Magnetic Resonance-Guided High Intensity Focused Ultrasound Generated Hyperthermia: A Feasible Treatment Method in a Murine Rhabdomyosarcoma Model

This protocol allows for novel preclinical testing of tumor targeted hyperthermia in combination with thermo sensitive drugs, which can be applied to improve survival and reduce systemic toxicity in sarcoma patients. The main advantage of this technique is that the chemotherapy release is confined to the heated region maximizing drug release within the tumor while minimizing the systemic release of the drug in healthy tissue. Our methods can be translated clinically to treat sarcomas.

Also, our work is learning how localized hypothermia can incite the immune system to help the body target cancer alongside standard chemotherapy. Demonstrating the procedure will be Dr.Warren Foltz, the lead MRI physicist at the Star Innovation Center, Dr.Adam Waspe, the lead HIFU physicist at the Hospital for Sick Children, and Suzanne Wong, a master's graduate in applied science from the Drake Lab at the hospital for Sick Children in Toronto. To begin shave the hind leg of the restrained mouse using clippers and wipe it with 70%ethanol.

Aspirate approximately 10, 000 cells in 10 microliters of cell suspension solution using a microliter syringe. Extend the right hind leg of the mouse and inject the cell suspension in a steady motion into the thigh musculature using a microliter syringe with a 26 gauge needle. The needle should be inserted parallel to the femur toward the knee, taking care not to hit the sciatic nerve.

Remove the needle and return the mouse to a second cage. Evaluate the animals daily and monitor their hind limbs for tumor growth through palpation. Image the anesthetized mouse using the MRI scanner.

On the T2 weighted image take note of in plaine dimensions and the number of axial slices that the tumor appears within. Note the location of the tumor in reference to the femur and lateral surface of the thigh where the ultrasound wave would enter. To prepare the small bore HIFU system, turn the generator on, put the membrane on the transducer and fill the transducer with enough deionized water until the membrane is expanded below the transducer.

But not so firm that it would compress the mouse. Degas the water in the transducer circuit for 30 minutes to remove dissolved oxygen from the medium. After anesthetizing the mouse and transferring it to a nose cone, apply a corneal lubricant to the eyes to prevent damage due to the lack of blink reflex under anesthesia.

Shave the right hind limb of the mouse. Apply depilatory cream to the shaved area including the entire right hind limb. Position the mouse under a heat lamp while in the biosafety cabinet to help with thermal regulation.

And remove the hair and depilatory cream one minute after application. Move the mouse to an MRI compatible nose cone on the MRI sled and place the mouse with the non-tumor bearing side down and the tumor superior inside a 3D printed mouse holder on the sled. Place a heat lamp near the mouse to keep it warm.

If needed, cut to compressed ultrasound gel pad segment to put under the mouse lining the bottom of the holder with a thickness to level the tumor to the top of the holder. Tuck the uninvolved leg away from the tumor leg, either under the mouse or extended with the tumor leg flexed. Ensure the feet are not in the near field or far field of the tumor and ultrasound beam path.

To insert the esophageal temperature probe thread the esophageal probe through the nose cone and scruff the neck of the mouse. Tilt the mouse's nose up to create a line from its mouth straight to its stomach by extending the head. Slide the thermal probe above the tongue about 0.5 centimeters into the mouse's esophagus and replace the nose cone around the mouse's nose.

Secure the esophageal probe and nose cone at the top of the sled. Next, insert the rectal temperature probe and secure it with tape. Insert another gel pad if needed and apply eye lubricant or ultrasound gel around the right hind limb to fill any gaps.

Insert a 27 gauge butterfly needle tail vein catheter into a lateral tail vein attached to a micro tubing with 40 microliters of dead space and tape securely. Place the respiratory monitor with the connecting cable toward the head of the mouse so it does not interfere with the placement of the ultrasound transducer, secure with tape. Use two people to carry the prepared mouse, mouse sled, anesthesia line, respiratory line, tail vein catheter, and thermal probe cords into the MRI scanner and place them into the MRI sled holder.

