Mesenchymal stromal cells therapy in radiation oncology regenerative medicine

Mesenchymal stromal cells (MSCs) are multipotent somatic cells resident in many tissues and organs. They have specific characteristics that distinguish them from other cell types. They are self-renewing cells with multi-lineage differentiation potential. In addition, they possess anti-inflammatory and immunomodulatory properties. Studies have shown that they could be used as vehicles to deliver certain therapeutic gene products as well. These cells possess secretory capabilities of certain cytokines and growth factors that mediate various paracrine effects. They increase the secretion of the anti-inflammatory interleukin-10 (IL-10) together with lowering the availabilities of tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), and interleukin -1-beta (IL-1β) by signaling to the immune system elements, e.g. dendritic cells, T-cells, B-cells, and natural Killer cells (NK cells). Recently, studies have investigated such anti-inflammatory properties of MSCs in the repair of radiation-induced normal tissue injury, also called radiation oncology regenerative medicine (RORM), supported by the recently known MSCs radiation resistance potential. In this review, we summarize MSCs radio-resistant mechanisms, anti-inflammatory properties, and their application in RORM with special attention to adipose tissue-derived MSCs (aMSCs). Correspondence to: Dr. Thierry Muanza, MD, MSc, FRCPC, Radiation Oncology Translational Research Lab, Department of Radiation Oncology, Jewish General Hospital and Lady Davis Institute Research Centre, McGill University, 3755 Côte-St.-Catherine Road, Suite G002, Montréal, Québec, Canada, H3T 1E2, Tel: +1 (514)-340-8288, Fax: + 1 (514)-340-7548, E-mail: tmuanza@yaoo.com


Introduction
Mesenchymal stromal/Stem cells (MSCs) are multipotent somatic progenitor cells that have been isolated from different tissues, such as bone marrow, adipose tissue, muscles and skin [1][2][3]. They can be expanded ex-vivo to hundreds of million cells, maintaining their phenotype and characteristics, and used as therapies in different diseases [1][2][3]. Another property of these cells is their homing to the site of tissue injury, an ability that widens the choices for their route of administration [2,4,5]. In addition to their multi-lineage differentiation potential [6], these cells possess anti-inflammatory and immunomodulatory properties and paracrine effects that qualified them for regenerative medicine applications ( Figure 1) [7][8][9][10][11]. Furthermore, MSCs could be genetically engineered and used as vehicles for delivering therapeutic gene products [12][13][14]. Studies in radiotherapy have shown that MSCs can be recruited to the radiation injury site where they secrete many cytokines and growth factors, e.g. prostaglandin-E2 (PGE2), nitric oxide (NO), hepatocyte growth factor (HGF), interleukin-10 (IL-10), tumor growth factor-beta (TGF-β), and indoleamine 2,3-dioxygenase (IDO) [15]. These soluble mediators inhibit the major components of the immune system and inflammation, e.g. dendritic cells, T-cells, B-cells, and natural killer cells (NK cells) [15]. The final result will be an increase in the secretion of the anti-inflammatory interleukin-10 (IL-10) together with lowering the availability of pro-inflammatory mediators and cytokines, e.g. tumor necrosis factor-alpha (TNF-α), interferon-gamma (INF-γ), and interleukin -1-beta (IL-1β) [15] (Figure 1).

