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.
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.
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 disease3,4. 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 limitation5–7. 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 ongoing8,9,10,11,12,13. 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 microenvironment19,20,21,22. 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 RMS1,25. 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.
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.
2. Mouse genotyping
3. Tumor model preparation (Figure 1)
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.
5. Screening MRI scan
6. Experiment: HIFU treatment day animal preparation
7. Experiment: Mouse model imaging and sonication procedure for acute studies
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).
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 temperature had to remain below 39 °C, based on the rectal probe or the starting rectal temperature plus the change in the esophageal probe temperature (Supplemental Figure 2). Once the sonication of the MRgHIFU was stopped, the tumor rapidly returned to the baseline temperature.
The tumors were targeted to 40.5 °C to reach a temperature for rapid drug release while avoiding cumulative temperature effects above 43 °C. The average temperature of the ROI in all treated tumors was 40.6 °C (n = 65), with an average difference between the 10th percentile and 90th percentile voxels of 4.3 °C. The standard deviation of the average temperature was 1.3 °C for the duration of the treatment for both the 10 and 20 min treatments (Figure 5). The success rate of the treatments for meeting the inclusion criteria noticeably improved over the duration of the study from 11% to 100% (Figure 6).
After optimizing the treatment protocol, the duration of hyperthermia was assessed for drug release efficacy in comparison to normothermic (NT) mice. To determine the optimal hyperthermia treatment time for further studies, two durations of treatment were tested: 10 min and 20 min. These durations were selected for the feasibility of consistently maintaining core normothermia and tumor hyperthermia. High performance liquid chromatography and mass spectrometry (HPLC-MS) was used to assess the amount of doxorubicin in the tumors and quantify the difference of doxorubicin accumulation between the tested durations. There was a significantly higher percent of the initial dose (%ID) of doxorubicin in the tumors in the 20 min HT + TLD treated mice compared to the TLD 20 min NT mice (Figure 7, q = 0.000108). There was no significant difference between the 10 min and 20 min HT + TLD groups; however, there was a larger standard deviation in the 10 min treatment group compared to the 20 min group (3.698 vs. 2.065 %ID/g of tumor). Notably, there were four near-zero values within the 10 min HT + TLD treatment group, which were all treated with a single batch of TLD. TLD was characterized prior to use in in vivo experiments, as previously described by Dunne et al.28. Briefly, TLD was characterized in terms of its size, zeta potential, melting phase transition temperature, and drug concentration, and liposomes were used within 72 h of storage at 4 °C. Although all batches of TLD were tested prior to use, it is possible that the liposomes had released the doxorubicin during experimental setup, prior to use.In addition, motion during the scan can result in falsely elevated temperature calculations in the software, thus underheating the tumor and resulting in lower drug release. Alternatively, falsely low values could also be caused if the drug was never injected, for example, if the tail vein catheter had been removed or improperly placed. As seen above, the setup of the MRI sled included temperature probe insertion (rectal and esophageal), tail vein catheter insertion, and respiratory monitor placement, followed by moving of the sled, mouse, tail vein catheter, three fiber optic temperature probes, respiratory monitor, and anesthesia lines into the MRI bore. There are multiple time points during this process that the tail vein catheter can get dislodged. This was controlled for by checking blood back flow into the line, bleeding from the catheter insertion site, and drug pooling underneath the tape after treatment, but error remains a possibility.
Figure 1: Experimental protocol for animal treatments and the associated treatment groups for the HT duration studies. The mice were injected with M25FV24C cells in their right hind limb, and were screened for tumor formation using MRI after 2-3 weeks. They were then divided into normothermic (Non-HT) or hyperthermic (HT) groups, with either TLD or FD at durations of either 10 or 20 min. Abbreviation: Dox = Doxorubicin. Please click here to view a larger version of this figure.
Figure 2: Mouse setup during HIFU treatment. (A) A 3D printed holder (white) with inner rubber lining (red) and a cutout to allow for ultrasound beam passage for mouse positioning. (B) Mouse setup inside a 3D printed mouse holder with a rectal temperature hold (green cable), tail vein catheter (white), and respiratory monitor (blue). (C) Mouse positioning on the MRI HIFU bed during the procedure. Please click here to view a larger version of this figure.
Figure 3: Mouse in the MRI during MRIgHIFU treatment. (A) The tumor (circled in orange) and the drift tube used to measure ambient temperature (circled in light blue) are visible. (B) During treatment, the thermometry temperature measurement is overlaid on the MRI image. Please click here to view a larger version of this figure.
Figure 4: Temperature (°C) monitored during treatment. Average (green), top 10th percentile (red), and top 90th percentile (cyan) temperatures of all voxels in the ROI. Please click here to view a larger version of this figure.
Figure 5: Average temperatures during treatment within the ROI for each mouse tested during the optimization phase with the standard deviation during the treatment. The overall average temperature and standard deviation during treatment is also shown (orange). Please click here to view a larger version of this figure.
Figure 6: Success rates of hyperthermia treatment improved over time. Treatment success was dependent on the inclusion criteria (systemic temperature, tumor temperature and variation with the ROI, and no distal heating). Blue line = % of mice for which treatment was successful. Orange bars = number of mice treated with HT. Each treatment (treatment 1-6) refers to a separate date that the experiments were conducted on. Please click here to view a larger version of this figure.
Figure 7: Amount of doxorubicin in the tumor after drug treatment. (A) Multiple Mann-Whitney tests with FDR correction for multiple comparisons of the HPLC-MS results demonstrate significance (q < 0.05) between the amount of doxorubicin in the tumor in the 20 min TLD + HT group compared to the NT control. (B) No differences were seen in the tumor in the FD groups. %ID = percent of initial dose. **** = q < 0.0001. Abbreviations: HT = hyperthermia, NT = normothermia. Please click here to view a larger version of this figure.
