluminescence (BLI) and ultrasound imaging (US), are non-invasive and cost-effective. However, their limited spatial resolution, particularly for internal organs, can lead to a poor correlation with tumor volume12,15,17,18. Bioluminescence requires genetically engineered cells expressing luciferase signal, which also depends on substrate delivery, blood flow, and tissue oxygenation. A computer tomograph provides fast imaging with minimal motion artifacts but poor soft tissue contrast without contrast agents. Iodinated contrast may cause toxicity in mice. Fluorescence imaging requires contrast agents/fluorescent probes, and autofluorescence from abdominal organs reduces sensitivity.
Because of the constraints associated with other imaging methods, magnetic resonance imaging (MRI) is generally regarded as the most reliable technique for evaluating deep-seated tumors, offering superior soft tissue contrast and molecular sensitivity for internal anatomical structures13,14,15,19. However, standard preclinical MRI protocols incur high operational costs and long acquisition times and can be a barrier for many research laboratories. Multianimal MRI can overcome this access barrier by reducing pre-scan animal preparation time (anesthesia), scanning time, and post-scan animal recovery. This protocol demonstrates the technical feasibility of PDAC tumor imaging (Figure 1, Figure 2, Figure 3, and Figure 4) and the use of image findings to recruit animals for chemotherapeutic treatment with gemcitabine (Figure 5). PDAC tumors exhibit multiple inherent mechanisms that contribute to resistance against standard-of-care (SOC) chemotherapy agents. such as gemcitabine, gemcitabine/nab-paclitaxel, or FOLFIRINOX2,3,4. This protocol provides a suitable and robust platform to evaluate new therapeutic agents and SOC chemotherapy to find promising combinations20,21,22,23,24,25,26,27 that could enhance treatment response in patients.

<table><tbody><tr><td>3D printer&amp;nbsp;</td><td>Stratasys</td><td>J750&amp;nbsp;</td><td>For multi-animal MRI bed inserts</td></tr><tr><td>Gemcitabine HCl</td><td>ApexBio</td><td>A1402-500</td><td>For ip injections&amp;nbsp;</td></tr><tr><td>HOROS</td><td>HOROS</td><td>https://horosproject.org/</td><td>For MRI analysis</td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>11695067772</td><td>For mouse anesthesia</td></tr><tr><td>Isoflurane vaporizer</td><td>SOMNI Scientific</td><td>VS6002</td><td>For mouse anesthesia</td></tr><tr><td>Lo-Dose U-100 Insulin Syringes</td><td>BD</td><td>14-826-79</td><td>For ip injections&amp;nbsp;</td></tr><tr><td>luer lock tube couplings, nylon straight plugs, barbed for 1/16&quot; tube ID x7</td><td>McMaster</td><td>51525K521</td><td>For multi-animal MRI bed assembly</td></tr><tr><td>luer lock tube couplings, plug, for 1/16&quot; bardbed tube ID x2</td><td>McMaster</td><td>7466N11</td><td>For multi-animal MRI bed assembly</td></tr><tr><td>male to male luer connector x2</td><td>SAI</td><td>MML</td><td>For multi-animal MRI bed assembly</td></tr><tr><td>MR-compatible small animal monitoring and gating system</td><td>SAII</td><td>Model 1030</td><td>monitor animals during MRI</td></tr><tr><td>MRI bed adapter</td><td>Bruker</td><td>T150743</td><td>vendor-provided MRI bed component</td></tr><tr><td>MRI coil, 72 mm ID</td><td>Bruker</td><td>T20209V3</td><td>vendor-provided MRI coil</td></tr><tr><td>Multimodality cradle medium rat</td><td>Bruker</td><td>T151629</td><td>vendor-provided MRI bed</td></tr><tr><td>ophthalmic lubricant&amp;nbsp;</td><td>Covetrus</td><td>75848</td><td>For use during anesthesia</td></tr><tr><td>ParaVision 360 software</td><td>Bruker</td><td>https://www.bruker.com/en/products-and-solutions/preclinical-imaging/paravision-360.html</td><td>For MRI analysis</td></tr><tr><td>PMOD 4.2</td><td>Bruker</td><td>https://www.bruker.com/en/products-and-solutions/preclinical-imaging/pmod.html</td><td>For MRI analysis</td></tr><tr><td>preclinical MRI system&amp;nbsp;</td><td>Bruker</td><td>BioSpec 70/30</td><td>For MRI</td></tr><tr><td>Stratasys Vero Clear resin</td><td>Stratasys</td><td>RGD810</td><td>For multi-animal MRI bed fabrication</td></tr><tr><td>Tee Socket-to-Socket Connectors, Luer Lock Tube x2</td><td>McMaster</td><td>51525K443</td><td>For multi-animal MRI bed&amp;nbsp;</td></tr><tr><td>tubing (inner diameter 1/16&quot;, outer diameter 1/8&quot;)</td><td>Bruker</td><td>T12330</td><td>For multi-animal MRI bed assembly</td></tr></tbody></table>

multianimal magnetic resonance imaging for tumor measurements in pancreatic cancer mouse models

