**2. Pathophysiological Mechanisms of Radiation Injury**

RT utilizes high doses of radioactive energy, known as ionizing radiation (IR), to kill cancer cells. Notably, IR also injuries the healthy cells around the tumor, causing various complications. However, the pathophysiological mechanisms of radiation injury remain mostly unclear. IR induces increased production of reactive oxygen species (ROS), referred to as oxidative stress, injuring cell components such as DNA, proteins, organelles, etc. [20]. The damages to DNA mainly comprise single- and double-stranded breaks and base lesions [21]. Incorrect DNA repair would give rise to mutagenesis or chromosomal instability resulting in cell apoptosis and carcinogenesis [22]. Excessive ROS activates unfolded protein response in the endoplasmic reticulum (ER), which further elicits Ca2+ release from ER, causing ER stress [23]. If the ER stress was uncontrolled, the unfolded protein response pathways trigger downstream signals such as c-Jun N-terminal kinase and Bcl-2 protein family members, initiating cell apoptosis or autophagy [24]. The enhanced ROS and imbalanced Ca2+ in the cytoplasm cause mitochondrial membrane permeabilization [25], leading to Bax's activation and the release of cytochrome c, promoting apoptosis development [26]. Moreover, mutated mitochondrial DNA, impaired PPAR-α pathways, and dysregulated ROS production induce mitochondrial dysfunction [26]. The proper functionality of cellular components is closely connected with the cell fate. Thus, clarifying the alterations of intercellular and intracellular signal cascades is beneficial for understanding the radiation injury.

Inflammatory responses, endothelial cell injuries, and fibrosis are vital radiation injury features [27–29]. At the acute phase after IR, inflammatory cytokines (tumor necrosis factor, TNF; interleukin-1, IL-1; IL-6; IL-8), chemokines (C-C motif chemokine ligand, CCL; C-C motif chemokine, CXC), and adhesion molecules (intercellular cell adhesion molecule, vascular cell adhesion molecule, E-selectin) are secreted, inducing vasodilation and vascular permeability [30]. Subsequently, coagulation cascade signals are triggered, and endothelial basement membrane is degraded, enabling clearance of damaged tissue and repairing initiation. This acute response may sustain from minutes to several days after IR [29]. Notably, chronic inflammation and oxidative stress would induce fibrosis at the later phase of diseases [31]. The transforming growth factor-β1 (TGF-β1)/Smad signaling has been recognized as the primary player that mediates myofibroblasts proliferation and regulates extracellular matrix and collagens deposition [32]. IR also upregulates the connecting tissue growth factor levels that can enhance the binding of TGF-β1 with its receptor (Smad2, Smad3), promoting fibroblast trans-differentiation [33]. By dissociating TGF-β from its complex, the enhanced ROS promotes TGF-β1/Smad signaling, which further modulates ROS generation via upregulating NADPH oxidase 4 transcriptional activity [34]. Moreover, myofibroblasts are also found to originate from the process named epithelial or endothelial to mesenchymal transition [35]. Other profibrotic cytokines, such as CCL3, CCL2, IL-1, and IL-6, are also essential for fibrosis progress. Elevated IL-6 levels post IR is correlated with radiation toxicity in breast cancer patients and the degree of fibrosis in the irradiated lung [36,37]. Fibrosis formation is usually a chronic but ongoing progressing process, and it lacks sensitive tools allowing for early detection.

Apart from these mechanisms, telomere erosion, miRNAs alterations, epigenetic regulations, and stem cell damage are also engaged in the pathophysiological development of radiation injury [38–41]. Moreover, these underlying mechanisms interconnect with each other and vary depending on the tissue/cell types, IR patterns (types, doses, and dose rates), and patient-related factors (individual comorbidities and risk factors, such as body mass index, smoking, and genetic predisposition). Thus, determining factors that promote radiation injury progression from asymptomatic remains challenging.

