Spatial Measurement of Tumor Interstitial Fluid Pressure: A Method to Measure the Interstitial Fluid Pressure

1. Spatial Measurements of Tumor Interstitial Fluid Pressure

1. To measure IFP connect the 25 G spinal needle to the pressure transducer and to the IFP acquisition system through 50 cm of PE20 polyethylene tubing. Flush the entire system with a heparin sulfate/saline solution (1:10). Sterilize the needle with 70% isopropyl before use.
2. Turn on the acquisition system and launch the IFP acquisition software and load the settings files to calibrate the system to acquire IFP measurements in mmHg. Click the acquire button to continuously collect IFP data.
3. Perform IFP measurements between 48 and 72 hr post-injection of CT-liposomes (this corresponds to the approximate time of peak accumulation of the CT-liposomes in the tumor), using the method described.
   1. Use the syringe pump to inject a bolus of CT-liposomes at a concentration of 400 mg iodine kg^-1. Set the pump to inject a volume of approximately 150 µl (assuming a 25 g mouse). Press the 'start' button on the pump to inject. Manually flush the catheter with 50 µl of saline (twice the volume of the catheter) to ensure the entire agent amount was injected and the catheter is clear.
      Attach the IFP needle to the CT-IFP robot.
4. Perform calibration scans to align the coordinate systems for the CT-IFP robot and the CT scanner. Add the fiducial marker attachment to the CT-IFP robot and perform a four volumetric CT scan with the fiducial marker in four different positions.
   1. Launch the CT-IFP robot controller software, initialize the robot, and move the robot to the three positions by entering the x,y,z targeting positions and clicking the 'go' button.
   2. Take a CT scan at the following x,y,z coordinates: (1) 0,0,0; (2) -10,0,0; (3) 0,7,0; and (4) 0,0,10. Select a 90 kV, 10 mA, 16 sec scan using the CT-scanner software and press 'Start' to initiate the scan. Reconstruct the scan as described. Perform a DCE-CT scan by setting the syringe pump to inject a volume of 100 µl of the free iohexol mixed with saline (9:1 ratio by volume).
      1. On the CT-scanner console select the 5 min dynamic scan that uses an x-ray energy setting of 80 kV, a tube energy of 90mA, and captures 416 image projections every sec for the first 30 sec and followed by an acquisition every 10 sec. Capture 5 sec of DCE-CT data and then press the start button on the injection pump.
      2. After the DCE-CT scan perform a volumetric anatomical micro-CT scan.
5. Launch the CT-IFP robot alignment software. Click the 'add' button loaded in the 'Registration Data' region and select the four reconstructed registration scans obtained in 1.3, then click 'open'.
   NOTE: The pixel location of the fiducial marker will automatically be entered into the software.
   1. Click the 'Calculate Transform' button and then click the 'Apply Transform' button. This generates alignment data that will be used to convert the CT-IFP robot coordinate system to the CT scanner coordinate system. After the calibration is complete, attach the animal platform to the CT-IFP robot.
6. Anesthetize each mouse using 2% isoflurane mixed with medical air or oxygen and confirm by pinching the toe and observing no reaction. Immobilize animal on the CT-IFP robot platform and position the mouse such that the tumor is accessible to the CT-IFP robot system. Immobilize the tumor using tape such that it does not move during IFP needle insertion.
7. Perform an anatomic micro-CT scan prior to inserting the IFP needle. Reconstruct the CT data using the steps described in 1.2.
8. Load the pre-needle insertion CT data into the CT-IFP robot alignment software. Adjust the window and level to visualize the tumor. Click on the rim of the tumor in any image, then click on a second rim location.
   NOTE: The software will calculate a series of positions along a linear line between the two positions. Note the x, y and z coordinates for a series of 5 to 8 evenly spaced positions from the list.
9. Prepare the IFP system by flushing the needle with heparin saline solution prior to insertion.
10. Enter the first pre-determined needle positions into the x,y,z, into the CT-IFP robot control software and press-move to 'go' button to move the robot to the desired position. Click the 'Insert Needle' button to insert the needle into the tissue.
    1. After inserting the needle ensure good fluid communication between the IFP needle and the tissue by pinching and releasing the PE20 tubing, noting that the IFP measurement increases and returns to pre-pinching value on the IFP acquisition software. Reject measurements that do not return to baseline.
11. Acquire an anatomic CT scan with the needle inserted, then click the 'Retract Needle' button on the CT-IFP robot control software to retract the needle from the tissue. Reject any IFP measurements where the IFP value does not return to the pre-needle insertion value after withdrawal of the needle. This signifies the needle may have been clogged during the measurement. Repeat steps 1.8 to 1.10 for each needle position.
12. Determine the needle position within the tumor volume by calculating the x, y, and z positions of the needle port relative to the center of mass of the tumor volume as identified in the post-insertion volumetric CT scan of the needle.
13. Return animals to their cage after all measurements are complete. Do not leave animals unattended, and take care to observe them until consciousness has been regained and they are able to maintain sternal recumbency.