**1. Introduction—Optimising the Therapeutic Window of Radiation Treatment**

With an ever-aging population the number of people diagnosed with cancer is constantly growing [1]. Therefore, there is an even greater onus on the need to develop both current and new methods to enhance the efficacy of cancer treatments. Traditional cancer treatments, such as radiotherapy, chemotherapy and surgery, are still the most common modalities, but newer treatments such as immunotherapy are becoming more and more prevalent. Radiotherapy (either alone or in combination with surgery and/or chemotherapy) is used to treat over half of all cancer patients, with a curative intent in the majority of these cases [2,3]. Furthermore, the number of patients undergoing radiotherapy is predicted to increase even further due to an aging and growing population, as well as rapid technological advances in radiotherapy delivery practices [4]. The primary goal of radiotherapy, as with all other forms of cancer treatment, is to maximise the therapeutic window. The therapeutic window describes the balance between the probability of increasing tumour cell kill while minimising the probability of normal tissue complications. This can be achieved by using drugs which target the intrinsic vulnerabilities of a tumour to make it more susceptible than healthy tissue, or alternatively by physically targeting the tumour with greater accuracy and minimising the co-irradiated normal healthy tissue (Figure 1).

**Figure 1.** Optimising the therapeutic window of radiotherapy. The therapeutic window describes the balance between the probability of tumour control (blue line) and normal tissue complications (yellow line). There are three main rationales behind broadening the therapeutic window in radiation treatment: (1) Increasing tumour sensitivity using radiosensitisers, reducing the dose required for tumour kill (blue line shifts to the left), (2) protecting normal tissue using radioprotectors or mitigators, thus increasing the tolerable dose of normal tissue (shifting the yellow line to the right) or (3) high precision dose delivery which can reduce the volume of co-irradiated normal tissue (effectively shifting the yellow line to the right) while in the case of charged particles an increased relative biological effectiveness reduces the dose required for tumour control. Abbreviations: PARP; poly-ADP ribose polymerase, ATM; ataxia telangiectasia mutated, TGF-β; transforming growth factor beta, IMRT; intensity-modulated radiation therapy, SBRT; stereotactic body radiation therapy, MRI; **Figure 1.** Optimising the therapeutic window of radiotherapy. The therapeutic window describes the balance between the probability of tumour control (blue line) and normal tissue complications (yellow line). There are three main rationales behind broadening the therapeutic window in radiation treatment: (1) Increasing tumour sensitivity using radiosensitisers, reducing the dose required for tumour kill (blue line shifts to the left), (2) protecting normal tissue using radioprotectors or mitigators, thus increasing the tolerable dose of normal tissue (shifting the yellow line to the right) or (3) high precision dose delivery which can reduce the volume of co-irradiated normal tissue (effectively shifting the yellow line to the right) while in the case of charged particles an increased relative biological effectiveness reduces the dose required for tumour control. Abbreviations: PARP; poly-ADP ribose polymerase, ATM; ataxia telangiectasia mutated, TGF-β; transforming growth factor beta, IMRT; intensity-modulated radiation therapy, SBRT; stereotactic body radiation therapy, MRI; magnetic resonance imaging. Created with BioRender.com.

magnetic resonance imaging. Created with BioRender.com.

The development of high precision means of dose delivery, such as intensity modulated radiation therapy [5], stereotactic radiation therapy [6] and charged particle radiotherapy [7], have allowed for substantial reductions in co-irradiated normal tissue during therapy. These strategies enable better sparing of crucial organs [8] or sub-regions [9] within organs during treatment or dose escalation to the tumour. Furthermore, real-time advanced imaging, such as magnetic resonance imaging (MRI), during radiation therapy has been suggested as a means to further optimise the delivery of radiation to the target tumour with an increased sparing of the surrounding healthy tissue. Initial in vitro studies showed no changes in survival in response to X-rays when a magnetic field of 1.5 T was applied [10]. Indeed, combining X-ray therapy with MRI-guidance has been successfully applied in clinical practice to increase the accuracy of dose delivery and thus spare a greater proportion of healthy tissue [11]. Particle therapies, such as proton therapies, can modulate the dose to encompass the whole tumour in a so-called "spread-out Bragg peak" with a minimised entrance dose and negligible exit dose, sparing healthy tissue [12,13]. Furthermore, MRI-guided proton therapy has also been proposed [14,15] and early in vitro findings suggest that a magnetic field perpendicular to the radiation beam has no effect on the radiobiological effectiveness of the dose The development of high precision means of dose delivery, such as intensity modulated radiation therapy [5], stereotactic radiation therapy [6] and charged particle radiotherapy [7], have allowed for substantial reductions in co-irradiated normal tissue during therapy. These strategies enable better sparing of crucial organs [8] or sub-regions [9] within organs during treatment or dose escalation to the tumour. Furthermore, real-time advanced imaging, such as magnetic resonance imaging (MRI), during radiation therapy has been suggested as a means to further optimise the delivery of radiation to the target tumour with an increased sparing of the surrounding healthy tissue. Initial in vitro studies showed no changes in survival in response to X-rays when a magnetic field of 1.5 T was applied [10]. Indeed, combining X-ray therapy with MRI-guidance has been successfully applied in clinical practice to increase the accuracy of dose delivery and thus spare a greater proportion of healthy tissue [11]. Particle therapies, such as proton therapies, can modulate the dose to encompass the whole tumour in a so-called "spread-out Bragg peak" with a minimised entrance dose and negligible exit dose, sparing healthy tissue [12,13]. Furthermore, MRI-guided proton therapy has also been proposed [14,15] and early in vitro findings suggest that a magnetic field perpendicular to the radiation beam has no effect on the radiobiological effectiveness of the dose [16], while a magnetic field longitudinal to the beam slightly changes the effectiveness [17], emphasising the potential of such advances in a clinical setting.

[16], while a magnetic field longitudinal to the beam slightly changes the effectiveness [17], emphasising the potential of such advances in a clinical setting. Further advances in radiation Further advances in radiation delivery include FLASH radiotherapy, which delivers ultra-high dose rates of ionising radiation which are believed to reduce normal tissue complications compared to conventional dose rates [18], although the therapeutic window of FLASH therapy still needs to be addressed [19].

All of these technological advances in the field of radiation beam delivery have significantly reduced the amount of co-irradiated healthy tissue during radiation treatment; however, none of these developments can completely eliminate dose to the surrounding tissue. Therefore, it is still necessary to develop in vivo and in vitro models to improve understanding of the mechanisms involved to better protect and/or regenerate normal tissue or to target intrinsic vulnerabilities of a tumour to enhance radiotherapy efficacy. These models should also take the therapeutic window into account as there is often an overlap between these mechanisms in both normal tissue and tumours, a feature which is regretfully often overlooked. Here we discuss one such in vitro model which could potentially allow the comparison of normal tissue and tumour responses at a patient-specific level, organoids, and the ever-growing role it has in radiobiological studies. We examine the strength of organoids in mechanistic studies in both normal and diseased tissue, but also examine the prospects of organoids in a more personalised medicine approach for patients. Finally, we discuss (potential) developments within the field of organoid research that could further benefit the radiobiology world.
