Sonodynamic Therapy for the Treatment of Glioblastoma Multiforme in a Mouse Model Using a Portable Benchtop Focused Ultrasound System

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 untouched^3,8.

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 experiments^4,9. 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 declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Amir Manbachi teaches and consults for BK Medical (GE Healthcare), Neurosonics Medical, and is an inventor on a number of patent-pending FUS technologies. Betty Tyler has research funding from NIH and is a co-owner of Accelerating Combination Therapies*. Ashvattha Therapeutics Inc. has also licensed one of her patents, and she is a stockholder for Peabody Pharmaceuticals (*includes equity or options).

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.