Cancer therapy-induced oral mucositis represents a compromised oral wound characterized by atrophy, erythema, ulceration, and, eventually, loss of the mucosal barrier functions secondary to impaired regenerative capacity of the epithelium [11
]. Using a murine model of chemotherapy-induced oral mucositis, we have demonstrated for the first time to our knowledge that spheroid-derived GMSCs are capable of enhanced therapeutic efficacy to reverse body weight loss, regenerate the epithelial lining, and recover mucosal disruption as compared with their adherent cells. The improved therapeutic benefits of spheroid-derived GMSCs for oral mucositis may be attributed to their enhanced capabilities for engraftment and survival at the injury sites, trans
-differentiation into epithelial cells, and preconditioning to hypoxic and oxidative challenges existing in the 3D spheroid cultures.
Despite overwhelming benefits of MSC-based therapeutics for a variety of diseases, several obstacles clearly remain for their large-scale clinical applications. It has been recognized that progressive subculturing of MSCs potentially leads to changes in cellular phenotypes affecting their regenerative and homing abilities [5
]. Currently, intravenous delivery of a large bolus of cells and direct tissue injection are the 2 major methodologies for preclinical studies and clinical trials of MSC-based therapy. However, intravenous delivery requires a relatively large dose of cells (from 1 to 5×106
cells/kg body weight) at significant production cost and increased risk of side effects such as pulmonary embolism and organ infarction, whereas direct tissue injection requires invasive, technique-sensitive, and precise infusion methodologies [5
]. Therefore, how to maximize therapeutic efficacy and safety of MSC-based therapy while minimizing adverse effects, overall manufacture, and procedure costs remains a great challenge. To date, transgenic approaches have been developed to force MSCs to overexpress certain defined factors with either trophic or tropic, survival, proangiogenic, immunomodulatory, or anti-inflammatory functions [5
]. In recent years, numerous nontransgenic approaches aiming to increase the multipotency of MSCs or to evoke the secretion of these functional factors by MSCs have attracted more and more attention due to the lack of viral and mutation risks. One of the common nontransgenic approaches for optimizing MSC functions involves priming or preconditioning MSCs in vitro with certain proinflammatory cytokines such as IFN-γ or TNF-α, which may be encountered by MSCs at the sites of inflammation or injured tissues [27
]. Another strategy for optimization of MSCs involves preconditioning cells under low oxygen tension or hypoxic condition, a common physiologic or metabolic milieu of stem cell niche, that potentially play a critical role in the maintenance of an undifferentiated state of both embryonic and adult stem cells, and in the regulation of proliferation and cell fate commitment [28
]. For instance, previous studies have shown that bone marrow MSCs cultured in hypoxia exhibit increased expression of Oct-4 and telomerase activity [29
] and decreased differentiation into adipogenic and osteogenic lineages [31
]. In addition, MSCs exposed to hypoxic conditions display a more migratory or proangiogenic phenotype, and improved survival due to an increased expression of a variety of hypoxia-responsive genes involved in angiogenesis and cell migration, such as VEGF, SDF-1α, and CXCR-4 [33
]. Therefore, hypoxic preconditioning of MSCs is an effective methodology to optimize MSC functions, thus enhancing their global functions and therapeutic benefits after migrate to sites of inflammation and injured tissues, where they may encounter an environment of hypoxia and oxidative stresses [36
Most recently, several studies have shown that 3D spheroid cultures of MSCs significantly enhance the stem cell-like properties and therapeutic effects. For example, Bartosh et al. have recently reported that aggregation of human bone marrow-derived mesenchymal stem cells (BMSC) into 3D spheroids not only enhanced their multipotent differentiation capacities, but also led to an increased expression of the TNF-α suppressing gene-6 (TSG-6), an anti-inflammatory molecule, as compared with their adherent counterparts [7
]. In addition, Li et al. have shown that growing cardiac-derived progenitor cells as 3D spheroid cardiospheres led to upregulated expression of stem cell related genes and improved cell survival after exposure to oxidative stress as compared with their monolayer counterparts [8
]. Most recently, Bhang et al. reported that spheroid cultures were more effective in preconditioning human adipose-derived stem cells to a hypoxic environment, leading to upregulation of hypoxia-adaptive signals and enhancing secretion of both angiogenic and antiapoptotic factors as compared with monolayer cultures [10
]. Consistently, in the present study we have demonstrated that 3D spheroid culture of GMSCs not only enhanced the expression of stem cell relevant genes and their multipotent differentiation capacity but also the expression of hypoxia responsive genes such as HIF-1 and −2α, VEGF, SDF-1α, and CXCR-4, as compared with the monolayer cultures. Meanwhile, we have demonstrated that spheroid GMSCs show an increased production of ROS and superoxide dismutase-2 (SOD2) as well as improved survival under oxidative stress conditions. These findings support the notion that the 3D spheroid culture condition recapitulates hypoxic and oxidative microenvironment of the inflammatory niche at the injured or mucositic sites and potentially act as an in vitro preconditioning of GMSCs to be more resistant to apoptotic stress and optimize their therapeutic effects in reversal of oral mucositis.
Of note, the regeneration of injured or damaged epithelium-lined organs requires the coordination of multiple types of cells, the epithelial, endothelial, mesenchymal, and immune cells [38
]. Recent studies have shown that mesenchymal stem cells (MSCs) participate in the regeneration and repair of a variety of diseased epithelial tissues, including injured epithelial layers in skin [39
], airway [40
], cornea [41
], gastric and intestine [42
], kidney [44
], and oral cavity [45
]. The mechanisms underlying MSC-mediated regeneration of injured epithelial tissues may involve not only the secretion of various factors with antioxidant, anti-inflammatory, antiapoptotic, or proangiogenic functions, but also the transdifferentiation of MSCs into epithelial-like cells possibly through MET [44
]. In the present study, we have shown that in comparison with their adherent counterparts, spheroid-derived GMSCs displayed increased cell plasticity and abilities to home to the mucositic lesions. The relatively smaller cell sizes and increased expression of CXCR-4 by spheroid GMSCs may facilitate their faster trafficking through the lung microvasculature and more efficient distribution into tissues. Meanwhile, our findings have revealed an enhanced potential for homed spheroid-GMSCs to transdifferentiate into epithelial-like cells. This might contribute, at least in part, to their enhanced therapeutic efficacy for the regeneration or repair of mucositic lesions. However, further studies are warranted to explore the deep mechanisms.
Despite the observed therapeutic benefits of spheroid-derived GMSCs in cancer therapy-induced mucositis, their potential impact on cancer development remains to be addressed. The literature on tumor tropism, pro-, and antitumorigenicity of MSCs [46
] is highly controversial. In some studies, MSCs have been reported to promote tumor proliferation, angiogenesis, epithelial–mesenchymal transition, as well as metastasis [46
], whereas other studies have shown the tumor suppressive effect of MSCs in different tumor models [46
]. The unique tumor tropism of MSCs renders them a unique delivery vehicle for antitumor agents [48
]. Understanding the interaction between MSCs and tumor cells and their underlying mechanisms will allow development of novel therapeutic approaches to target the stromal effects on tumor growth while reversing the detrimental effect of chemotherapy-induced epithelial injuries.