Multianimal Magnetic Resonance Imaging for Tumor Measurements in Pancreatic Cancer Mouse Models

Advanced in vivo imaging modalities, such as magnetic resonance imaging (MRI), are essential research tools to effectively detect tumors in preclinical mouse models. MRI provides detailed anatomical information on tumor location and size. However, this imaging modality is expensive and time-consuming. To overcome this access barrier without compromising imaging quality, a multianimal MRI protocol was implemented. This protocol uses a four-chamber bed insert to obtain high-resolution multianimal anatomical MRI scans within a single acquisition session. Simultaneously imagining multiple mice reduces the time and cost of this procedure. This protocol describes a multianimal MRI workflow designed to increase the efficiency of tumor detection and longitudinal tumor monitoring in a genetically engineered Kras-driven, p53-deleted (KPC) mouse model of pancreatic ductal adenocarcinoma. This well-established clinically relevant KPC model has provided invaluable insight into the molecular mechanisms underlying pancreatic carcinogenesis, tumor progression, and treatment resistance. As proof-of-concept, this protocol is applied to validate the therapeutic benefit of the standard-of-care chemotherapeutic agent gemcitabine in the KPC model. Future applications of this MRI-guided preclinical study design are briefly discussed to evaluate the therapeutic efficacy of combination therapies.

