Here, we describe a protocol that details how to perform sonodynamic therapy in an in vivo mouse glioblastoma model using magnetic resonance-guided focused ultrasound.
Sonodynamic therapy (SDT) is an application of focused ultrasound (FUS) that enables a sonosensitizing agent to prime tumors for increased sensitivity during sonication. Unfortunately, current clinical treatments for glioblastoma (GBM) are lacking, leading to low long-term survival rates among patients. SDT is a promising method for treating GBM in an effective, noninvasive, and tumor-specific manner. Sonosensitizers preferentially enter tumor cells compared to the surrounding brain parenchyma. The application of FUS in the presence of a sonosensitizing agent generates reactive oxidative species resulting in apoptosis. Although this therapy has been shown previously to be effective in preclinical studies, there is a lack of established standardized parameters. Standardized methods are necessary to optimize this therapeutic strategy for preclinical and clinical use. In this paper, we detail the protocol to perform SDT in a preclinical GBM rodent model using magnetic resonance-guided FUS (MRgFUS). MRgFUS is an important feature of this protocol, as it allows for specific targeting of a brain tumor without the need for invasive surgeries (e.g., craniotomy). The benchtop device used here can focus on a specific location in three dimensions by clicking on a target on an MRI image, making target selection a straightforward process. This protocol will provide researchers with a standardized preclinical method for MRgFUS SDT, with the added flexibility to change and optimize parameters for translational research.
Glioblastoma (GBM) is a form of highly aggressive brain cancer that has an incidence of 3.21 per 100,000 people and is the most common malignant brain tumor1. The current standard of care includes surgical resection, radiation, and chemotherapy2. Due to the tumor's invasive and infiltrative nature, complete tumor resection is rare. Residual tissue at the tumor margins results in a high rate of tumor recurrence and a low survival rate of less than 6% after 5 years1.
Due to this prognosis, researchers are exploring new therapeutic options to combat this deadly disease. Sonodynamic therapy (SDT) is a noninvasive treatment that combines low-intensity focused ultrasound (FUS) and sonosensitizing agents to produce a cytotoxic effect in targeted cells3. As an example, porphyrin-based sonosensitizers such as 5-aminolevulinic acid (5-ALA) are preferentially taken up by tumor cells and increase reactive oxidative species (ROS) production to damaging levels when exposed to focused ultrasound. Overexpressed levels of ROS in cells can damage cellular structures and trigger apoptosis. Since 5-ALA is preferentially taken up by tumor cells, healthy tissue within the treatment region is unharmed3,4. Preliminary in vitro studies have revealed that many cancer cells are lysed by SDT treatment, though the rate of cell death is dependent on the cell line. Preliminary in vivo studies yield similar results, confirming that SDT can trigger apoptosis5.
This protocol aims to describe effective techniques and parameters for the SDT treatment of rodent models with intracranially implanted GBM cells using a benchtop FUS research platform. Researchers can use this protocol to perform and optimize SDT for translational FUS research.
All animal studies were approved and conducted in accordance with the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC). Athymic nude female mice (age: 10 weeks) were obtained from commercial sources (see Table of Materials). All Biosafety Level 2 (BSL-2) regulations were followed, including the usage of masks, gloves, and gowns.
1. Tumor implantations and bioluminescence imaging
2. Treatment day setup
3. Preparation of requisites for experiments
4. MRgFUS system setup
5. Initialization
6. Animal preparation for SDT
7. MRI procedures
8. Focused ultrasound treatment (Figure 4)
9. Post-treatment steps
Tumor size declines in animals treated with SDT 24 h post-treatment.
On the day of SDT treatment, the original average bioluminescence signal for the control and treatment groups (N = 4 each) was 2.0 x 106 ± 3.1 x 106 and 2.3 x 106 ± 1.3 x 106 p/s/cm2/sr, respectively. The average bioluminescence values corresponding to tumor size before treatment between the two groups were not statistically significant (p = 0.89). The average bioluminescence signal of the treatment group was 3.57 x 106 ± 2.3 x 106 24 h following SDT, while the bioluminescent signal of the control group was increased to 5.5 x 106 ± 8.2 x 106 p/s/cm2/sr. As shown in Figure 5, this corresponds to a growth rate of 83.4% ± 78% and 172% ± 34%, respectively, assuming exponential growth (p = 0.08). Of the four treated animals, three had lower growth rates post-treatment compared to controls. There was one outlier in the treatment group that showed comparable growth to controls, skewing the deviations.
