Novel Therapeutic Approaches to Treat Brain Cancer Combining 3D Cell Culture Models, Cold Atmospheric Plasma and Airborne Acoustic

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

3D mammalian cell culture models in toxicology testing

3D cell culture can be successfully used as an alternative to laboratory animals, and as a cost effective and time-saving tissue culture technique, which also reduces the trial period for drug testing

U-251MG Spheroid generation using low attachment plate method protocol

3D cell culture is a process used to grow cells in vitro to mimic an in vivo environment. 3D cell models are very useful for understanding disease mechanisms and exploring drug therapeutics. 3D cultures can be grown from cells taken from cancer organoids in patients. Once grown, they can be used to screen for small molecule drugs or they can be genetically modified in order to analyse disease pathways or predict the toxicity or efficacy of a drug treatment. These cultures decrease the need to use animals in research and provides more reliable results as it uses human physiology. This protocol describes the in vitro generation of spheroids using the low attachment plate method. This method uses low-adhesion plates that are coated with hydrophilic polymer to allow cells to cluster together, forming their own extracellular matrix, rather than sticking to the plate surface. The scaffold-free 3D cell culture models produced can more accurately reflect an in vivo microenvironment making them useful in the study of oncology, hepatotoxicity, neurology, nephrology and stem cell biology

U-251MG Spheroid generation using a scaffold based method protocol

3D cell culture is a technique that is used to grow cells in vitro that will mimic an in vivo environment. 3D cell models are a helpful learning tool for researchers to better understand disease mechanisms and to explore different therapeutic properties of drugs. 3D cell cultures can be developed using patient derived cancer cells. Once they have been grown, these 3D cells can be used to screen for small molecule drugs or for genetic modification in for analysis of disease pathways or to predict drug treatments toxicity or efficacy. 3D cell cultures are a big step towards the more ethical testing of drug toxicity and efficacy as they decrease the need to use animals in research as well as providing more reliable results as the cells used are of human physiology. Cellusponge are 3D porous hydroxipropylcellulose scaffolds that are designed for use with cells that do not require specific ligands. As well as the standard non-coated cellusponge, there are two more of the same type of scaffold available for use that are made with two different coatings to allow for improved adaptation of different cell types, these are called Cellusponge-Gal and Cellusponge-Col. Cellusponge is a no-coating approach that is intended for use in the development of general soft tissue 3D culture. It has been used as soft matrix for 3D cell culture and 3D tumour model

U-251MG Spheroid Generation Using Hanging Drop Method Protocol

The use of 3D cell culture has been a major step in developing cellular models that can mimic physiological tissues. Traditional 2D cell cultures are often unable to accurately represent the cellular functions and responses that are present in tissues, as a result, research findings based on 2D cultures tend to be skewed with limited predictive capability. 3D cell cultures can be grown from cells obtained from cancer organoids in patients. These models are useful for understanding disease mechanisms and exploring drug therapeutics in areas such as toxicity and efficacy. In order to gather more physiologically relevant data, a variety of 3D cell culture techniques have been developed to mimic the in vivo characteristics of physiological tissues. This protocol describes in vitro generation of U-251MG spheroids using the hanging drop method. Advantages of using hanging drop plate method are, able to produce uniform size spheroids, low cost, comfortable to handling and suitable for short term culture. The main downside of this method is medium change, different drug treatment at different time points are impossible and labor intensive. This method uses the Perfecta3D hanging drop plate, a novel cell culture device that simplifies the process of spheroid formation, testing and analysis. Rather than having to invert the plates which often results in spillage or detachment, these plates are designed to create hanging drops using a plateau structure at the bottom of the plate

Three-Dimensional (3D) In Vitro Cell Culture Protocols to Enhance Glioblastoma Research

Three-dimensional (3D) cell culture models can help bridge the gap between in vitro cell cultures and in vivo responses by more accurately simulating the natural in vivo environment, shape, tissue stiffness, stressors, gradients and cellular response while avoiding the costs and ethical concerns associated with animal models. The inclusion of the third dimension in 3D cell culture influences the spatial organization of cell surface receptors that interact with other cells and imposes physical restrictions on cells in compared to Two-dimensional (2D) cell cultures. Spheroids’ distinctive cyto-architecture mimics in vivo cellular structure, gene expression, metabolism, proliferation, oxygenation, nutrition absorption, waste excretion, and drug uptake while preserving cell–extracellular matrix (ECM) connections and communication, hence influencing molecular processes and cellular phenotypes. This protocol describes the in vitro generation of tumourspheroids using the low attachment plate, hanging drop plate, and cellusponge natural scaffold based methods. The expected results from these protocols confirmed the ability of all these methods to create uniform tumourspheres

Advances in 3D culture systems for therapeutic discovery and development in brain cancer

This review focuses on recent advances in 3D culture systems that promise more accurate therapeutic models of the glioblastoma multiforme (GBM) tumor microenvironment (TME), such as the unique anatomical, cellular, and molecular features evident in human GBM. The key components of a GBM TME are outlined, including microbiomes, vasculature, extracellular matrix (ECM), infiltrating parenchymal and peripheral immune cells and molecules, and chemical gradients. 3D culture systems are evaluated against 2D culture systems and in vivo animal models. The main 3D culture techniques available are compared, with an emphasis on identifying key gaps in knowledge for the development of suitable platforms to accurately model the intricate components of the GBM TME

Plasma induced reactive oxygen species-dependent cytotoxicity in glioblastoma 3D tumourspheres

The aim of this study was to determine the effects of a pin‐to‐plate cold atmospheric plasma (CAP) on U‐251 MG three‐dimensional (3D) glioblastoma spheroids under different conditions. 3D tumorspheres showed higher resistance to the CAP treatment compared to 2D monolayer cells. A single CAP treatment was able to induce cytotoxicity, while multiple CAP treatments augmented this effect. CAP was also able to induce cytotoxicity throughout the tumoursphere, and we identified that reactive oxygen species(ROS) plays a major role, while H2O2plays a partial role in CAP‐induced cytotoxicity in tumour-spheres. We conclude that ROS‐dependent cytotoxicity is induced uniformly throughout glioblastoma and epidermoid tumourspheres by direct CAP treatment

Ultrasound 96 Probe Device Protocol for cancer cell treatment

Ultrasound is a sound wave with frequencies ranging between 20 kHz and 20 MHz. Ultrasound is able to temporarily and repeatedly open the BBB safely and enhance chemotherapeutic delivery without adverse effects. This novel technique in drug delivery benefits from the powerful ability of ultrasound to produce cavitation activity. Cavitation is the generation and activity of gas-filled bubbles in a medium exposed to ultrasound. As the pressure wave passes through the media, gas bubbles expand at low pressure and contract at high pressure. This leads to oscillation which produces a circulating fluid flow known as microstreaming around the bubble with velocities and shear rates proportional to the amplitude of the oscillation. At high amplitudes the associated shear forces can cut open liposome