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

Summary

Presented here is a protocol to use controlled hyperthermia, generated by magnetic resonance-guided high intensity focused ultrasound, to trigger drug release from temperature-sensitive liposomes in a rhabdomyosarcoma mouse model.

Abstract

Magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) is an established method for producing localized hyperthermia. Given the real-time imaging and acoustic energy modulation, this modality enables precise temperature control within a defined area. Many thermal applications are being explored with this noninvasive, nonionizing technology, such as hyperthermia generation, to release drugs from thermosensitive liposomal carriers. These drugs can include chemotherapies such as doxorubicin, for which targeted release is desired due to the dose-limiting systemic side effects, namely cardiotoxicity. Doxorubicin is a mainstay for treating a variety of malignant tumors and is commonly used in relapsed or recurrent rhabdomyosarcoma (RMS). RMS is the most common solid soft tissue extracranial tumor in children and young adults. Despite aggressive, multimodal therapy, RMS survival rates have remained the same for the past 30 years. To explore a solution for addressing this unmet need, an experimental protocol was developed to evaluate the release of thermosensitive liposomal doxorubicin (TLD) in an immunocompetent, syngeneic RMS mouse model using MRgHIFU as the source of hyperthermia for drug release.

Introduction

Rhabdomyosarcoma (RMS) is a skeletal muscle tumor that most commonly occurs in children and young adults¹. Localized disease is often treated with multimodal treatment, including chemotherapy, ionizing radiation, and surgery. The use of multi-drug chemotherapy regimens is more prevalent in pediatric patients, with improved outcomes compared to their adult counterparts²; however, despite ongoing research efforts, the 5-year survival rate remains at around 30% in the most aggressive form of the disease^3,4. The chemotherapy standard of care is a multidrug regimen that includes vincristine, cyclophosphamide, and actinomycin D. In cases of relapsed or recurrent disease, alternate chemotherapies are used, including standard (free) doxorubicin (FD) and ifosfamide¹. While all these chemotherapies have systemic toxicities, the cardiotoxicity of doxorubicin imposes a life-long dose limitation^5-7. To increase the amount of the drug delivered to the tumor and to minimize systemic toxicity, alternative formulations have been developed, including liposomal encapsulation. These can be non-thermosensitive doxorubicin, which has been approved for the treatment of breast cancer and hepatocellular carcinoma, or thermosensitive doxorubicin, for which clinical trials are ongoing^{8,9,10,11,12,13}. Alternative methods for delivering liposomal encapsulated drugs such as multi-vesicular liposomes and ligand-targeted liposomes have been evaluated and show promise for the treatment of tumors⁹. In this study, the addition of heat has multifactorial impacts, including drug release¹⁴. The combination of hyperthermia (HT) generated with magnetic resonance-guided high intensity focused ultrasound (MRgHIFU) and thermosensitive liposomal doxorubicin (TLD) is a novel multimodal therapeutic approach for using this toxic yet effective drug to treat RMS, while minimizing dose-limiting toxicity and potentially increasing the immune response to the tumor.

Doxorubicin releases rapidly from TLD at temperatures >39 °C, well above the average human body temperature of 37 °C but not high enough to cause tissue damage or ablation; this starts to occur at 43 °C, but occurs more rapidly as temperatures approach 60 °C¹⁵. Various methods have been used to generate HT in vivo, including lasers, microwaves, radiofrequency ablation, and focused ultrasound, many of which are invasive heating methods¹⁶. MRgHIFU is a noninvasive, nonionizing heating method that facilitates precise temperature settings within the target tissue in situ. Magnetic resonance (MR) imaging crucially provides real-time imaging, where computer software can be used, to calculate a thermometry measurement of the tissue throughout treatment; subsequently, this data can be used to control the ultrasound therapy in real time to reach and maintain a desired temperature set point¹⁷. MRgHIFU has been tested in various tissue types and can be used for a wide range of temperature treatments, from mild HT to ablation, as well as clinically to successfully treat painful bone metastases¹⁸. Additionally, HT has been shown to cause tumor cytotoxicity, modulate protein expression, and alter the immune response in the tumor microenvironment^19,20,21,22. One study combined mild HT with TLD, followed by ablation with MRgHIFU, in a synergetic R1 rat model²³, resulting in necrosis in the tumor core and drug delivery to the periphery. Traditionally, radiotherapy has been used as an adjunct therapy to damage tumor cells and decrease local disease recurrence. However, its use is limited by lifetime dosing and off-target damage¹. Thus, HT is unique in that it can cause some of the same effects without the same toxicities or limitations.