MSCs radio-biological response
The exposure of MSCs to ionizing radiation (IR) induces direct and indirect double stranded DNA breaks (DSB) which are detected by Poly (ADP-ribose) polymerase (PARP) and heterodimeric Ku protein complex (Ku70/80) sensor proteins [27,28]. At the DSB location, PARP started the signal amplification upon formation of the Mre11, RAD50, and NBS-1 protein complex which leads to recruitment and auto-phosphorylation of Ataxia Telangectasia mutated protein (ATM). Phosphorylated ATM (p-ATM) is a main station that leads to multiple downstream signals. P-ATM enhances the phosphorylation of histone H2X (to γ-H2AX) and DNA-PK (to p-DNA-PK), phosphorylates P53 (a tumor suppressor regulatory protein), activates the cell cycle checkpoint effector protein kinases (Chk-1 and Chk-2), and prepares for cell cycle arrest (G2/M). In addition, the Chk1 activation is augmented by the replication stress-mediated ATR pathway (through replication protein A, RPA), while the Chk2 activation is enhanced directly through Ku70/80-mediated p-DNA-PK signaling [27,28]. Cell division cycle phosphatase (Cdc25) is crucial for removing the inhibitory phosphorylation on specific residues on the cyclin-dependent kinase (Cdk). Chk1 phosphorylates Cdc25 in the presence of DNA damage resulting in the inhibition of Cdc25 activity. Chk1 and Chk2 are main inhibitors of Cdc25A and Cdc25C resulting in Cdk/cyclin-mediated cell cycle arrest [29]. It has been suggested that DSB in MSCs are repaired by activation of both the homologous recombination (HR, during S and G2 phases) and the non-homologous end-joining (NHEJ, during all cell cycle phases) DNA repair pathways [27,28,30]. Our recent study showed the activation of HR and NHEJ repair pathways in irradiated aMSCs [31]. In addition, p-ATM enhances the stabilization of the tumor suppressor regulatory protein and transcription factor P53 which up-regulates the expression and enhances the stabilization of the transcription factor and inhibitory regulatory protein p21, which potently inhibits Cdks which are needed for the G1/S transition leading to inhibition of the entry into S phase [27].
The application of MSCs in radiation oncology regenerative medicine (RORM) was enhanced by their efficient radiation-induced DNA repair machinery and their relative radiation resistance [30][31][32][33][34]. Such radiation resistance was mediated by many mechanisms, e.g. the ATM phosphorylation, activation of cell cycle check points (G2/M arrest), and activation of single and double stranded DNA repair by both homologous and non-homologous recombination mechanisms and other pathways [30,31] (Figure 2). DSB resulting from the direct and indirect radiation injury stimulate the phosphorylation of ATM which is the proximal step for cell cycle check point's activation (G2/M arrest). In addition, the nuclear apoptotic factor P84 (P84/53E10 = the nuclear protein encoded by the N5 gene) is up regulated, which participates in the apoptotic response of the aMSCs. It has been documented that irradiated aMSCs showed p-ATM dependent and p-ATM independent (P84-mediated) G2/M arrest [31]. Phosphorylated histone-2AX (γ-H2AX) stimulated both the HR and the NHEJ of the dsDNA breaks and other repair mechanisms [35]. Rad-51 is considered one of the mandatory proteins for HR to occur. DNA-PK is the major protein in the NHEJ repair pathway. Studies have shown that both proteins (Rad-51 and DNA-PK) were up regulated in irradiated MSCs ( Figure 2) [28,30,31].

MSCs applications in radiation oncology regenerative medicine (RORM)
Adding up all their beneficial characteristics, MSCs have been investigated in RORM preclinical and clinical studies (Table 2). Nevertheless, the few clinical data representing the therapeutic benefits of the application of MSCs in radiation-induced normal tissue injury are promising. Among these, in radiation-induced bone injury, MSCs therapy caused early hematopoietic recovery with improved osteonecrosis. In radiation-induced intestinal injury, MSCs therapy produced significant repopulation of intestinal epithelium with reduced pain, diarrhea, and hemorrhage. In radiation-induced skin injury, MSCs therapy showed significant improvement and repopulation of skin tissue [29]. The following are the clinical studies that have been investigating the potential application of MSCs in RORM.

Skin repair application after radiation exposure
MSCs have been used in the repair of radiation-induced skin injuries where they were administered systemically and led to decreased radiation-induced skin fibrosis through enhancing the secretion of IL-10 and increasing the infiltration of anti-inflammatory regulatory CD163(+) macrophages, in addition to decreasing the secretionof IL-1 beta and the number of infiltrated pro-inflammatory CD80(+) macrophages [36]. It was suggested that the autologous grafting of MSCs is more efficient than the allogenic grafting in cutaneous radiation syndrome [20]. MSCs secrete growth factors and anti-inflammatory mediators that can be combined with other external growth factors, e.g. basic fibroblast growth factor (b-FGF) in order to improve the healing in radiation-induced skin damage [37]. The improved migration of fibroblasts and collagen production will protect the fibroblasts from the oxidative stress of UVB radiation [37].

Intestinal repair application after radiation exposure
MSCs have been applied for the repair of radiation-induced intestinal injury [26,38]. When MSCs were given before irradiation, treated mice showed higher body weight, thicker intestinal submucosal and muscle layer, significant higher survival rates and stromal derived factor-1 (SDF-1) expression, and lower numbers of radiation-induced ulcers [25,38]. Another study reported that MSCs therapy showed better maintenance of epithelial homeostasis, neovascularization, high anti-inflammatory IL-10, increased expression of VEGF, b-FGF and EGF in irradiated intestine, and increased the homing of CD31positive hematopoietic stem cells or hematopoietic progenitor cells to the irradiated intestine [39]. MSCs therapy showed decreased activation and proliferation of T-lymphocytes together with increased local corticosterone secretion at the intestinal mucosa that highlighted an immunosuppressive effect of MSCs mediated by glucocorticoid receptors [40]. It was found that MSCs reparative and paracrine effects in radiation-induced intestinal injury were enhanced by pretreating them with TNF-alpha, IL-1 beta, and nitric oxide [41].