Sequence Name | Ax_Screen | Ax_Loc | Sag_Loc | Cor_TestShot | Therm |
Sequence Type | T2w RARE | T2w RARE | T2w RARE | FLASH | FLASH |
Orientation | Axial | Axial | Sagittal | Coronal | Axial/Sagittal |
Echo time (ms) | 40 | 72 | 72 | 6 | 6 |
Repetition time (ms) | 3200 | 4500 | 4500 | 39.06 | 39.06 |
Flip angle (degrees) | 90/180 | 90/180 | 90/180 | 10 | 10 |
Field-of-view (mm) | 28.8 x 28.8 | 36 x 36 | 35 x 35 | 35 x 35 | |
Matrix size | 128 x 128 | 128 x 128 | 128 x 128 | 128 x 128 | 128 x 128 |
Resolution (mm) | 0.225 x 0.225 | 0.281 x 0.281 | 0.281 x 0.281 | 0.273 x 0.273 | 0.273 x 0.273 |
Slice number | 20 | 20 | 20 | 3 | 2 |
Slice thickness (mm) | 1 | 1 | 1 | 1.5 | 1.25 |
# Averages | 3 | 1 | 1 | 1 | 1 |
# Repetitions | 1 | 1 | 1 | 9 | 9 or 300 |
Scan time | 4 min 0 s | 1 min 12 s | 1 min 12 s | 45 s | 25 min |
Table 1: Parameters for MRI capture with associated sequence names.
Supplemental Figure 1: A 3D model mouse holder (white) with inner rubber lining (red). Dimensions: length = 43 mm, outer radius = 15 mm, inner width = 20.7 mm. Please click here to download this File.
Supplemental Figure 2: Temperature (°C) monitored during treatment. Core temperature measured by rectal (blue) and esophageal (orange) probes. Please click here to download this File.
Supplemental Coding File 1: 3D printing file for the mouse holder. Please click here to download this File.
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 into the target tissue1. Secondly, mouse positioning is vital to a successful treatment; the tumor should be placed superiorly in the mouse holder to be in closer contact with the ultrasound transducer. In addition, bony structures should be positioned out of the ultrasound beam path without injuring the mouse. Bone has been shown to absorb ultrasound waves efficiently, subsequently acting as an in situ heating source. It can affect the heating profile while blocking ultrasound transduction into the region of interest4. The contralateral limb should also be placed out of the way of the ultrasound path, either by tucking the leg up under the rest of the body or by extending it and filling the air between the legs with ultrasound gel or a gel pad. The rectum must also be out of the path of the ultrasound to avoid off-target heating and reflection from the temperature probe. Careful tumor positioning is the most important step to completing a successful treatment.
After correct positioning, placement of the esophageal temperature probe must be performed carefully to avoid tracheal occlusion. When inserting the mouse into the MRI bore, the metal connection hub between the catheter in the tail and the injection pump catheter should be secured with tape distal to the area of imaging to avoid artifact creation. The ultrasound transducer should be placed so that it is in contact with the fur-less area of the leg and not displacing the respiratory monitor. Careful monitoring of the mouse’s core body temperature during treatment and subsequent adjustment of the convection heating system is required for mouse survival. Due to the proximity of the rectum and the tumor in some mice, the addition of the esophageal probe was important to determine core temperature change, as the rectal temperature could only reflect local heating as opposed to core body heating.
In designing and implementing this protocol, extensive troubleshooting was successfully executed by a multidisciplinary team. For mouse positioning, a mouse holder was designed and 3D printed to use on a rat MRI sled to allow for flow of the warmed air around the mouse for intra-procedural body temperature adjustment. The materials for this holder were chosen based on their ability to hold the mouse securely while allowing for ultrasound transduction. A rubber insert inside the printed holder allowed for individual mouse adjustments, while the cut out in the bottom prevented ultrasound wave reflection and unintended heating.
There are limitations associated with the model, such as the proximity of the tumors to nearby structures-bone (femur) and the rectum-which can absorb or reflect ultrasound waves, respectively. Unintentional heating of the femur can result in bone marrow destruction and pain, while ultrasound reflection from air in the rectum can cause local heating and tissue damage. Additionally, there were instances of trapping of the ultrasound wave due to skin regrowth after treatment in the survival mice, causing localized heating of the skin. It is suspected that this is due to air trapping around the hair follicle that is not displaced with the ultrasound gel between the transducer and the skin. In each case, the skin appeared darker than the surrounding hairless skin. On immunohistochemistry sections of these mouse limbs, hairs were seen within the epidermis, but no tumor fibrosis or other explanation was found for why the ultrasound would be unable to pass through the skin and subcutaneous tissues.
With the development of this protocol, further studies are planned to extend the model systems to test other pediatric solid tumors, such as osteosarcoma and myxofibrosarcoma, for treatment with HT and TLD. This is promising as these patients can face debilitating pain with limited treatment options in this clinical context. This protocol can be extended to other solid tumor types located in the extremities that are targetable with MRgHIFU29,30. In conclusion, the data supports that the combination of thermosensitive liposomes can be extrapolated to encapsulate other forms of chemotherapy or drugs where targeted drug delivery would be beneficial and having a noninvasive form of heating, such as MRgHIFU, would be ideal.
The authors have nothing to disclose.
We would like to acknowledge our sources of funding for this project and the personnel involved including: C17 Research Grant, Canada Graduate Scholarship, Ontario Student Opportunity Trust Fund, and James J. Hammond Fund.
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 |