Preclinical animal models for RMS include syngeneic immunocompetent models and patient derived xenografts (PDX) in immunocompromised hosts. While the immunocompromised models allow for growth of the human tumors, they lack the appropriate tumor microenvironment and are limited in their ability to study immune response²⁴. FGFR4-activating mutation is a promising marker for poor prognosis and a potential therapeutic target in adult and pediatric RMS^1,25. In the syngeneic RMS models developed in the Gladdy lab, the tumors are able to grow in an immunocompetent host, which develops innate and adaptive immune responses to the tumor²⁶. As HT influences the immune response, observation of the change in the murine immune response is a valuable advantage of this tumor model. To test both the tumor response to TLD in comparison to FD, as well as the change in the immune response of the tumor to both chemotherapy and HT, a protocol was developed and employed to treat syngeneic murine RMS tumors in vivo using MRgHIFU and TLD, which is the focus of this study.

Protocol

Research was performed in compliance with the animal care committees with approved animal use protocols under a supervising veterinarian at The Centre for Phenogenomics (TCP) and University Health Network (UHN) Animal Resource Centre (ARC) animal research facilities. All procedures, excluding the MRgHIFU, involving the animals were done in a biological safety cabinet (BSC) to minimize animal exposure to external air or susceptible infection.

1. Mouse breeding

NOTE: A total of 65 mice (strain B6.129S2-Trp53tm1Tyj/J) were included in the pilot study (male: n = 23; female: n = 42). Both male and female mice were used at 7-9 weeks of age. Their pups were weaned and genotyped, and the p53 heterozygous mice were used for the experiments.

1. House two female mice with each male mouse to create breeding cages. Count the ages of their pups from birth (birth = day 0).
2. At day 10, identify the pups with an ear notch. Collect tail snips for genotyping prior to cell line injection.

2. Mouse genotyping

1. Extract DNA from the collected 2 mm tail clippings using a commercial DNA extraction kit (see Table of Materials), following the manufacturer's instructions.
2. Determine the DNA concentration and purity by measuring the absorbance at 260-280 nm on a spectrophotometer (see Table of Materials).
3. Perform polymerase chain reaction (PCR).
   1. Create a master mix containing a commercial PCR mix (containing Taq polymerase, dNTPS, and MgCl₂; see Table of Materials), primer, and dH₂O in a 12.5:0.25:10.75 (µL) ratio for the required number of samples. Add 1 µL of DNA sample to each PCR tube and include dH₂O, a null sample (homozygous for p53 mutation), a heterozygous sample (heterozygous for p53 mutation), and a wild type (homozygous for normal p53) sample as PCR controls.
   2. Add 24 µL of the master mix to each PCR tube containing DNA. Pipette the solution in each PCR tube up and down to distribute the DNA throughout the master mix.
   3. Place the reaction tubes in a thermal cycler and cycle according to the following specifications: 95 °C for 2 min, 40 cycles of 95 °C for 15 s, 60 °C for 15 s, and 72 °C for 1 min, and then maintain at 4 °C until ready to analyze on the gel.
4. Analyze the PCR products using agarose gel electrophoresis.
   1. Prepare a 2% gel (50 mL of 1x TAE and 1 g of agarose) by heating the agarose in the TAE and mixing until dissolved. When cooled and still liquid, add 2.5 µL of DNA gel stain to the agarose and mix. Cast the gel in a gel box with a comb. Place the gel in the electrophoretic apparatus (see Table of Materials) and cover with 1x TAE.
   2. Load 10 µL of 1 kB DNA Ladder on the gel. Load 12.5 µL of each sample. Run the gel for 25 min at 135 V.
   3. Image the gel using appropriate settings for the used DNA gel stain on a gel imager (see Table of Materials), according to the manufacturer's instructions.

