**3. Organoids and Regeneration of Radiation-Induced Damaged Tissue**

Since the identification of Lgr5 as a marker for intestinal stem cells [42], one of the most studied and established organoid models are the gastrointestinal "mini-gut" organoids. Originally established from mouse small intestinal stem cells [43], organoid "mini-gut" models have subsequently been established from human stem cells [44], as well as from various different locations along the gastrointestinal tract, including stomach [45], colon [44] and oesophagus [46]. Furthermore, pluripotent stem cells have been utilised to successfully generate intestinal [47] and oesophageal [48] organoid cultures. These models have opened novel avenues of study for intestinal development, cancer progression [49] and other diseases, such cystic fibrosis [50].

While there have been only a limited number of studies using organoids to investigate radiation-induced gastrointestinal injury, some recent studies have used organoids to complement and reinforce important insights from in vivo mouse studies [51–53]. Wang et al. [51] demonstrated using intestinal crypt organoids that selective inhibition of radiation-induced p53-mediated apoptosis using CHIR99021, an inhibitor of glycogen synthase kinase-3 (GSK-3), can protect intestinal stem cells against radiation due to an increased survival of Lgr5+ cells. This was recapitulated in vivo, indicating a pivotal role for p53 post-translational modifications in intestinal stem cell responses to irradiation [51]. More recently, using intestinal organoids from mouse jejunum and human colon, Bhanja et al. [52] revealed the potential of BCN057, an anti-neoplastic small molecular agent, to mitigate radiation-induced gastrointestinal syndrome in normal tissue. Interestingly, BCN057 did not have a radiomitigative effect in tumour-derived organoids with these findings again mimicking in vivo findings. The same group also investigated the potential of repurposing auranofin, an anti-rheumatoid drug containing gold, as a radioprotective agent against intestinal injury [53]. In both in vivo mice and ex vivo human colon organoids treatment with auranofin significantly reduced the toxicity of radiation [53]. Furthermore, Martin et al. [54] recently demonstrated that the profile of the Lgr5+ stem cell population of the large and small intestines following irradiation of organoids could act as a marker for predicting the sensitivity of these organs to radiation. The authors validated their approach using organoids with a well-established in vivo microcolony assay which quantifies the number of regenerating crypts per small intestinal circumference [54,55]. This assay is regarded as a benchmark assay for establishing the radiosensitivity of intestinal stem cell survival and highlights the potential of intestinal organoids to predict radiation responses [54]. These studies demonstrate the strength of "mini-gut" organoids as a model for radiation studies of the gastrointestinal tract and also the opportunities for radiobiological studies in other organoid systems, particularly in tissues which lack accurate in vitro models for radiobiological studies.

Radiotherapy is used to treat the majority of head and neck cancer patients, either alone or in combination with surgery and/or chemotherapy [56]. Frequently, irradiation of head and neck tumours leads to the unavoidable co-irradiation of salivary glands, with almost half of head and neck cancer patients subsequently suffering from radiation-induced xerostomia due to hyposalivation. This drastically impacts on the quality of life of patients due to impaired chewing, swallowing, speaking and an increased risk of oral infections [57]. In vivo studies using rats have shown that sparing a region of the salivary gland which contains a high density of tissue specific stem/progenitor cells has been shown to reduce the effects of salivary gland irradiation [9]. Therapeutic options are available to stimulate salivary gland flow post-irradiation but are limited in their effectiveness [57]. Therefore, a need for a more long-term strategy for salivary gland regeneration following radiotherapy

remains [58]. While in vivo animal models have provided a wealth of knowledge as to the mechanisms behind salivary gland regeneration following injury, including radiation-induced damage [59–64], there is a limited number of in vitro systems to accurately study salivary glands following irradiation. Thus there is a growing niche for new models such as organotypic slice cultures [65] and organoids in the area of salivary gland radiation research.

Recently, our group has established protocols for the isolation and expansion of both murine [66] and human [67] submandibular salivary gland stem/progenitor cells. Using these protocols, we have shown that transplantation of enriched murine or human stem/progenitor cell populations improved functional readouts of irradiated mice salivary glands [37,67,68]. However, this effect may not only be directly from the expansion of the stem/progenitor cells in the transplanted tissue, but also due to paracrine effects of the transplanted cells acting on the recipient tissue [67]. Another recent study by Tanaka et al. has demonstrated the ability to derive salivary gland stem cells from embryonic stem cells [69]. Upon transplantation into parotid gland-defective mice, the induced salivary gland cells (transplanted either alone or together with mesenchymal cells) were capable of generating mature salivary gland tissue. The newly generated tissue was also shown to be functional as demonstrated by an increased saliva secretion in transplanted mice [69]. Combined, these studies hold significant preclinical promise for studying the mechanisms behind salivary gland regeneration and amelioration of salivary gland damage, both irradiation and non-irradiation induced damage [67,69]. However, the translation of any embryonic stem cell derived treatment [69] to a clinical application is always likely to be hindered by ethical concerns [70] and safety concerns regarding tumorigenicity [71].

Our models have been successfully utilised to study the survival responses of salivary gland stem/progenitor cells [26]. The salivary gland stem/progenitor organoids demonstrated a disproportionate sensitivity to low dose of radiation which was recapitulated in a functional low dose sensitivity in vivo [72]. While low dose hypersensitivity is not a new phenomenon [73,74], this was the first study to show the relevance of this phenomenon in stem/progenitor cells, with a potential clinical relevance. Furthermore, we have recently developed a protocol for the culturing of parotid salivary gland organoids and demonstrated that parotid gland stem cells display a similar radiosensitivity as those of submandibular salivary glands [75]. Importantly, as organoids are derived from stem/progenitor cell populations, they allow for the study of a more stem/progenitor specific response. As stem/progenitor cells play a prominent role in tissue regeneration following irradiation, models which allow for the understanding of these cells are crucial to protecting these tissues.

Another tissue in which the use of radiation is highly limited due to radiation-induced toxicity is the liver. Along with lung, breast, colorectal and pancreatic cancers, liver cancer deaths are one of the highest of all cancer-related deaths each year [76], while the prognosis is extremely poor due to limited treatment options [77]. The use of radiation treatment for liver cancer is severely hindered by the development of radiation-induced liver disease [78], a consequence which can also impede the utilisation of radiotherapy for other abdominal tumours in proximity to the liver, such as gastrointestinal cancers [79]. Much of what is known regarding radiation-induced liver disease is from retrospective clinical studies [79], as current lab models for studying it are limited with in vitro studies generally limited to cell lines lacking cellular heterogeneity and functionality. The recently developed models of both mouse [80] and human [81] derived liver organoid cultures from tissue resident stem cells, as well as pluripotent stem cell-derived liver cultures [82–85], may represent an ideal model for studying radiation-induced liver disease in the future. These models display cellular, functional activity and have structural organisation, while they have been successfully utilised to study genetic liver disorders mimicking the clinical pathology [81] and drug-induced liver injury [86]. Understanding the mechanisms of radiation-induced liver disease may eventually allow for increased treatment options for liver cancers.
