Spatial Measurements of Perfusion, Interstitial Fluid Pressure and Liposomes Accumulation in Solid Tumors

The overall goal of this experiment is to relate the intratumoral accumulation of nanotherapeutics to the properties of the tumor microenvironment, including the tumor microcirculation and the elevated interstitial fluid pressure, or IFP. This method lets us answer important questions about nanomedicines. Questions like what drives heterogeneous uptake of nanoparticles inside a tumor?

The main advantage of this technique is that allows for co-localized spatial mapping of the properties of the tumor microenvironment and the intratumoral distribution of nanoparticles. After confirming the appropriate level of anesthesia by topenge, apply ointment to the mouse's eyes and tape the animal's limbs in the prone position on a thin plastic board. Next, insert a custom 27-gauge catheter connected to a 20-centimeter piece of PE10 tubing into the lateral tail vein and secure the tubing with several pieces of tape.

Now, fill one one milliliter syringe with at least 200 microliters of computed tomography, or CT liposomes and one one milliliter syringe with at least 150 microliters of a 9-1 ratio by volume of free iohexol mixed with saline. Place the CT liposome syringe in a syringe pump and attach the catheter to the syringe, setting a pump rate of 600 microliters per minute, equivalent to 10 microliters per second. Then place the mouse on the micro CT scanner bed and use the laser positioning system to adjust the tumor so that it will be approximately the same orientation for each scan.

Using the CT scanner console software for each imaging protocol of interest, select bright dark from the drop-down menu and click the scan button to initiate the calibration and to initialize the system. To obtain a volumetric anatomical micro CT of the tumor before any contrast agent is injected, first check the CT scanner console software indicator to confirm that the CT scanner safety interlocks have been cleared. Then select the scan that uses an x-ray energy of 80 kilovolts, a tube current of 70 milliamps and captures 1, 000 image projections.

Then, initiate the scan. When the scan is finished, set the pump to inject approximately 150 microliters of the liposome solution and press the start button to inject the bolus of CT liposomes at a 55 milligram of iodine per milliliter of solution concentration. Manually flush the catheter with 50 microliters of saline to ensure the entire amount of liposome agent was injected and that the catheter has been cleared.

After 10 minutes, perform a second anatomical scan of the tumor, as just demonstrated. To perform a DCE-CT, place a syringe of free iohexol solution into the syringe pump and set the pump to inject 100 microliters of the iohexol at the same injection rate. These injection volumes are higher than the standard of 200 microliters, but the animals are monitored during recovery and no adverse events have been observed.

Then, on the CT scanner console, select a five minute dynamic scan using an x-ray energy of 80 kilovolts and a tube current of 90 milliamps, as just demonstrated, that captures 416 image projections every one second for the first 30 seconds followed by 416 image projections every 10 seconds. Capture five seconds of DCE-CT data, then start the injection pump. At the end of the scan, perform a third volumetric anatomical micro CT scan.

48-70 hours later, capture anatomical CT images of the liposomes using the same volumetric settings as just demonstrated. To measure the IFP, tape the animal on the CT IFP robot platform such that the tumor is immobilized and accessible to the CT IFP robot system. Obtain an anatomic micro CT scan as just demonstrated.

Then load the pre-needle insertion data into the CT IFP robot alignment software and adjust the window and level to visualize the tumor. Click on the rim of the tumor in any image followed by the selection of a second rim location on the adjacent side of the tumor. The software will calculate a series of positions along a linear line between the two points.

Then, select the X, Y and Z coordinates for a series of five to eight evenly-spaced positions from the list. Next, flush the IFP system needle with a saline heparin solution and enter the first predetermined needle positions into the X, Y, Z coordinate windows of the CT IFP robot control software. Press the go button to move the robot to the desired location.

Then, for each needle position in turn, click the insert needle button to insert the needle into the tissue. Pinch and release the PE20 tubing to confirm good fluid communication between the IFP needle and the tissue and observe that the IFP measurement increases and returns to the pre-pinching value on the IFP acquisition software. Finally, acquire an anatomic CT scan with the needle inserted, clicking the retract needle button at the end of the scan to remove the needle from the tissue.

Selecting a region of interest within the tumor yields a time/intensity curve that can be used to yield quantitative estimates of select hemodynamic parameters in the tumor. Segmenting the tumor volume into equally-sized multiple regions of interest allows the quantification of the spatial distribution of these parameters within the tumor volume. The biodistribution of the CT liposomes at 48 hours post-injection can also be observed.

As indicated, the agent is still circulating in the vascular system with a substantial uptake observed in the spleen and liver. The intratumoral accumulation of these CT liposomes is heterogeneous with a predominantly peripheral accumulation compared to the center of the tumor tissue. The needle can clearly be identified by high resolution micro CT, allowing the spatial localization of the IFP measurements within the tumor volume.

Spatially co-localized measurement of the profusion and the plasma volume fraction demonstrates a significant correlation with the intratumoral accumulation of CT liposomes in subcutaneous tumors. Further, the radial distribution of the IFP correlates with the other hemodynamic measurements, suggesting a complex spatial/temporal relationship between the tumor microcirculation, the IFP and the intratumoral accumulation of the liposomes. While attempting this procedure, it's important to secure the animal on the scanner bed, ensuring minimal tumor motion between IFP measurements.

The implications of this work extend to the development of new therapies. The transport of nanoparticles is a key first step in building efficacious therapeutics based on nanomedicine. After watching this video, you should have a good idea of how to spatially map tumor interstitial fluid pressure, the tumor microcirculation, and nanoparticle distribution.