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

Overview

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.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Magnetic Resonance Imaging

1. 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).
2. 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).
3. 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.
4. Anesthetize animals using ventilation with ~1 - 2% of isoflurane mixed with oxygen (2 L/min) throughout preparation and imaging. Confirm the depth of anaesthesia by toe pinch reflex. Apply veterinary ophthalmic ointment on eyes to prevent dryness while under anaesthesia. 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.
5. Place the animal in supine position in an animal bed equipped with circulating warm water (or warm air) to regulate body temperature during imaging. Set the temperature on the bed to 37 °C. Insert a rectal temperature probe to monitor the body temperature during imaging.
6. 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.
7. Tape a respiration pad transducer (SA Instrument, Inc., Stony Brook, NY) 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).
8. Perform matching and tuning for both the receiver and transmitter, followed by shimming.
9. 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.
10. 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, 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 instead.
11. 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).
12. 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.
13. After completing DCE-MRI, take off the needle and other probes, and place the animal in an empty cage bedded with paper towels.  Massage softly the lower abdominal area.  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.  Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency.

Materials

Name	Company	Catalog Number	Comments
9.4T MR scanner	Bruker Biospin Corporation	BioSpec 94/20 USR
Gadoteridol	Bracco Diagnostics Inc	NDC 0270-1111-03
Micro-polyethelene tube	Strategic Applications, Inc	#PE-10-25
30 G blunt tip needle	Strategic Applications, Inc	89134-194
Monitoring and gating system	SA instruments, Inc	Model 1030	This is an MR compatiable system to measure respiratory rating and body temperature of small animals at the same time.
Syringe pump	New Era Pump Systems, Inc.	NE-1600