Glioblastoma (GBM), an adult-type diffuse grade 4 glioma (IDH wild type), is the most prevalent, aggressive, fatal, highly vascularized, malignant primary brain tumour in adults with a poor prognosis. Despite existing therapies such as surgical resection, radiation therapy and chemotherapy such as temozolomide (TMZ), patient survival remains largely unchanged over the last three decades. There is an urgent need for novel and effective therapeutic strategies that can overcome drug resistance, cross the blood brain barrier, and minimise off-target side effects that can negatively impact a patient\u27s quality of life. The high failure rate of clinical trials is due to inefficient treatment methods and imperfect pre-clinical models, which limit our ability to predict efficacy and toxicity in humans. Thus, the aim of this project is to investigate the effectiveness of non-thermal therapies, such as cold atmospheric plasma (CAP), ultrasound (US), and plasma microbubble (PMB) (alone or in combination) for the treatment of GBM using in vitro three-dimensional (3D) tumour spheroid models to closely mimic the natural in vivo environment, shape, and cellular response.
In an effort to mitigate the issue of inaccurate pre-clinical therapeutic outcomes resulting from imperfect pre-clinical models, we have optimized and integrated the usage of 3D tumour spheroid models into our research using the low attachment plate, hanging drop plate, and scaffold-based approaches. During efforts to address the issue of inefficient treatment methods, we found out that the use of novel therapeutic methods such as CAP (alone), US (alone), and the combination of US and CAP / PMB treatments can effectively induce 3D GBM and epidermoid tumour spheroid cell death in a time-, dose-, treatment frequency-, and reactive oxygen species- dependent manner. Additionally, these single or synergistic treatments were also able to significantly reduce 3D GBM spheroid regrowth cell proliferation, growth metabolic and while induce, cytotoxic effects, DNA double strand breaks, damage to the tumour sphere\u27s cell membrane, spheroid shrinkage, and damage to the tumour microenvironment (TME). We also found out that CAP (alone), PMB (alone) and in combination of US treatments were able to induce cytotoxicity throughout the tumour sphere, likely via long-lived reactive oxygen and nitrogen species (RONS) (H2O2, NO2-, and NO3-) and also other reactive species, with multiple treatments augmenting this cytotoxic effect. The combination of US and CAP has a synergistic effect that leads to higher cytotoxicity in 3D tumour sphere models compared to either CAP or US alone, and this effect is dependent RONS. Single treatments of CAP and US activate the JNK signaling pathway, while multiple treatments can trigger multiple cell demise pathways, including caspase-dependent, JNK-dependent, and calpain-mediated cell death. Our study on drug delivery demonstrated that combining US and TMZ enhances the cytotoxicity of GBM and epidermoid carcinoma in 3D tumour spheres compared to two-dimensional (2D) cells. We used doxorubicin as a reporter to show that US improves drug diffusion in 3D models and drug uptake into cells in tumour spheres, leading to enhanced cytotoxicity that is not observed in 2D culture models, where the cells are exposed to drug directly and the effects of sonoporation are minimal.
These findings set an important limitations on the likely approach needed when translating CAP / US / PMB into a clinical settings and also emphasize the importance of using 3D cell culture models in pre-clinical research, as relying solely on 2D cell culture models followed by animal testing and clinical trials has resulted in a 95% failure rate due to inadequate prediction of human efficacy and toxicity