Additionally, the animals underwent subsequent contrast-enhanced MR imaging the day following treatment for pre- and post-treatment comparison of the tumor. The average grayscale intensity of contrast agent in tumors was conducted across each MRI slice for each animal to measure how much contrast agent was entering tumors following treatment, as an estimate of tumor size. Pre-treatment, the average tumor grayscale intensity between control and treated groups was similar. On average, this greyscale intensity increased in the control group to a larger magnitude than in treated groups, although this was not significant (p = 0.47). This data can be seen in Table 2. The high variability of these results is potentially due to the fact that MRIs were taken only 24 h post-treatment, at which time the therapeutic potential of SDT is only beginning to occur. Even so, Figure 6 shows an example of the lesions created by SDT.
Figure 1: FUS system setup. (Top) MRgFUS System with labeled components. (Front) 1. Platform. 2. Axis motorized transducer arm. 3. FUS transducer. 4. Water bath. 5. MRI bed. 6. Monitor. 7. Transducer impedance matching box. 8. Power amplifier box. 9. Function generator. 10. Function generator channel 1 BNC port. 11. Desktop computer. (Bottom) MRgFUS system with color-coded wiring schematic with the following connections. (Back) 1. Power cords. 2. Monitor HDMI to desktop HDMI. 3. Port B USB B to desktop USB A. 4. Oscilloscope LAN ethernet to desktop ethernet. 5. Motion interface ethernet to desktop ethernet. 6. Oscilloscope aux in/out BNC to AWG input BNC. 7. Oscilloscope channel 1 (Front) BNC to SYNC BNC. 8. Matching box output BNC to RF input coaxial. 9. RF output coaxial to matching box coaxial. Please click here to view a larger version of this figure.
Figure 2: Phantom registration. (A) System setup and software during phantom registration. (B) Screenshot of the phantom registration screen, where the red circle is the selected circumference of the axial cross section. (C) Phantom placed on the MRI bed, top view. (D) Side view of the phantom, where the red line is in the axial slice corresponding to the circle in C. Please click here to view a larger version of this figure.
Figure 3: Animal placement. (A) MRI bed and cradle, with various parts labeled: 1. MRI cradle. 2. Stereotactic MRI bed. 3. Ear bars. 4. Bite bar. 5. Nose cone. 6. MRI bed peg hole. 7. Isoflurane anesthesia tubes. (B) Illustration representing the placement of the mouse on the MRI bed and placement on the cradle, with the RF coil (Orange) (illustration modified using Biorender 2022 template). (C) Illustration representing the placement of the mouse on the MRI bed during a FUS treatment (illustration modified using Biorender 2022 template). Please click here to view a larger version of this figure.
Figure 4: Focal point selection. Example of a sonication point selection in a single animal. Each column represents a T1-weighted post-contrast MRI slice where each slice is 0.5 mm in the proximal (slice 1) to distal (slice 5) direction. The tumor boundary was manually segmented and is outlined in red (row 1), and the corresponding sonication locations (row 2) are represented by a light green square (focal max center) and blue circle (half max focal circumference). Each location was sonicated for 2 min. Please click here to view a larger version of this figure.
Figure 5: Growth rate following SDT. The growth rate of GBM tumors from pre- to 24 h post-SDT treatment in treated and untreated (control) animals with intracranial M59 tumors based on the measured luminescence. Error bars indicate standard deviation. A two-sample student's T-test was performed to determine significance. Please click here to view a larger version of this figure.
Figure 6: SDT generated lesion. Pre- and post-contrast enhance T1-weighted MRI scans from an animal model featuring a representative axial slice showing a lesion in the tumor created by SDT. (Left) MRI scan taken prior to SDT treatment, with the tumor outlined in red. (Middle) FUS focal point selection where the maximum pressure is represented by light blue circles and the half-maximum pressure regions are represented by blue circles. (Right) Post-SDT MRI scans, where the tumor is outlined in red. SDT-created lesions and the syringe hole for implantation are shown. Please click here to view a larger version of this figure.
Sequence | T1 |
Repetition time | 3000 ms |
Echo time | 30 ms |
Slice thickness | 0.5 mm |
Number of slices | 25 |
Pixel spacing | 0.187 mm x 0.187 mm |
Acquisition matrix | 133 x 133 |
Averages | 4 |
Table 1: MRI settings.
Control | SDT Group | P-Value | |
Pre-Treatment | 7.49 x 103 ± 2.2 x 103 | 7.48 x 103 ± 1 x 103 | 0.99 |
Post-Treatment | 8.79 x 103 ± 7.7 x 102 | 7.95 x 103 ± 1.1 x 103 | 0.33 |
Percent Difference | 16% ± 16% | 7% ± 12% | 0.47 |
Table 2: Post-contrast enhanced T1-weighted MRI greyscale.
New therapeutic and efficacious treatment options are necessary for patients with GBM. This protocol has outlined a preclinical FUS-mediated treatment for GBM that is currently undergoing extensive investigation for clinical translation. Although SDT has exciting potential, there is still much to understand and optimize in the preclinical setting.