#### **3. Characteristics of MSCs**

Currently, there is no absolute definition of MSCs. To facilitate the development of MSCs-based study, the International Society for Cellular Therapy proposes several minimal criteria identifying MSCs [42–44]. Firstly, surface CD antigens are the most primary and necessary verification method. MSCs positively express stro-1, CD44, CD73, CD90, and CD105. Different from hematopoietic stem cells, MSCs lack CD34, CD45, CD14 (or CD11b), CD79α (or CD19), and HLA-DR. Secondly, MSCs are considered to be plastic-adherent when cultured under standard conditions. Lastly, MSCs must possess the capability of differentiating into osteoblasts, adipocytes, and chondroblasts. This report largely standardizes the definition of MSCs and instructs investigators to estimate the authenticity of their cells.

MSCs can be obtained from multiple tissues (bone marrow, adipose tissue, peripheral blood, umbilical cord, and placenta), providing researchers with great convenience and increasing its clinical application popularity [45]. MSCs derived from differed tissues show distinct characteristics, including proliferation and differentiation potential, paracrine effect, immunophenotypes, and immunomodulatory capacity [46,47]. For example, umbilical cord blood-derived MSCs (UC-MSCs) show more significant proliferation and slower senescence compared with that from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) [48]. However, BM- and AT-MSCs are capable of tri-lineage differentiation (osteogenic, adipogenic, and chondrogenic) under respective culture conditions, while placenta- and UC-MSCs only differentiate into two cell lineage [46]. Additionally, discrepant paracrine activity reflected by the expression of various cytokines and growth factors was observed in UC- and AT-MSCs [49]. All these differences may influence the function of MSCs from multiple sources. A comprehensive understanding of these features would promote a more efficient clinical application of MSCs.

In most MSCs-based therapy studies, immunomodulation is regarded as the leading factor of the therapeutic property. MSCs can interact with immune system cells (T cell, B cell, natural killer cells, etc.) and regulate immune response depending on direct cell-cell contact and various immunomodulated factors [50]. High inflammation levels would stimulate MSCs to release anti-inflammatory cytokines, inhibiting overactivated inflammation and immune responses. The involved molecules include inducible nitric oxide synthase (iNOs), TGF-β, IL-10, prostaglandin E2 (PGE2), and hepatocyte growth factor (HGF) [51]. T cells would be deactivated by inducing apoptosis or suppressing proliferation [52]. On the contrary, the silent immune system would induce the pro-inflammatory phenotype of MSCs to ensure basic self-defense against the external pathogen. Such plastic immunomodulation function protect tissue against pathogen invasion or self-attack, making MSCs a popular object in the study of tissue repair and regeneration [53].

#### **4. Current Attempts of MSCs for Mitigating Radiation Injury**

Considerable progress in medications has dramatically reduced the mortality and morbidity of cancer patients. The increased number of cancer survivors enables clinicians to realize the side effects of related treatments such as RT. To date, it has gained remarkable improvements in achieving high-precision RT. For instance, breast cancer patients receiving IMRT exhibited significantly lower occurrence, severity, and persistent of radiodermatitis than those receiving conventional RT [8]. A significant reduction in gastrointestinal toxicity was observed in IMRT than conventional two-dimensional RT (IMRT vs. RT: 33% vs. 77%) [6]. Moreover, the combination of IGRT and IMRT (IG-IMRT) showed more significant superiority than conventional three-dimensional conformal RT in the treatments of rectal cancer and hepatocellular carcinoma [7,9]. With IG-IMRT, hepatocellular carcinoma patients showed longer median survival (IG-IMRT vs. RT: 44.7 vs. 24.0 months) [7,9]. Although modern RT doses have been minimized and are precise, radiation complications

still typically occur acutely or chronically. Here, we mainly discuss the latest advances in MSCs therapy application mitigating radiation injury involving the skin, intestine, brain, lung, liver, and heart.
