# A High-Throughput In Vitro Radiobiology Platform for Megavoltage Photon Linear Accelerator Studies
## 1 Introduction  
Radiotherapy is an important cancer treatment modality as more than half of cancer patients will require some form of radiotherapy during their illness [1]. Preclinical radiobiology experiments can provide valuable information about tumours and normal tissue radiation effects at the molecular level and serve as a framework to study radiation-drug combinations. These preclinical explorations may assist in developing clinical trial strategies, which can ultimately improve the outcomes of radiotherapy treatments.  
Various approaches have been used to improve the therapeutic ratio in radiotherapy [2]. A large number of novel anticancer agents targeting cancer cell signalling pathways and several immunotherapy agents are now in routine clinical use. Combining these novel agents with radiation offers many therapeutic opportunities [3]. The challenge is to suggest optimal clinical radiation-drug schedules given a large number of potential combinations, doses, and schedules of each agent.  
Practical models can help to explore this large experimental space of radiation-drug combinations, but each of them has its advantages and disadvantages [4]. In vitro models and assays are commonly employed for the preliminary screening [5, 6], and large tissue culture vessels and laboratory irradiators are widely used for this purpose [7]. These allowone to culture large numbers of cells and enable homogeneous radiation dose distributions across the tissue culture vessel. However, their large size may also mean that only a limited number of experimental conditions are possible per tissue culture vessel, which may have resource and cost implications [8]. Furthermore, when multiple tissue culture plates are used for replicate experiments, variations such as differences in incubation and drug exposure times, and exposure to different environmental conditions can lead to unintended variations between experiments. A high-throughput in vitro platform that is efficient, quick, and predictive would help to address some of these challenges.  
A high-throughput in vitro platform would help to examine human-derived tumours, which, compared to animal models, avoid variabilities due to interspecies differences, and offer an opportunity to test other cells in the tumour microenvironment, such as vascular endothelial cells, which may be important to the radiation response. Additionally, this platform would enable testing various radiation response modifiers and potentially personalising radiotherapy through mathematical modelling [9].  
Intensity-modulated radiotherapy (IMRT) is a well-established clinical radiotherapy technique where the delivery of radiation beams is dynamically modulated to create nonuniform radiation beam intensities. This nonuniform radiation beam profile potentially offers the ability to deliver different radiation doses to a single multiwell tissue culture plate, which may help to expedite the screening of numerous radiation-drug combinations. There are concerns regarding radiation beam homogeneity between and within, individual wells when multiwell tissue culture plates are used due to a lack of full scatter conditions due to air cavities and multiple tissue-air cavity interfaces that potentially introduce variability between experiments.  
We aimed to develop and validate a high-throughput in vitro radiobiology platform using IMRT and widely available clinical radiotherapy facilities for conducting radiobiology experiments within a clinical environment and to investigate beam inhomogeneity issues when using multiwell tissue culture plates.