3. Tumor model preparation (Figure 1)

1. Grow the M25FV24C cell line (passage 12-15) 1 week prior to the injection date in complete growth media (Dulbecco's Modified Eagle Medium [DMEM] with additives: 10% FBS, 1% Penicillin/Streptomycin, and 2 mM L-alanyl-L-glutamine dipeptide) in a 75 mL flask, at 37 °C and 5% CO₂. Once the cells are ~80% confluent, aspirate the media and wash the cells 1x with 5 mL of Dulbecco's phosphate-buffered saline (DPBS).
   NOTE: M25FV24C is a murine cell line engineered to overexpress mutant FGFR4^V550E, which is observed in pediatric and adult RMS^1,26.
2. Lift the cells by adding 0.5 mL of 0.25% trypsin solution to the side of the plate and incubating the vessel for 2-3 min at room temperature. Once the cells appear detached, add 2.5 mL of complete growth media at room temperature to inactivate the trypsin. Use a 10 µL sample aliquot to determine the cell concentration of viable cells using a hemocytometer and trypan blue exclusion.
3. Prepare the correct volume of DPBS-suspended cells for injection and place into a 1.5 mL microcentrifuge tube: volume to centrifuge = (number of mice × number of cells per mouse)/ (concentration of cells), where the number of mice = the mice to be injected + 10 extra mice for error, and the number of cells per mouse = 10⁴.
4. Centrifuge for 5 min at 153 x g. Resuspend the cell pellet in the appropriate volume (10 µL per mouse × number of mice) of myo-injection buffer (F10 media + 0.5% FBS) and inject the mice within 1 h of preparation of this suspension.

4. Intramuscular cell injection

NOTE: M25FV24C cells are injected into the right hind limb of mice between 4 and 6 weeks of age. Injection at 4 weeks produces a small mouse with a tumor that can be harder to treat as there is less surrounding tissue for HT dispersion; waiting until 6 weeks yields a larger mouse, making it easier to treat the tumor.

1. Invert the cell suspension several times prior to aspiration to help evenly distribute the cells within the solution. Aspirate 10 µL (10⁴ cells) using a microliter syringe (see Table of Materials). Scruff the mouse; once restrained, access to the caudal thigh muscles can be gained by extending the hind leg. Shave the leg using clippers and wipe with 70% ethanol.
2. Inject the M25FV24C cell suspension (10 µL, 10⁴ cells) into the right hind limb thigh musculature of a 4-6-week-old mouse using a gas-tight microliter syringe with a 26s G needle.
   NOTE: The needle should be inserted parallel to the femur toward the knee, taking care not to hit the sciatic nerve. Insert only the point of the needle (approximately 2 mm) due to the small muscle mass of the hind limb.
3. Administer the solution in a steady motion. Remove the needle and ensure bleeding does not occur. Return the mouse to a second cage.
4. Evaluate the animals daily and monitor their hind limbs for tumor growth through palpation. Euthanize the mice using carbon dioxide if any of the following early end points are encountered: tumor size exceeding 1.5 cm in diameter, tumor ulceration, or systemic signs of illness (piloerection, hunched posture, inactivity, or decreased intake of food or water).

5. Screening MRI scan

1. Anesthetize the mouse, to a level where there is no movement with paw squeeze, with isoflurane under the following parameters: induce in a chamber with 4% at 1.5 LPM, then transfer to the nose cone on the MRI scanner sled and continue isoflurane maintenance on the nose cone with 1.5%-2% at 0.75 LPM. Attach the respiratory monitor. Use vet ointment on eyes to prevent dryness while under anesthesia.
2. Image the anesthetized mouse using the MRI scanner (see Table of Materials). On the T2-weighted image (Ax_Screen acquisition, Table 1), take note of in-plane 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.
   NOTE: The tumor appears as a hyperintense mass within the muscle that is asymmetric from the opposite side. A good starting tumor size for multiple treatments is 2 mm x 2 mm x 2 mm for either acute or survival studies. If it is much larger, it will only be good for acute studies as the tumor will reach a size end point prior to completing three weekly treatments. Exclusion criteria for HIFU treatment include: wrapped around the femur, too close to the femur, too posterior on the mouse, medial to the femur, too close to the rectum.
3. Remove the mouse from the scanner and obtain a baseline weight. Shave the mouse from their mid body down to their feet under anesthesia with an electric shaver.
   NOTE: Ideally, shaving is done 1 day prior to treatment, as it allows the mouse to perform grooming which allows the depilatory cream to work more efficiently.
4. Recover the mouse in the BSC, using a heating pad under one end of the cage. Return the mouse to its cage when it regains sternal recumbency.

