**7. Discussion and Conclusions**

Although irradiation techniques have improved over time, the incidence of radiation cystitis still poses a real problem for clinical management. Indeed, the management of radiation cystitis, especially in the late-stage, is based largely on symptomatic treatments. This was historically explained by the (theoretical) irreversibility of late-stage histological fibrotic lesions. Despite encouraging results, evidence that radiation cystitis can be modulated pharmacologically is insufficient and requires further confirmation as these findings are based only on small sample sizes or on retrospective analyses. The pathogenesis of fibrotic diseases remains a major challenge, due not only to the variety and multiplicity of initiating events but also to a large number of profibrotic mediators involved. Nevertheless, MSCs appear to be a promising therapeutic alternative for the treatment of fibrosis

in chronic radiation cystitis. The pro- or antitumor effects of biotherapies using MSCs have been widely discussed in the literature and are one major parameter that must be better understood before clinical application [108–110]. Other stem cells may be promising treatments of hemorrhagic cystitis. The placenta is a potential source of stromal cells, with decidual stromal cells (DSCs). These stem cells are easily amplified in vitro and have greater immunosuppressive potential than BM-MSCs. DSCs inhibit alloreactive T cell proliferation better than stromal cells from other sources and induce coagulation more effectively than BM-MSCs. Iterative infusions can be considered in patients with inflammatory pathologies [111–113]. In preclinical studies, compared to bone marrow-derived MSCs, DSCs had better viability [114]. Their clinical use must, however, be optimized. It should be noted that stromal cell injections must be carried out only in a patient with a long complete remission to limit their potential implantation near dormant cancer cells. Such safety issues may limit the use of MSC in clinics. MSC paracrine action is widely described in the literature [115,116]. To limit the implantation of these cells, it is possible to use the extracellular microvesicles (Evs-MSCs) that they secrete to reduce radiation-induced lesions, including fibrosis, without exposing patients to the risk of cancer reactivation [117–119].

To increase the likelihood of finding a potential preclinical treatment for radiation cystitis, it is imperative to test novel mitigators of the radio-induced inflammatory reaction. For example, TCDO/WF10 is a chemically stabilized chlorite matrix that has previously been shown to have a positive effect in the context of chronic inflammatory conditions. It induces natural immunity and stimulates cellular defense mechanisms through its actions on natural killer cells, cytotoxic T lymphocytes, and modification of the monocytemacrophage system. It reduces inflammation quickly so that healing can begin [120,121]. In an early-stage clinical study, 20 patients with grade 3 radiation cystitis received intravenous TCDO treatment for 5 consecutive days. From 1 to 9 months after TCDO treatment, patients had no recurrent bleeding, and no side effects from treatment with TCDO were observed [122]. In a phase-II study involving 100 patients, Veerasarn et al. evaluated the effectiveness of WF10 in combination with standard care compared to standard care alone. The complete resolution rate for hematuria was comparable in both arms (74% vs. 64% in the experimental arm and in the standard arm, respectively). However, a significant reduction in the relapse of hematuria was noted among responders in the experimental arm (47% vs. 77%, *p*-0.01). No severe adverse events were reported [123,124].

To date, no preclinical treatment without reconstructive surgery appears to fully restore the function and structure of the bladder after radiation exposure. A number of preclinical studies have described strategies for limiting fibrosis (cf. Table 1). The majority of studies have been carried out in mice on a model of radio-induced pulmonary fibrosis. Those were mitigating and continuous treatments (isoflavone mixture gavage, oral clarithromycin) that were evaluated with respect to the inflammation and migration of immune cells, including macrophages, and the development of fibrosis [81,125]. Clarithromycin (CLA) administration, before and after lung radiation exposure, reduced expression levels of TNF-α, TNFR1, TNFR2, TGF-β1, CTGF and type I collagen, and inhibits both the increased acetylation of NF-κB p65 and the elevated expression of COX-2 with reduction of both fibrosis and macrophage infiltration [81]. To reduce fibrosis, as described by Chung et al., it is necessary to target type 2 cytokines such as IL13, limiting recruitment and polarization of Ym1/Chi3L3-positive macrophages, which are alternatively activated in the lungs following thoracic irradiation. Intraperitoneal injection of IL-13 neutralizing antibody does not completely suppress radiation-induced TGF-β expression, suggesting that sustained IL-13 or TGF-β neutralization therapy may be necessary to durably mitigate fibrotic progression [84]. Rapamycin is a potent immunosuppressive drug used in solid organ transplantation for the prevention of allograft rejection. In oncology, mTOR (mammalian target of rapamycin) inhibitors are currently being evaluated in several types of cancers. Targeting mTOR signaling may provide a therapeutic option for radiation-induced lung injury. Indeed, mTORC2 activity has been described in the non-canonical signaling of TGF-β, and mTORC2 inhibitors could thus be of interest in the case of fibrosis [126].

In rats, melatonin or metformin administration before irradiation using a <sup>60</sup>Co source of gamma rays in the thoracic area (i.e., heart and lungs) helped prevent the infiltration of macrophages and lymphocytes, as well as the upregulation of IL-4, IL4ra1, Duox1, andDuox2 [83,127]. Furthermore, metformin treatment could stimulate the activity of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione (GSH) [128]. Other antioxidants, such as fucoidan, have been orally administered preclinically after whole lung irradiation at 10Gy, mitigating different proteins (TIMP-1, CXCL1, MCP-1, MIP-2, and IL-1Ra) expression in pleural fluid, decreasing pleural fluid accumulation and reducing neutrophil and macrophage infiltration in lung tissues. Fucoidan changed the expression patterns of inflammatory cytokines, which may consequently attenuate lung fibrosis [129,130].

Many molecular mechanisms still need to be better understood in order to develop a targeted treatment for fibrosis and radiation cystitis. It is, therefore, necessary to broaden our knowledge of myeloid and lymphocytic dynamics in the development of this fibrosis induced after pelvic radiotherapy, based on data obtained in other models (ex: lung fibrosis). It is also necessary to follow a step-by-step translational development to ensure that patient outcome may be improved, through the integration of robust biomarkers of toxicity, as well as through the implementation of modern radiotherapy tools in clinical research to minimize the doses to organs at risk, including the bladder, and therefore improve the therapeutic index. The current knowledge on the biological processes involved in late radiation toxicity clearly suggests that it is indeed easier to prevent late toxicity than to reverse [131].

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

