*6.1. Preclinical Studies of Radiation-Induced Cystitis*

trated in Table 1, the radiation exposure is mostly delivered as a single dose via an X-ray or gamma-ray irradiator. However, it appears more appropriate to use X-rays to study the effects of radiotherapy on tissues. For example, the SARRP (Small Animal Radiation Animal models of radiation cystitis are preferably performed in rodents. As illustrated in Table 1, the radiation exposure is mostly delivered as a single dose via an X-ray

or gamma-ray irradiator. However, it appears more appropriate to use X-rays to study the effects of radiotherapy on tissues. For example, the SARRP (Small Animal Radiation Research Platform) is one of the X-ray irradiators used in preclinical research. These small animal radiotherapy devices enable state-of-the-art image-guided therapy (IGRT) research to be performed by combining high-resolution cone-beam computed tomography (CBCT) imaging with an isocentric irradiation system [69]. This radiation fractionation is, however, not clinically relevant, as most pelvic therapeutic irradiations are delivered through fractionated schemes in patients. Among the factors inducing fibrosis, Th-2 cytokines were among the first to be recognized to have strong profibrotic properties. Typical cytokines released from Th-2 cells are IL-4, IL-5, IL-10 and IL-13. Three of them, IL-4, IL-5 and IL-13, are linked to fibrosis development [70]. A few months after irradiation of the bladder, degenerative epithelial tissue, urothelial swelling, pseudo-carcinomatous epithelial hyperplasia, fibrous tissue in the lamina propria and between muscle cells, a mild increase in inflammatory cells, disruptions in tight junction formation, edema, loss of endothelial cells, urothelial hyperplasia, and bleedings (in the most severe cases) were detected [71]. As described in Table 1, three preclinical treatments were shown to reduce the development of radiation cystitis, including Hyperbaric oxygen therapy (HBOT), liposomal and tacrolimus instillations and also vasculogenic and angiogenic localized therapies [72–74]. For HBOT, 14 days after radiation, rats were treated in the chamber (95% oxygen and pressurized to 200 kPa for 90 min), twice daily, for a period of two weeks. This therapy reduced radiation oxidative stress and TGF-Beta and consequently lowered levels of IL-10. Using endothelial cells as a vasculogenic therapy and vascular endothelial growth factor (VEGF) as an angiogenic therapy wasbeneficial in the early chronic phase. But this angiogenic therapy using endothelial cells could promote tumor revascularization, although routine endothelial cells culture is still limited [75,76]. It is well documented that ionizing radiations activate the Nuclear Factor κB (NF-κB) signaling cascade directly or via induction of double-strand breaks and oxidative stress [77]. The NF-κB pathway is a link to the immune system in radiation response [78]. Thalidomide, an immunosuppressive drug thatinterferes with the activation of NF-κB, may be a valid treatment option for patients with inflammatory diseases refractory to other first- and second-line treatments. Considering the immunomodulatory effect of thalidomide, Kowaliuk et al. recently investigated the role of NF-κB and the functional effects of this treatment on radiogenic bladder dysfunction. Early thalidomide infusion after pelvic irradiation using a YXLON MG325 X-ray device showed beneficial and promising effects on the incidence and severity of bladder dysfunction [79]. The late administration of thalidomide showed no significant effect on functionality with possible neurological side effects, limiting its use [80]. Oral administration of clarithromycin or isoflavone before and after irradiation results in the anti-inflammatory macrophage subtype switch and reduction of macrophage infiltrate, respectively [81,82]. Intraperitoneal injection of Melatonin before radiation reduces lymphocytic and macrophagic infiltrates [83]. Moreover, Intraperitoneal injection over 8 weeks of Purified murine anti-IL-13 IgG antibody, starting 3 Week post radiation exposure participate ininhibition of polarization of alternatively M2-macrophages, also after Iterative IV infusion 5 × 10 Adipose -MSCs/infusion [84,85]. To increase the chances of finding a potential preclinical treatment for radiation cystitis, it is imperative to explore novel mitigators of radio-induced inflammatory reactions.

**Table 1.** Recent animal models for preclinical studies of radiation cystitis (RC) and preclinical studies targeting immune cells to limit the development of radio-induced fibrosis.


#### *6.2. Stem Cell Therapy: A New Therapeutic Avenue*

Because of their ability to migrate to the irradiated site and of their immunomodulatory and antioxidant properties in promoting tissue repair, mesenchymal stem (or stromal) cells (MSCs) are a potential antifibrotic therapeutic candidate [86–90]. Preclinical studies have described their beneficial effects, in particular their ability to limit the development of pulmonary and colorectal after irradiation by modulating the polarization of macrophages. From these investigations, it seems that MSCs could not only replace damaged epithelial cells but also promote tissue repair through the secretion of anti-inflammatory and antifibrotic factors [85,91,92]. However, it is important to note that these studies were performed on non-cancerous models. In a recent preclinical study of radiotherapy to treat colorectal cancer, it was shown that treatment with bone marrow (BM)-derived MSCs significantly reduced both cancer initiation and cancer progression by increasing the number of tumorfree animals as well as decreasing the number and the size of the tumors by half, thereby extending their lifespan. The attenuation of cancer progression was mediated by the capacity of the MSCs to modulate the immune component. The MSCs reprogrammed the macrophages to become regulatory cells involved in phagocytosis, thereby inhibiting the production of proinflammatory cytokines. Thus in the long term post-radiotherapy, this biotherapy allows the maintenance of tissue homeostasis and inhibits tumor progression [93]. MSCs inhibit fibrosis by reducing the expression of TGF-*β*1, modulating the inflammatory response, apoptosis, oxidative stress and remodeling of the extracellular matrix. In particular, preclinical studies have shown that MSCs could act on fibrosis by directing the polarization of macrophages and the differentiation of CD4+ T lymphocytes [94–96]. In response to signals derived from tissue damage, macrophages undergo reprogramming, which leads to the emergence of a spectrum of distinct functional phenotypes (Figure 2). A study by Chen et al. showed that MSCs couldpromote M2 macrophage polarization by secreting TGF-β3 and TSP1 [97]. Recent publications have shown that MSCs could induce M2 macrophages through the secretion of exosomes, and these effects could be due to the activation of transcription factors Stat6, MafB [98] and the secretion of miR-223 targeting PKNOX1 in macrophages [99]. These regulatory mechanisms are involved in acute inflammation. However, in the case of chronic radiation cystitis, fibrosis is triggered by chronic inflammation. MSCs could inhibit chronic inflammation by altering the polarization of macrophages to resolve chronic inflammation through the secretion of exosomes containing miR let-7b [100]. Moreover, HGF and TSG-6 have been shown to be major effectors of the antifibrotic activity of MSCs in several models (e.g., cutaneous and renal fibrosis [101–103]. HGF has been shown to be up the urine of prostate cancer survivors with a radiation history [104]. HGF could potentially play a dual role in radiation cystitis whereby it promotes angiogenesis and is protective against fibrosis [105]. TSG-6 is able to form hyaluronan polymers, which trigger the activation of NF-κB and the subsequent acquisition of the M1 phenotype [82]. Thus, TSG-6 could act as a negative regulator of M2 activity by promoting the availability of hyaluronan. As prolonged M2 activity has previously been associated with worsening fibrosis, newly secreted TSG-6 could be a major regulator of inflammation after MSC transplantation [106,107].