6. Experiment: HIFU treatment day animal preparation

1. To prepare the small bore HIFU (see Table of Materials) system, turn the generator on 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 min to remove dissolved oxygen from the medium.
2. Prepare the associated computer system.
   1. Turn on the controlling computer and ensure it is connected via ethernet to the HIFU generator and via USB to the thermal probe display. Start the software and click Home to home the transducer prior to inserting the mouse.
   2. Calibrate the fiberoptic thermal probes: Get the baseline room temperatures and note the temperature change in the MRI room. Note the magnitude of temperature drift for each probe due to the magnetic field strength. Insert the drift tube temperature probe into a gadolinium filled glass tube for temperature calibration during scanning and secure the drift tube with tape.
      NOTE: The baseline room temperature (drift tube) is added manually as a thermometry parameter in the GUI in the software. A region of interest (ROI) is set within the drift tube in the MR image to detect any temperature drift and will automatically correct the thermometry images.
   3. Draw up the drug to be injected into a 1 mL syringe and place it into the automatic delivery pump (see Table of Materials). Prime the line that will connect to the mouse's tail vein catheter until the drug has completely filled the line by pressing the manual delivery button on the automatic delivery pump.
   4. Use a heat lamp to warm the mice in the cages for ~20 min prior to transfer to the anesthetic chamber.
      NOTE: Preheating promotes vasodilation, which will be encountered as soon as the mouse is anesthetized and aids in catheter placement.
3. Anesthetize the mouse with isoflurane (induction: 4% at 1.5 LPM; maintenance: 1.5%-2% at 0.75 LPM) and transfer to a nose cone. Apply a corneal lubricant to the eyes to prevent damage due to the lack of blink reflex under anesthesia.
4. Apply depilatory cream to the shaved area, including the entire right hind limb, and follow the manufacturer's instructions for hair removal.
   NOTE: Position the mouse under a heat lamp while in the BSC to help with thermoregulation during hair removal under anesthesia.
5. After washing off the depilatory cream with warm water, weigh the mouse on a digital scale and record for drug dosing.
6. Move the mouse to an MRI-compatible nose cone on the MRI sled. Position a heat lamp on the mouse to keep it warm while preparing for the MRI. Place the mouse in the lateral decubitus position with the non-tumor bearing side down and the tumor superior inside a 3D-printed mouse holder on the sled (Supplemental Figure 1 and Figure 2). Ensure proper positioning of the tumor (i.e., in the center of the coil horizontally and vertically, with the height just above the edges of the mouse holder to account for compression by the ultrasound transducer).
   NOTE: If needed, cut a 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.
7. 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. Position the heat lamp 15 cm from the tail to warm for catheter insertion into the tail vein.
8. Insert the esophageal temperature probe.
   1. Thread the esophageal probe through the nose cone and scruff the neck of the mouse. Tilt the mouse 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 cm into the mouse esophagus and replace the nose cone around the mouse's nose. Secure the esophageal probe and nose cone at the top of the sled.
      NOTE: Monitor for signs of respiratory distress immediately after insertion as it can be improperly inserted into the trachea.
9. Insert the rectal temperature probe.
   NOTE: The rectal and esophageal temperature probes should be within 3 °C of each other.
10. 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.
11. Insert a 27 G butterfly needle tail vein catheter into a lateral tail vein attached to microtubing with 20 µL of dead space and tape securely. After taping, ensure that the catheter is still flushing well.
12. 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 into the MRI sled holder.
13. Have the HIFU software (see Table of Materials) operator move the meniscus of the transducer directly over the tumor by visual inspection for an initial alignment²⁷. Apply eye lubricant or degassed ultrasound gel to the hairless skin above the tumor and couple the HIFU transducer to the tumor area.
14. Connect the drug delivery line from the automatic pump to the tail vein catheter. 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.
15. Set the amount of drug infusion on the pump, depending on the drug type and concentration and the weight of animal, and add the amount of dead space. Set the pump to a rate of infusion of 200 µL/min.
    NOTE: In this study, FD and TLD were used at a concentration of 2 mg/mL and a dose of 5 mg/kg body weight.
16. Record the baseline thermal probe temperatures.
17. Place the air convection warming device (see Table of Materials) on the warmest setting. Point the tube blowing air toward the mouse in the center of the MRI bore and secure with tape. The warming device will later be turned to its lowest setting (32 °C) to prevent overheating of the mouse during sonication.
18. Acquire the survey MR images (Ax_Loc, Sag_Loc; Table 1) 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 (Figure 3A). Also note the location of the drift tube. Repeat as necessary.
19. Determine the location of the focal spot of the transducer in the coronal plane by performing a brief 5 s x 50 mV amplitude continuous 'test shot' sonication during the Test_Shot thermometry acquisition (Table 1).
20. Align the MR survey images with the coronal view of the focal spot within the HIFU software. Review the images for tumor location, relative to bony structure and the rectum, and revise the transducer positioning as deemed necessary.
21. Repeat the test shot sonication during nine-repetition Therm imaging (Table 1) 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.
22. Using the HIFU treatment monitoring software, define the 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 around the drift tube for drift correction. Enter the baseline temperature based on the rectal probe temperature for thermometry measurements. The HIFU system is used to initiate HIFU treatment sonication and for thermometry monitoring.
23. Open the 20 min hyperthermia treatment specifications in the software and start sonication once the reference MR images are collected and the thermometry begins.
24. Perform a 20 min treatment (Figure 3B) during Therm imaging (Table 1) using the built-in proportional-integrative-derivative (PID) controller software. Inject the selected drug at 1.5 min, after the temperature in the ROI warms to the desired temperature (40 °C).
25. Monitor the core temperature throughout treatment. If the rectal temperature is increasing rapidly during treatment, mouse repositioning may be required to avoid rectal warming over the 10 or 20 min treatment duration. Stop the treatment if the rectal temperature increases to >40 °C.