Lung tissue repair application after radiation exposure
MSCs therapy was shown to reduce radiation-induced lung tissue injury. Administration of MSCs resulted in decreased radiationinduced inflammatory response in terms of reduced pro-inflammatory mediators (IL-1 beta, IL-6, TNF-alpha), increased anti-inflammatory mediators (IL-10), reduced expression of TGF-β, alpha-smooth muscle actin (Alpha-SMA) and type 1 collagen level, and control of the proand anti-apoptotic mediators (Bcl-2, Bax, and caspase-3) protecting the lung tissue from apoptosis [42]. Moreover, MSCs therapy reduced bronchial epithelium senescence and lowered the risk of metastatic spread in lung tissue [43]. In addition, MSCs therapy decreased the mortality rate in mice with radiation-induced lung injury [44]. These cells showed a proven beneficial therapeutic effect in radiation pneumonitis as well [45].

Hematopoietic system homeostasis radiation injury
MSCs therapy has been shown to reduce the radiation-induced bone marrow apoptosis, and enhancemegakaryopoiesis and platelet recovery [46]. Moreover, MSCs therapy resulted in improved recovery of the hematopoietic system through decreased apoptosis and radiation-induced oxidative stress [47,48].

Radiation-induced cardiac injuries
A case report of a patient suffering from late radiation cardiomyopathy and radiation exudative pericarditis after radiotherapy of Hodgkin lymphoma showed that systemically transplanted MSCs partially differentiated to cardiomyocytes [49].

Radiation-induced salivary gland injury
In irradiated mice, systemically transplanted MSCs resulted in improvement of the saliva flow rate, lower salivary gland damage and atrophic acini, and higher mucin and amylase production [50].

Radiation-induced oral mucositis
Bone marrow-derived mesenchymal stromal cells (bmMSCs) therapy have been applied in fractionated radiation-induced oral mucositis where the administration of a systemic single dose of 6 million MSCs resulted in a significant decrease in ED50 (the RT dose that produces ulcer in 50% of irradiated mice) [51]. The first MSCs therapy for RIOM was done in 2014 by Schmidt et al. and concluded that transplantation of bone marrow (BM) or bmMSCs could modulate RIOM in fractionated RT, depending on the time of plantation [52]. Nevertheless, in another study they also concluded that bmMSCs plantation had no therapeutic benefits on RIOM in single dose RT when compared to the therapeutic gain by the mobilization of endogenous BM stem cells [53]. Further studies are needed in this field since the initial studies showed significant clinically relevant therapeutic effects.

Liver tissue protection
MSCs therapy reduced the radiation-induced liver injury by anti-oxidative, vascular protection, hepatocyte differentiation, and    trophic mechanisms. There was decreased expression of Nrf2 and superoxide dismutase (SOD) in MSCs-treated irradiated liver which showed decreased apoptotic cells as well.These findings suggested that, these effects were mediated by an anti-oxidative mechanism. The increased expression of VEGF and Angiopoietin-1 (Ang-1) in the perivascular region, associated with an increased expression of VEGFr1, r2 suggested the vascular protection mechanism in the livers of MSCs-treated animals. After engrafting, MSCs showed expression of cytokeratin CK18 and CK19 and alpha-fetoprotein (AFP) genes which suggested hepatocyte differentiation. The increased secretion of nerve growth factor (NGF), hepatocyte growth factor (HGF), and anti-inflammatory molecules IL-10, IL1-RA suggested MSCs' trophic effects [40,54]. MSCs conditioned media improved the viability of liver sinusoidal endothelial cells (SECs) in vitro. Infusion of MSCs conditioned media significantly reduced the radiation-induced SECs apoptosis and improved the histopathological picture of irradiated livers. In addition, there was increased secretion of anti-inflammatory cytokines and decreased secretion of pro-inflammatory cytokines [40,55].

Studies with gene-modified MSCs for RORM
Genetically modified MSCs have been applied in RORM studies. HGF-expressing MSCs have improved the radiation-induced intestinal injury where they increased the expression of anti-inflammatory mediators and improved the histopathological picture of irradiated intestine [12]. Hepatocyte growth factor gene-modified adiposederived mesenchymal stem cells improved the radiation induced liver damage in a rat model [13]. A similar picture was noted with TGFbeta-expressing MSCs therapy in radiation-induced lung injury [14].

Summary
Although limited data are available for the clinical application of MSCs in radiation-induced normal tissue injury, promising therapeutic benefits have been shown in a small number of isolated clinical studies [29].
Isolated clinical case reports showed promising beneficial effects of MSCs therapy; e.g. regenerating hematopoiesis and osteoradionecrosis, improved breathing parameters and lung immune function, improved intestinal mucosal inflammation, hemorrhages, fistulization, pain and diarrhea, and regenerated skin ulceration, in ionizing radiationinduced injury of bone, lung, intestine, and skin, respectively [29,40,56,57]. Table 2 summarizes the recent preclinical and clinical studies conducted in RORM applying MSCs therapies.