Everybody has been screened and nothing is brought into the MR suite, which is ferromagnetic or could be damaged within the strong magnetic field of the system. After the HIFU software operator moves the meniscus of the transducer directly over the tumor for an initial alignment, apply eye lubricant, or degassed ultrasound gel, to the hairless skin above the tumor and couple the HIFU transducer to the tumor area. Connect the drug delivery line from the automatic pump to the tail vein catheter and calculate the amount of dead space in the tail vein line and the connecting line.

Slide the mouse HIFU sled on MRI rails into the center of the MRI and place the air convection warming device on the warmest setting. Point the tube blowing air toward the mouse in the center of the MRI bore and secure it with tape. The warming device will later be turned to its lowest setting to prevent overheating of the mouse during sonication.

Acquire the survey MR images of both axial and sagittal to determine the tumor location for sonication targeting, including depth. Adjust the transducer position accordingly using the HIFU software by inserting the desired movement distance as measured on the image, and then clicking the arrow direction to move. Also, note the location of the drift tube.

Repeat as necessary. Determine the location of the focal spot of the transducer in the cornal plane by performing a brief 50 millivolts continuous test shot sonication for five seconds during the test shot thermometry acquisition. Align the MR survey images with the cornal view of the focal spot within the HIFU software.

Review the images for tumor location relative to the bony structure and the rectum, and revise the transducer positioning as deemed necessary. Repeat the test shot sonication during nine repetition thermal imaging to confirm whether there is even and accurate heating in the tumor volume with minimal off-target heating. Adjust the slice location, transducer location and depth of steering and confirm heating performance with repeat test shots as deemed necessary.

Using the HIFU treatment monitoring software, define the region of interest, or ROI, for thermometry monitoring within the final heating profile by measuring the distance to move and then altering the grid coordinates in the program. Set an ROI within the fluid signal from the drift tube for drift correction of the thermometry output. If this fluid signal is not well defined within the axial planning image at the level of the tumor then the direct output from the fiber optic temperature probe within the drift tube is used for drift correction.

Enter the baseline temperature based on the rectal probe temperature for thermometry measurements. Open the 20 minute hyperthermia treatment specifications in the software and start sonication once the reference MR Images are collected and the thermometry begins. Perform a 20 minute treatment during thermal imaging using the built-in proportional integrative derivative, or PID, controller software.

Inject the selected drug 1.5 minutes after the temperature in the ROI warms to the desired temperature. If the rectal temperature is increasing rapidly during the treatment and exceeds 40 degrees Celsius, halt sonication and allow the rectal temperature to drop before restarting. After treatment completion, remove the mouse from the MRI bore, ensuring hemostasis at the tail vein catheter insertion site and transfer it to the biosafety cabinet.

Wash off the ultrasound gel and lubricant from the hind leg using water. Transfer the mouse to its cage and monitor its recovery. Point the heat lamp at one end of the cage to assist with thermal regulation during recovery.

Using this hyperthermia protocol, the tumors in the hind limb were able to be consistently heated to the desired set temperature for the duration of the treatment. The temperature monitored during the representative treatment is shown in this figure. Average top 10th percentile and top 90th percentile temperatures of all voxels in the ROI are presented here.

Average temperatures during treatment within the ROI for each mouse were tested during the optimization phase with the standard deviation. The overall average temperature is also shown. The success rates of hyperthermia treatment were also improved over time.

Treatment success was dependent on the inclusion criteria. That is the systemic temperature, the tumor temperature and variation with the ROI and no distal heating. Here, the blue line represents the percentage of mice for which the treatment was successful and the orange bars represent the number of mice treated.

Each treatment refers to a separate date on which the experiments were conducted. To determine the optimal hypothermia treatment time for further studies, two durations of treatment were tested, 10 minutes and 20 minutes. High performance liquid chromatography and mass spectrometry, or HPLC MS, was used to assess the amount of doxorubicin in the tumors and quantify the difference in doxorubicin accumulation between tested durations.

The results demonstrate the significance between the amount of doxorubicin in the tumor in the 20 minute thermosensitive liposomal doxorubicin plus hyperthermia group compared to the normal thermia control. No differences were seen in the tumor in the free doxorubicin groups. When preparing and positioning the mass it is imperative that the HIFU beam is unobstructed.

Air bubbles in the gel or bone in the beam path must be avoided. We collect tissue and blood to analyze the pharmacokinetics and toxicity levels in various organs as well as examine the immediate and delayed response of the immune system to treatment.