7. Experiment: Mouse model imaging and sonication procedure for acute studies

1. After completion of the treatment, remove the mouse from the MRI bore, ensuring hemostasis at the tail vein catheter insertion site. Transfer the mouse to the BSC and place on the nose cone for continued anesthesia.
2. Place the mouse on its back on a blue absorbent pad with its limbs restrained and the heart exposed.
3. Euthanize the mice through exsanguination via cardiac puncture followed by removal of the heart. Take the blood immediately and centrifuge for plasma separation at 10,621 x g for 10 min.
4. Perform necropsy and store the organs as required for analysis. Freeze the organs in liquid nitrogen and store at -80 °C for several months or long-term in a liquid nitrogen tank.
5. Mechanically homogenize the tumor tissue by adding a nine-fold excess (w/w) of deionized water and breaking down the tissue using a bead-beating homogenizer. Extract doxorubicin from 600 µL of homogenized tissue by sequentially adding 75 µL of 300 mg/mL silver nitrate, 75 µL of 10 mM sulfuric acid, and 2.5 mL of 1:1 isopropanol:chloroform. Vortex for 20 min and store at −20 °C overnight.
6. To prepare samples for high performance liquid chromatography (HPLC), centrifuge the solution from step 7.5 at 4,500 x g, remove the organic solvent layer, and dry the isopropanol:chloroform under a stream of nitrogen gas. Resuspend in 100 µL of 2:1 MeOH:H₂O. Measure doxorubicin concentration using HPLC-MS/MS²⁸.

8. Experiment: Mouse model imaging and sonication procedure for survival studies

NOTE: For survival studies, follow the HIFU treatment day animal preparation procedure (step 6.1 to 6.25).

1. After completion of the treatment, place the mouse under a heat lamp to allow it to recover, and monitor its breathing and movement until it regains sternal recumbence. Then, return the animal to its cage.
   NOTE: Ensure half of the cage is in line with a heat lamp, as the thermal regulation of the animals is affected by the anesthesia and HT treatment.
2. Monitor the mice daily for behavior, feeding patterns, and respiratory rate for any signs of distress.
3. Perform treatments once weekly following steps 6.1 to 6.25 for 3 consecutive weeks.
4. Twice weekly, perform MRI imaging of the mice for tumor measurement. For the weeks during treatment, perform one MRI scan and one ultrasound every week. After treatment is completed, perform biweekly ultrasound imaging.
5. Euthanize the mouse 60 days after completing the series of treatments, or when a humane endpoint has been reached (tumor size >1.5 cm³ or morbidity from the tumor), followed by necropsy with tumor and organ removal for analysis.