Adipose tissue-derived MSCs (aMSCs)
Adipose tissue-derived mesenchymal stem/stromal cells (aMSCs) are multipotent progenitor cells located in the stromal vascular fraction (SVF) of adipose tissue [2]. They are characterized by expressing cell surface antigens Sca1, CD106, CD105, CD73, CD29, and CD44, and lacking the expression of hematopoietic stem cells (HSCs) surface antigens (e.g. CD11b and CD45) [2,3,58]. In addition to their multilineage differentiation potential, they have anti-inflammatory/ immune-modulatory and paracrine effects [59][60][61]. In addition, MSCs can home to the site of tissue injury that is caused by irradiation and inflammation [2,5,62].These advantages, in addition to their source abundance, ease of isolation and high cell count after expansion, render aMSCs promising for cellular therapies [63]. Table 3 lists 22 clinical trials using aMSCs therapy for various disorders, with no trial yet found for their application in RORM, following a search on the clinical trials website of the NIH, i.e. https://clinicaltrials.gov/, in Nov. 2015.

MSCs mechanisms of action in RORM
There are proposed mechanisms of action of MSCs radio-protective properties in radiation-induced normal tissue injury repair. Homing and paracrine effects with anti-inflammatory/immunomodulatory mechanisms are supported by in-vitro data from radiation-induced intestinal injury studies and [59][60][61][62]. MSCs therapy in radiationinduced intestinal injury showed the homing of systemically administered MSCs in measurable numbers at the intestinal injury site [25,26,41]. There were increased levels of IL-10, VEGF, b-FGF, and EGF. Histopathological studies showed improved intestinal epithelial homeostasis that may be due to MSCs overexpressing stromal cellderived factor receptor CXCR-4 [29]. These findings suggest that the paracrine and the anti-inflammatory effect of MSCs is the expected radio-protective mechanism of action of MSCs in RORM [29].

Challenges facing MSCs therapy
The fear of MSCs-mediated radioprotection of tumor tissues has been a raised concern after the availability of in-vitro data suggesting that breast cancer cells grow and proliferate more with MSCs-therapy owing to high insulin-like factor production [53]. Also, MSCs have some angiogenic properties evident by increased secretion of platelets derived growth factor (PDGF), VEGF and TGF-β at the tumor perivascular area and parenchyma in low dose irradiated mice owing to MSCs infiltration at the tumor site [53]. MSCs angiogenic properties might counteract the anti-angiogenic cancer therapies, a question that needs to be answered with solid in-vitro and in-vivo studies [28,29].
Another challenge appeared in MSCs therapies. MSCs have been found to have heterogeneous radiation resistant populations, both in human and mouse MSCs [53]. A finding that might interfere with the overall radio-protective and tissue regenerative properties of MSCs.  Nevertheless, studies may find molecular biomarkers for isolating homogenous populations of MSCs with uniform high RT resistance profile [28,29].
A further challenge that has been found to be more frequent in mouse MSCs than in human MSCs, is MSCs in-vitro transformation (the tumorigenic potential of MSCs) [53]. Such challenge carries a significant worry for MSCs therapies, since MSCs are radio-resistant cells. Thus, their transformation may signify the generation of a severe form of radio-resistant tumor that is extremely hard to control. Tight and fine validation of MSCs before each single dose therapy is recommended for preventing the use of any potentially transformed cells [28,29,34].

Conclusion
MSCs have been widely used in preclinical studies of radiation oncology regenerative medicine. MSCs have been shown to be reliable candidates in radiation oncology regenerative medicine translational and clinical research. The strong potential of MSCs therapy in RIOM is supported by their relative radiation resistance and robust DNA repair mechanisms, multi-lineage differentiation potential, and antiinflammatory/immunomodulatory properties. Nevertheless, few but considerable challenges in MSCs therapies are requiring more research in order to develop solid solutions. However, the overall data collected from preclinical and clinical studies with MSCs therapy promise with cell therapy choices competing the traditional therapies. Adipose-tissue derived mesenchymal stromal/stem cells are reliable candidates for radiation oncology regenerative medicine applications owing to the advantages they possess, e.g. source abundance, enhanced anti-inflammatory effects, robust IL-10 secretion, easy isolation, high expansion.

Authorship and contributions
Osama Maria: Conception and design, collection and/or assembly of data, review writing, final approval of the review.
Nicoletta Eliopoulos: Conception, design and final approval of the review.
Thierry Muanza: Conception and design, financial support and final approval of the review.