One of the most important components of this protocol is utilizing MR-guided FUS to target the tumor for maximal efficacy. Using a phantom, a 3D coordinate space can be created, where each pixel of axial MRI slices can be assigned a coordinate. Then, a simple procedure of selecting the sonication location on the MR image informs the transducer where to aim. The preclinical FUS system used is highly versatile and applicable when needing to target locations of specific pathology such as a tumor, including deeper seated tumors which would be hard to target without imaging confirmation. Using gadolinium as a contrast agent, there is clear visualization of the tumor, allowing the user to make informed decisions when choosing targets. The advantage that SDT has over many other treatments is that it is a tumor-specific therapy. Low-intensity FUS should only target the tumor tissue, while leaving the healthy brain parenchyma relatively untouched3,8.
The results of this experiment highlight how the advantages of this protocol can lead to therapeutic results that are similar to other findings in the literature for SDT. Figure 5 shows that within as little as 24 h following the day of treatment, there was a slowdown of tumor growth in the treated cohort. Although insignificant using this small sample size, significance might result with a larger number of animals. This delay in tumor growth is similar to what was shown in the pioneering paper on this subject by Wu et al. (2019), which exhibited slowed tumor growth over time in treated animals, as well as increased survival times9.
Considerations that were made when designing this protocol included animal strain, tumor type, and sonosensitizing agent selection. Athymic nude mice were chosen for this protocol for multiple reasons. First, the nude mouse is easier to sonicate as the lack of hair prevents any attenuation. Also, the lack of an immune system allows for the implantation of patient-derived xenografts (PDXs) so that the tumor model more closely resembles the clinical situation. The downside of using an athymic model is that the immune system cannot be characterized, so any SDT-generated immune response will not be measured in these studies10. The tumor line chosen is an aggressive and fast-growing PDX line. The time of treatment is very important because the establishment of the tumor must be verified, but the tumor burden should not fill the cranial hemisphere. Different cell lines require different incubation times to achieve an optimally sized tumor for preclinical experimentation. In this protocol, 5-ALA was used as the sonosensitizer because of its preferential uptake in GBM tumors, which has been confirmed in vitro for this cell line in previous experiments (unpublished data). Other sonosensitizers can be substituted and tested to determine the compound most suitable for efficacy and safety. Finally, treatment was commenced 3 h post 5-ALA injection, as previous literature has shown that this is the optimal time with that injection dosage5.
The FUS parameters chosen in this protocol (10 W/cm2 for 2 min at 515 kHz at each target location) were decided based on a review of previous literature and initial experiments4,9. A grid of sonication points covering the entire tumor was chosen in order to generate the ROS effect throughout the entire tumor. The intensity used here is higher than other publications, but at a short time span, this is not expected to lead to any adverse temperature-related effects, as intensities up to 25 W/cm2 have been successfully used in a mouse model without significant side effects11. Importantly, no standardized or optimized set of FUS parameters has been published in the literature. Therefore, the specific values that are reported here can be adjusted to determine the optimal set of parameters, leading to the maximal reduction of tumor tissue while maintaining safety. Additionally, as different cell lines have varying levels of vascularization and hypoxia, this treatment may need to be adjusted. We have shown overall decreased tumor growth (Figure 5) within 24 h of SDT treatment, although the parameters need to be optimized and more animals need to be tested to determine the maximal effect of this treatment. Post-treatment MRI scans show no appearance of lesions created by FUS treatment in healthy tissue, with the effect localized to tumor tissue (Figure 6). There is also the opportunity to combine SDT with other FUS techniques, such as transiently permeabilizing the blood-brain barrier, to maximize 5-ALA uptake in the tumor12. This protocol can be further supplemented by performing various histology techniques to check for safety and efficacy at the structural level. A hematoxylin and eosin (H&E) stain can be performed to check for structural or tumor damage13, while a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stain can be performed to check for cellular apoptosis14. Regardless, this protocol presents a safe and tumor-specific treatment where changes are noticeable even 24 h post-treatment, which is apparent by comparing the growth rate of tumors treated with SDT and untreated tumors, as well as comparing tumor slices before and after sonication.
With any protocol, there are always disadvantages or limitations that need to be weighed. The main limitation of the current protocol is time and expense. Meanwhile, one of the advantages of this protocol is its automated focused aim. To accomplish this focused procedure, MRI scans need to be taken for each individual animal to ensure that the targeting of the tumor is correct, a process which can be both time-consuming and expensive. Additionally, depending on the number of focal spots desired, the amount of time to perform this protocol could be hours for even just a few animals, resulting in low experimental animal numbers. Despite these drawbacks, this targeted noninvasive protocol remains a feasible preference when compared to open surgery options.