Results

Using the MRgHIFU-generated 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 (Figure 4 shows a representative treatment, 10 or 20 min, n = 65). To consider a treatment to be successful, the ROI had to be maintained above 39 °C for the entirety of the treatment, with <6 °C variation throughout the treatment and without heating of off-target tissue. Additionally, the core temper...

Discussion

The protocol developed herein was used to target hind limb tumors using MRgHIFU for mild HT treatment and release encapsulated drugs from liposomes in vivo. Several critical steps were encountered in this protocol during the pilot study, and optimizing these critical steps accounted for the improved treatment success over the pilot study. First is the complete removal of the hair on the area to be sonicated. Any gas trapping within the fur prevents the ultrasound beam from passing and blocks ultrasound passage i...

Materials

Name	Company	Catalog Number	Comments
1.5mL Eppendorf tubes	Eppendorf	22363204
1kb plus DNA Ladder	Froggabio	DM015-R500
2x HS-Red Taq (PCR mix)	Wisent	801-200-MM
7 Tesla MRI BioSpec	Bruker	T184931	70/30 BioSpec, Bruker, Ettlingen, Germany
C1000 Thermal cycler	Biorad	1851148
Clippers	Whal Peanut	8655
Compressed ultrasound gel	Aquaflex	HF54-004
Convection heating device	3M Bair Hugger	70200791401
Depiliatory cream	Nair	61700222611	Shopper's Drug Mart
DMEM	Wisent	219-065-LK
DNeasy extraction kit	Qiagen	69504
DPBS	Wisent	311-420-CL
Drug injection system	Harvard Apparatus	PY2 70-2131	PHD 22/2200 MRI compatible Syringe Pump
Eye lubricant	Optixcare	50-218-8442
F10 Media	Wisent	318-050-CL
FBS	Wisent	081-105
Froggarose	FroggaBio	A87
Gel Molecular Imager	BioRad	GelDocXR
Glutamax	Wisent	609-065-EL
Heat Lamp	Morganville Scientific	HL0100	Similar to this product
Intravascular Polyethylene tubing (0.015" ID x 0.043" OD, 20G)	SAI infusion	PE-20-100
Isoflurane	Sigma	792632
M25FV24C Cell line	Gladdy Lab	N/A
Microliter Syringe	Hamilton	01-01-7648
Molecular Imager Gel Doc XR	Biorad	170-8170
Mouse holder	The 3D printing material used was ABS-M30i, and it was printed on FDM Fortus 380mc machine	N/A	Dimensions: length = 43 mm, outer radius = 15 mm, inner width (where the mouse would sit) = 20.7 mm.
MyRun Machine	Cosmo Bio Co Ltd	CBJ-IMR-001-EX
Nanodrop 8000 Spectrophotometer	Thermo Scientific	ND-8000-GL
p53 primers	Eurofins	N/A	Custom Primers
PCR tubes	Diamed	SSI3131-06
Penicillin/Streptomycin	Wisent	450-200-EL
Proteus software	Pichardo lab	N/A
Respiratory monitoring system	SAII	Model 1030	MR-compatible monitoring and gating system for small animals
Small Bore HIFU device, LabFUS	Image Guided Therapy	N/A	LabFUS, Image Guided Therapy, Pessac, France Number of elements 8 frequency 2.5 MHz diameter  25 mm radius of curvature 20 mm Focal spot size 0.6 mm x 0.6 mm x 2.0 mm Motor: axes 2 Generator: Number of channels 8 Maximum electrical power/channel Wel 4 Maximum electrical power Wel 32 Bandwidth 0.5 - 5 MHz Control per channel: Freq., Phase and. amplitude Measurements per channel: Vrms, Irms, cos(theta) Duty Cycle at 100% power % 100% for 1 min. Transducer: Number of elements 8 frequency  2.5 MHz diameter 25 mm radius of curvature 20 mm Focal spot size  0.6 mm x 0.6 mm x 2.0 mm
SYBR Safe	ThermoFisher Scientific	S33102
TAE	Wisent	811-540-FL
Tail vein catheter (27G 0.5" )	Terumo Medical Corp	15253
Thermal probes	Rugged Monitoring	L201-08
Trypan blue	ThermoFisher Scientific	15250061
Trypsin	Wisent	325-052-EL
Ultrasound Gel	Aquasonic	PLI 01-08