KPC animals develop pancreatic tumors after 12 weeks of age9. Using a multichamber MRI bed, tumor screening of KPC mice was initiated at 10 weeks of age. This enabled recruiting animals presenting with tumors of approximately 200 mm3 for the preclinical study with the gemcitabine treatment. Differences in tumor kinetics and variations in tumor presentation were observed. Most KPC tumors showed a solid growth pattern with a single or predominant focus (Figure 4A), which facilitated animal recruitment for treatments and tumor volume assessment. However, animals with multiple small solid tumors spread throughout the entire pancreas were also observed (Figure 4A). Some PDAC cases may also exhibit intratumoral fluid-filled cystic lesions, accompanied by peritumoral non-neoplastic cystic lesions and fluid retention resulting from ductal blockage of the gallbladder and/or pancreas. (Figure 4A).
Following recruitment, animals were randomly assigned to two experimental groups: a control group that received no treatment and a treated group that received intraperitoneal (i.p.) administration of gemcitabine at a dose of 100 mg/kg twice a week. Of the total 23 eligible animals, 4 females and 7 males were allocated to the control group, and 6 females and 6 males to the treated group. Weekly MRI scans were conducted using the multianimal bed system (Figure 5A), which enabled us to continuously monitor the changes in tumor growth kinetics in both the control and treated groups (Figure 5B).
The processed imaging data demonstrate that tumors in the gemcitabine-treated KPC model had a slower growth pattern compared to the untreated control group (Figure 5C), which was associated with a statistically significant (Log rank test, p = 0.04) increase in the overall survival of the treated cohort (Figure 5D). The median survival for untreated KPC was 20 weeks, whereas the treated group showed a prolongation of the median survival of over 23 weeks (Figure 5D).
<img alt="Preclinical MRI workflow diagram: preparation, multi-animal bed setup, MR imaging, image analysis." class="xfigimg" src="/files/ftp_upload/69927/69927fig1.jpg" data-altupdatedat="1784001604000" data-alt="Figure 1"> 
Figure 1: Workflow of multianimal magnetic resonance imaging procedure, image processing and analysis. Key steps of the multianimal MRI protocol are highlighted from prescan preparation to image-guided preclinical studies. Up to 4 animals are anesthetized in an induction chamber (prescan preparation) and prepared for placement in a multianimal bed (Multianimal MRI bed setup). A prescan image of the animals in a 2 x 2 configuration (2 in the top compartment, 2 in the bottom compartment) followed by a magnetic resonance scan (MR imaging). Each animal's imaging data are saved as individual files and analyzed independently (Image Analysis). Image finding and tumor size are used to select animals for preclinical studies and monitor treatment response (preclinical trials). <a href="https://www.jove.com/files/ftp_upload/69927/69927fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Anesthesia system diagram and setup, features top-bottom inserts, ports, and structural details." class="xfigimg" src="/files/ftp_upload/69927/69927fig2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 2"> 
Figure 2: Key steps for multianimal MRI from setup to scan. (A) Top panel, assembly of the custom multianimal MRI bed system. Custom-designed 3D printed bed inserts with integrated anesthesia ports fit into a standard single-rat PET/MRI bed holder. Bottom panel, these 3D printed inserts divide the bed into two chambers, allowing simultaneous imaging of up to four animals: animals 1 and 2 on top of the top insert, and animals 3 and 4 on top of the bottom insert. Transverse plane and frontal modified plane of the 3D printer inserts from STL viewer are shown. (B) Sequential placement of mice into a multichamber MRI bed (from top to bottom). Top image, two animals placed in ventral position on the bottom part (left and right) of the 3D printed insert, which is inside the vendor-provided PET/MRI bed. Middle image, the other two mice are placed in ventral position on the top part (left and right) of the 3D printed insert; the mouse positioned on the left side of the top insert has a respiratory monitor attached. Bottom image: completed assembly of four