*4.2. MSCs in Radiation-Induced Intestinal Injury*

Radiation-induced intestinal injury (RIII) or radiation enteropathy develops in RTtreated patients with abdominal or pelvic tumors. About 60–80% of patients have nausea, abdominal pain, and diarrhea within 2–3 weeks of RT [70]. Such symptoms usually disappear within 1–3 months of completing therapy. However, a few patients may experience delayed RIII, including disorders in intestine motility and nutrient absorption. Some severe chronic RIII may progress to intestinal obstruction or perforation and fistulae formation. The pathological changes in acute RIII involve inflammation reaction and consequent crypt cell death [71,72]. On the other hand, chronic RIII is more complex and is characterized

by mucosa atrophy, intestinal wall fibrosis, and microvascular sclerosis [70]. Numerous preclinical studies utilizing natural products [73], peptides [72], and small molecules [74] to alleviate RIII have been carried out. However, researcher have not yet reached a consensus on the clinical application. Amifostine, a free-radical scavenger, is the earliest drug proved by the FDA to mitigate radiation therapy-related injury [75]. Nevertheless, the narrow treatment time window and lingering concerns of amifostine hinder its clinical uses [76]. Moreover, the US FDA has approved Neupogen and Neulasta in 2015 and leukine in 2018 for acute radiation syndrome [77]. Thus, novel therapeutic strategies are eagerly needed, especially drugs specific for each radiation-induced organ injury.

MSCs were initially found to migrate and settle in the injured intestine after RT [78]. Lately, studies revealed that the transplanted MSCs can reverse the disrupted intestinal function by RT [79,80]. Such benefits were attributed to the MSCs secretome-mediated intestinal regeneration via inflammation inhibition, neovascularization, and epithelial homeostasis maintenance [81]. Additionally, there exist specific stem cells in the intestinal crypt responsible for intestinal repair and regeneration [82]. BM-MSCs (1 <sup>×</sup> <sup>10</sup><sup>6</sup> ) transplantation via tail vein injection was found to increase Lgr5<sup>+</sup> intestinal stem cell populations, thus facilitating the repair of radiation-induced intestinal injury via activated Wnt/β-catenin signaling [83]. Based on the excellent paracrine effect, MSCs-CM were also applied to preclinical experiments of RIII. Repeated injection of AT-MSCs-CM (abundant angiogenic factors such as IL-8, angiogenin, HGF, and vascular endothelial growth factor) promoted intra-villi microvascular recovery in the irradiated intestine via activating the PI3K/AKT signal pathway [84]. Nevertheless, MSCs cultured under normal conditions only secrete slight cytokines that may possess unsatisfactory therapeutic potential. Given this, Chen et al. pretreated BM-MSCs with pro-inflammatory factors (TNF-α, IL-1β, nitric oxide) and found an enhanced paracrine effect of MSCs, primarily represented by the secretion of IGF [85]. The pretreated BM-MSCs-CM exhibited a more significant therapeutic efficacy in modulating inflammatory responses and mediating epithelial regeneration [85]. Moreover, other modifications such as carrying foreign genes (HGF, CXCL12) or cytokines (R-Spondin1) and engineered MSCs (hydrogel loaded) have also been tested for their capacity in alleviating RIII [86–88]. Preclinical studies have shown the therapeutic potential of MSCs (modified or not) in treating radiation injury. MSCs was also tested for clinical treatment of RIII, in which reduced intestinal inflammation and hemorrhage were exhibited after systematic usage of MSCs [89]. However, a detailed treatment strategy remains unknown.

## *4.3. MSCs in Radiation-Induced Brain Injury*

Radiation-induced brain injury (RIBI) is mainly presented as cognitive dysfunction in patients experiencing head and neck RT [90]. The degree of tissue injury is unequal based on different periods (acute, early delayed, late delayed) [91]. Acute response is sporadic under current RT techniques. Early RIBI involves angioedema and manifested clinically as headache and drowsiness [92]. Acute and early RIBI are generally recovered within 1 to 6 months. However, late RIBI often represents severe irreversible lesions such as vascular injury and demyelination, leading to ultimate white matter necrosis and brain atrophy [93,94]. Apart from the vascular endothelial cells, neurons and glial cells are also susceptible to IR [95]. In all, RIBI is intractable due to the complex dynamic process [91]. Early epidemiological data showed 11% of morbidity of severe dementia in cancer patients receiving whole brain radiation [96]. In fact, sensitive neurocognitive tests suggested that 90% of irradiated patients had neurological impairment [97]. With regard to the treatment of RIBI, anti-inflammatory drugs have been applied to counteract RIBI, such as eicosapentaenoic acid and fenofibrate [98,99]. Moreover, traditional Chinese medicines are also beneficial for neuroprotection against radiation [100]. In preclinical studies, intrahippocampal transplantation of human neural stem cells restored neural plasticity of irradiated rats by improving the expression of activity-regulated cytoskeletal [101]. At present, MSCsbased cell transplantation and secretome administration are also considered as therapeutic

strategies preclinically. UC-MSCs (1 <sup>×</sup> <sup>10</sup><sup>6</sup> ) transplantation via caudal vein infusion showed anti-inflammatory and anti-apoptotic effects on mice with RIBI [102,103]. The RT-triggered inflammation was inhibited, reflected by the decreased IL-1, TNF-α, and the increased IL-10 [102]. On the other hand, the downregulation of pro-apoptotic proteins (p53, Bax) and the upregulation of anti-apoptotic Bcl-2 confirmed apoptosis reduction. This anti-apoptotic benefit was further enhanced through the combined administration of UC-MSCs and nimodipine [103]. MSCs-mediated regulation of both inflammation and apoptosis rescued neurons and astrocytes from necrosis. Additionally, microglia were activated during RIII and initiated inflammation reaction by cytokine and chemokine secretion [104]. Intensive inflammation further accelerated microglia pyroptosis related to the increased expression of NLRP3 inflammasome and caspase-1 [105]. Human trophoblast-derived MSCs (1 <sup>×</sup> <sup>10</sup><sup>5</sup> ) transplantations via brain cortex are able to reverse the microglia pyroptosis, promoting tissue repair [105]. Others also identified that the intranasally administered human MSCs (5 <sup>×</sup> <sup>10</sup><sup>5</sup> ) restored neurological function by reducing inflammation and oxidative stress via declined damage-induced c-AMP response element-binding signals [106]. Unfortunately, only a few researches on applying MSCs therapy in RIBI have been reported so far. The finding that MSCs are also homed to gliomas would encourage more efforts to be devoted to this area [107].