In conclusion, this protocol showed the ability of SDT treatment to decrease tumor growth in the brain within 24 h of treatment while maintaining healthy neural tissue in a preclinical mouse model. Studies of the effectiveness of SDT and optimizing the various parameters to increase ROS production are necessary to make this treatment clinically suitable. New avenues should be explored for the use of SDT as a noninvasive therapeutic model.
The authors have nothing to disclose.
The authors acknowledge funding support from the National Science Foundation (NSF) STTR Phase 1 Award (#: 1938939), by ASME Defense Advanced Research Projects Agency (DARPA) Award (#: N660012024075), and Johns Hopkins Institute for Clinical and Translational Research's (ICTR's) Clinical Research Scholars Program (KL2), administered by the National Institutes of Health (NIH) National Center for Advancing Translational Sciences (NCATS). The cells were purchased from and provided by the Mayo Foundation for Medical Education and Research.
0.5% Trypsin-EDTA | Thermo Fisher Scientific | 15400054 | |
1 mL Syringes | BD | 309597 | |
10 µL Hamilton syringe | Hamilton Company | 49AL65 | |
10 µL Pipette tips | USAScientific | ||
1000 mL Flask | Corning | MP-34514-25 | |
15 mL conical tubes | Corning | CLS430791 | |
200 Proof ethanol | PharmCo | 111000200 | |
5 mL pipettes | Falcon | 357543 | |
50 mL Conical tubes | Corning | 430290 | |
500 mL filter | Corning | 431097 | |
5-Aminolevulinic acid hydrochloride | Research Products International | A11250 | |
7T PET-MR system | Bruker | Biospec 70/30 | |
Aluminum foil | Reynolds Brand | ||
Amplifier | FUS Instruments | 2175 | |
Athymic nude mice | Charles River Laboratories | Strain Code 490 | |
Bone drill | Foredom | HP4-917 | |
Centrifuge | Thermo Fisher Scientific | 75004261 | |
Charcoal isoflourane waste container | Patterson scientific | 78909457 | |
Computer | FUS Instruments | 2269 | |
Cover glass | Fisherbrand | 12-545J | |
Desktop monitor | ASUS | VZ239H | |
D-Luciferin | Gold Biotechnology | LUCK-1G | |
DMEM | Thermo Fisher Scientific | 11965092 | |
Electronic shaver | Wahl | 93235-002 | |
Eppendorf tubes | Posi-Click | 1149K01 | |
Fetal bovine serum | Thermo Fisher Scientific | 16000044 | |
Formalin | Thermo Fisher Scientific | SF100-20 | |
Function generator | Siglent | QS0201X-E01B | |
Gadolinium contrast agent (Gadavist) | McKesson Corporation | 2068062 | |
Gauze | Henry Schein | 101-4336 | |
Heat lamp | |||
Heat pad | Kent Scientific | RT-0501 | |
Hemocytometer | Electron Microscopy Sciences | 63514-12 | |
Induction chamber | Patterson scientific | 78933388 | |
Isofluorane vaporizer | Patterson scientific | 78916954 | |
Isoflurane | Covetrus | 29405 | |
Isoflurane system | Patterson Scientific | 78935903 | |
IVIS spectrum | Perkin Elmer | 124262 | |
Lightfield microscope | BioTek | Cytation 5 | |
Nair | Church and Dwight Co. | 42010553 | |
Ophthalmic ointment | Puralube vet ointment | ||
P-20 pippette | Rainin | 17008650 | |
Patient derived xenographs | Mayo Clinic | M59 | |
Penicillin/Streptomyosin | Thermo Fisher Scientific | 10378016 | |
Phosphate buffered saline | Thermo Fisher Scientific | 70-011-069 | |
Pippetter | Drummond | 4-000-101 | |
Povidone-iodine | Covetrus | PI050CV | |
RK-50 MRgFUS system | FUS Instruments | 2182 | |
Scale | |||
Scalpel blade | Covetrus | 7319 | |
Scalpel handle | Fine Science Tools | 91003-12 | |
Screwdriver set | Jakemy | JM-8160 | |
Skin marker | Time Out | D538,851 | |
Staple remover | MikRon | ACR9MM | |
Stapler | MikRon | ACA9MM | |
Staples | Clay Adams | 427631 | |
Stereotactic frame | Kopf Instruments | 5000 | |
Stereotactic MRI prototype plastic imaging fixture | FUS Instruments | ||
T-25 culture flask | Corning | 430641U | |
Transducer and matching box | FUS Instruments | T515H750-118 | |
Ultrasonic degasser | FUS Instruments | 2259 | |
Ultrasound gel | ParkerLabs | 01-08 | |
Water bath | FUS Instruments | ||
Xylazine | Covetrus | 1XYL006 |