animals set with the cover on top. All animals have heads secured in a nose cone to maintain anesthesia (1.5-2% isoflurane, 0.8-2 L/min oxygen). Final arrangement of all four animals in the MRI bed holder with anesthesia delivery, warm water heater, and vitals monitoring system to allow MRI scanning and data acquisition of all animals in a single session. <a href="https://www.jove.com/files/ftp_upload/69927/69927fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="MRI cross-section analysis, tumor identification, comparative imaging, diagnostic methodology." class="xfigimg" src="/files/ftp_upload/69927/69927fig3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 3"> 
Figure 3: Data collection and processing from multianimal MRI scans. (A) Representative view (medial plane) of the initial MRI scan of all four animals. (B) MRI scan (dorsal plane) of all four mice in a multichamber bed: two mice positioned on the bottom (3, 4) and two mice on the top (1, 2) of the 3D printed inserts. (C) Final high-resolution MRI file of each individual mouse scan. <a href="https://www.jove.com/files/ftp_upload/69927/69927fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="MRI pancreas analysis; H&amp;E staining; tumor classification; pathology imaging; diagnostic process." class="xfigimg" src="/files/ftp_upload/69927/69927fig4.jpg" data-altupdatedat="1784001604000" data-alt="Figure 4"> 
Figure 4: Diverse presentations of PDAC tumors: an integrated MRI and histology analysis. (A) Representative MRI image (dorsal plane) illustrating the anatomical variability of PDAC tumors (from left to right): normal pancreas, multifocal tumors with fluid-filled cystic lesions, and solid tumor mass. Anatomical organs are color-coded as follows: stomach (yellow), spleen (green), tumor (purple), fluid accumulation due to duct blockage (blue). White scale bar = 1 cm. (B) Histological sections of H&amp;E-stained tumors alongside cell classes based on computer-assisted trained classifier overlays, highlighting the histopathological features of the tumors aligned with the MRI findings. Tumor (T) area outlined in black for all samples when the tumor is present. Cell classes are shown in color: normal pancreatic tissue (grey), tumor (red), and immune cells (yellow). Black scale bar = 0.5 cm. <a href="https://www.jove.com/files/ftp_upload/69927/69927fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Pancreatic tumor study; MRI imaging, treatment timeline, growth kinetics graph, survival chart." class="xfigimg" src="/files/ftp_upload/69927/69927fig5.jpg" data-altupdatedat="1784001604000" data-alt="Figure 5"> 
Figure 5: Multianimal MRI application for animal recruitment and tumor growth monitoring during gemcitabine treatment. (A) Timeline of the experimental design: Animals (14+ weeks old) were recruited after an MRI scan detected a tumor ≥ 200 mm3. Mice from the treated group received gemcitabine (100 mg/kg, i.p., b.i.w.) until they reached euthanasia criteria. Multianimal MRI scans were performed throughout the experiment for all animals to monitor tumor growth kinetics. (B) Representative MRI images (dorsal plane) showing tumor progression for one animal from the untreated and one animal from the treated group, from Day 0 until the final day of the experiment (survival endpoint). White scale bar = 1 cm. (C,D) Graphs illustrating tumor growth kinetics (C, n = 4 [2 F, 2 M] vs. 4 [2 F, 2 M]) and overall survival (D, n = 11 [4 F, 7 M] vs. 12 [6 F, 6 M]) in control untreated and treated group. Statistical significance for Kaplan-Meier survival curves was determined by the Log-rank (Mantel-Cox) test (* p &lt; 0.05). <a href="https://www.jove.com/files/ftp_upload/69927/69927fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental File 1: STL file for 3D printing of the bottom insert with integrated anesthesia ports that fits in a standard single-rat PET/MRI bed holder. <a href="https://www.jove.com/files/ftp_upload/69927/MSU%204%20mouse%20bed%20bottom.stl" target="_blank">Please click here to download this File.</a>
Supplemental File 2: STL file for 3D printing of the top insert with integrated anesthesia ports that fits in a standard single-rat PET/MRI bed holder. <a href="https://www.jove.com/files/ftp_upload/69927/MSU%204%20mouse%20bed%20top.stl" target="_blank">Please click here to download this File.</a>

This protocol uses a multichamber bed for parallel magnetic resonance imaging (MRI) of up to four animals to detect pancreatic adenocarcinoma tumors in genetically engineered mouse models. This multianimal MRI protocol is fast and cost-effective for detecting and measuring tumors, facilitating animal selection for preclinical studies and longitudinal monitoring of tumor growth.

Advanced in vivo imaging modalities, such as magnetic resonance imaging (MRI), are essential research tools to effectively detect tumors in preclinical ...

Pancreatic ductal adenocarcinoma is a highly lethal disease. PDAC is usually diagnosed at later stages with locally advanced or metastatic disease, making it even more difficult to treat. PDAC is a heterogeneous disease characterized by highly aggressive cancer cells, extensive desmoplastic reaction, and hypovascularity.KRAS-driven, genetically engineered mouse models are well suited for preclinical therapeutic evaluation as the carry mutation similar to human pancreatic cells and spontaneously develop pancreatic tumors with pathophysiology and molecular features resembling those of human PDAC. The KRAS-driven P53 deleted KPC model is a well-characterized PDAC genetically engineered mouse model. To effectively monitor tumor development and assess treatment responses in the KPC model, advanced in-vivo imaging modalities, such as magnetic resonance imaging are essential research tools.MRI is widely considered to be the gold standard imaging modality for deep tumor evaluation, exhibiting the highest degree of soft tissue anatomical contrast and molecular sensitivity for deep tissue targets. However, standard MRI protocols incur high operational cost and long accusation times. This protocol uses a multi-animal chamber for parallel magnetic resonance imaging of up to four mice for detecting pancreatic adenocarcinoma tumors as a fast, economic, and precise method for tumor detection and measurement for preclinical trial, animal recruitment, and monitoring of treatment response.Briefly, we begin by preparing the animal for the procedure and setting up the multi-animal MRI bed with our 3D printed inserts. Then we proceed with the MRI scanning, followed by data analysis to determine the success of the treatment for preclinical trials. Transport experimental KPC mice from the animal facility to the imaging facility.Anesthetize up to four mice in a pretty warm sealed induction chamber with isoflurane. Apply ophthalmic lubricant to both eyes of each mouse to prevent corneal desiccation. Place two anesthetized mice in a ventral position on the bottom part of the 3D printed bed insert, which is attached to the vendor-provided standard single rat PET MRI scan holder.Secure the head and a nose cone to maintain anesthesia. When scanning treated mice, place a protective barrier to prevent the potential transfer of chemotherapeutic agents through excretions. Cover the bottom with the top of the 3D-printed bed inserts.Place another two anesthetized mice in a ventral position on the top part of the 3D printed bed insert. Secure the head in a nose cone to maintain anesthesia. The vendor-provided single rat PET MRI bed is equipped with circulating warm air to maintain body temperature of the animals.Attach the respiratory monitor to the one mouse positioned on the left side of the top 3D bed insert. Cover the bed to complete multi-animal MRI chamber. Ensure the mice will be located in the center of the MRI coil by aligning the red laser with the center of the mice.Assign a label to the scan using the mouse ID and scan date in the following format. Strain, mouse ID, scan date. Acquire a localizer image to define the appropriate imaging area for all four mice positioned in the bed.The localizer scan is a T1 weighted flash scan with one axial slice, two coronal slices, and two sagittal slices to visualize the mice in the four mouse bed. To set up the T2 rare scan, adjust the slice packages based on the localizer image as needed to optimize visualization of the pancreas region and other abdominal organs for the top and bottom set of animals. Confirm that all four mice are correctly aligned in the multi-animal bed setup before proceeding with the scanning.Monitor respiration throughout the imaging sequences and adjust isoflurane level if necessary. When the scan is completed, remove the bed from the MRI. Open the top of the multi-animal bed and remove the respiratory monitor from the top left mouse.Transfer animals from the top and bottom of the 3D printed inserts into the recovery cage. Place the recovery cage over a 42 degrees Celsius heating pad for post anesthesia recovery. Monitor respiratory rate and movement to ensure full recovery from anesthesia before returning mice to home cages.Export the DICOM images from ParaVision 360 to an image analysis program to crop the images from one image with four mice to one image for each individual mouse. In PMOD, open the image for the top set of animals in the View module. In the VOI tab, draw a box around the top right mouse, then crop the image around that box.Edit the DICOM metadata to the correct mouse id, then save the crafted image with the correct file name. Repeat for the top left mouse using the same image, then open the image for the bottom set of mice and repeat the cropping and saving. Use MRI viewer software to analyze images stored in DICOM files.We use Open Source MRI Viewer for our software package. Analyze imaging slices from dorsal to ventral or ventral to dorsal. Locate organs like spleen and stomach for proper localization of the pancreas and pancreatic tumors.Define region of interest in all slices where tumor is present and perform semi-automated segmentation to calculate tumor volume. Take note of fluid cysts, and other hyperchromatic symptoms. In representative MRI image, we can detect the stomach, spleen, and tumor.Most KPC tumors showed a solid growth pattern with single or predominant focus, which facilitated recruitment for treatments and tumor volume assessment. However, we also observed animals with multiple small solid tumors spread throughout the entire pancreas. PDAC tumors may also demonstrate intratumoral cystic appearance accompanied by paratumoral, non-neoplastic cystic lesions or ductal blockage, which can result in fluid containing lesions.Correlative tissue studies show concordance between anatomical tumor feature by MRI and histological feature of H&E stain tumor tissues collected immediately after imaging session. Hyperchromatic region on MRI consistent with fluid-filled lesion corresponds to cystic lesion and histological analysis. We use this multi-animal MRI for monitoring pancreatic tumor progression in the KPC model treated with chemotherapeutic agent gemcitabine as a proof of concept preclinical trial.Gemcitabine alone or in combination is the first line treatment for patients with local or metastatic pancreatic cancer. KPC animals develop pancreatic tumors after 12 weeks of age. Following recruitment, animals were designated into two experimental groups.One, receiving intraperitoneal administration of gemcitabine and a control group without any treatment. Weekly MRI scans were conducted using the multi-animal bed system, which enabled us to continuously monitor the changes in tumor growth kinetics in both the treatment and control groups. Our data demonstrates that tumors in the gemcitabine-treated KPC model had a slower growth pattern compared to untreated control group.A result with gemcitabine treated animals are consistent with previous reports and conferring a survival benefit. This protocol provides a suitable and robust platform to study combination therapies of new therapeutic agents in standard of care chemotherapy. We are using this cost-effective MRI protocol to investigate the role of specific mRNAs and PDAC with complementary genetic and pharmacological approaches.But these combination therapies may also be small molecule inhibitors, immunotherapy, siRNAs, and other therapeutic oligonucleotides against key molecular targets. While the focus of this protocol is the imaging of the pancreas and other abdominal cavity organs, this multi-animal protocol and described MRI sequences can be applied for anatomical characterization of other mouse models of cancer, such as breast cancer and glioblastoma or other diseases involving the kidney and or liver. Overall, this multi-animal MRI protocol offers a fast, reliable, and cost effective platform for high-quality, longitudinal tumor monitoring and preclinical therapeutic evaluation in pancreatic cancer models.

multianimal-magnetic-resonance-imaging-for-tumor-measurements

Research

JoVE Journal

Cancer Research

This video describes the technique of DCE-MRI for orthotropic 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. This method will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

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

This video demonstrates an orthotopic model of pancreatic cancer that utilizes Matrigel for localized cell delivery and in vivo 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.

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

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 of an Orthotopic Pancreatic Cancer Mouse Model

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.



Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

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.

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

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

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.

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

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.

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

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.

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

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Learn multianimal MRI for efficient pancreatic tumor detection in KRAS-driven mouse models. This protocol enables simultaneous imaging of up to four mice, improving throughput for preclinical therapeutic evaluation.

Multianimal Magnetic Resonance Imaging for Tumor Measurements in Pancreatic Cancer Mouse Models

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