br Materials and methods br Results br

Materials and methods
Results
Discussion The effect of donor MSCs in tumor growth remains controversial (Cho et al., 2009). Discrepancies concerned to whether they promote or suppress it may be due to disparities in cell source, isolation and expansion conditions, via and timing of administration, cancer model, timing of assessment, etc. (Ikebe and Suzuki, 2014). Up to our knowledge, the impact of donor MSCs in precancerous lesions has not been reported. We decided to test it in the OSCC model settled in Syrian golden hamster because it mimics the etiology (DMBA is present in cigarrette smoke), the progression and the response to chemopreventive agents of human OSCC (Nagini et al., 2009). Together, changes are grossly visible and patognomonic of each stage (Nagini et al., 2009). When we administered 0.1×106 or 1×106 MSCs to OSCC lesions at papilloma stage we observed a membrane transporter in tumor size (Supplementary Fig. 2). This effect was more significant when we administered 3×106 MSCs (Fig. 3). The observed anti-tumorigenic effect of MSCs correlates with a decrease in cell proliferation and an increase in cell apoptosis. Both constrain tumor size enlargement. These results are consistent with previous works reporting that MSC administration inhibits established tumor growth through proliferation inhibition and apoptosis induction (Ramasamy et al., 2007; Qiao et al., 2008; Glennie et al., 2005; Beyth et al., 2005). Neovascularization plays a central role in the development and progression of tumors including OSCC (Li et al., 2007). It has been previously shown that MSCs differentiate and secrete vasculogenic growth factors supporting tumor vasculature (Roorda et al., 2009). In a glioma animal model it has been shown that the systemic administration of MSCs reduced the number and caliber of tumor vessels (Ho et al., 2013). Thus, depending on yet undefined variables MSCs can promote or inhibit neovascularization. Under the setting here presented, donor MSCs does not modify the vasculature of OSCC lesions (Fig. 5). Since neoplastic microenvironment is similar to that of an injured tissue. Man (2010) donor MSCs delivered around OSCC papilloma could trigger a non-specific allogeneic immune response that might impedes tumor growth. At the end of the study period we observed no differences in the infiltration of leukocyte or mast cells (first host line defense in the oral mucosa (Coussens et al., 1999)). Thus, chronic immunomodulation does not explain MSC anti-tumorigenic effect. We cannot rule out that acute immunomodulation is involved. Cytokeratins are epithelial specific intermediate filament proteins, underlying distinct cellular properties and differentiation stages (Schweizer et al., 2006). In the hamster buccal pouch, the epithelial basal cell layer expresses CK1 while the suprabasal cell layer express CK4 (Balasenthil et al., 2002a; Balasenthil et al., 2002b). Alterations in CK1 and CK4 expression are sensitive markers for epithelial tumor stratification (Ogden et al., 1996). Thus, the observed recovery of CK1 expression and the normalization of CK4 tissue distribution after MSC administration reflect an avoidance of epithelium dedifferentiation. Accordingly, it has been reported that MSCs stabilize cell phenotype of keratinocytes, endothelial cells, pericytes and monocytes in wound repair (Sasaki et al., 2008). Further support to the preclusion of dedifferentiation is the normalization of gene expression profile after MSC administration (Fig. 7B). The later has been observed in other oral pathological conditions (Dowdall et al., 2015). The preservation of a differentiated phenotype may be attributed to the potential of MSCs to sustain a homeostatic microenvironment, particularly scavenging oxidative stress (Valle-Prieto and Conget, 2010; Ramasamy et al., 2007). Taken together, our results support an anti-tumorigenic effect of MSCs in epithelial precancerous lesions. Thus, the local administration of allogeneic MSCs should be envisioned as a preventive strategy for the development of OSCC since donor cells modify its natural progression and improve prognosis due to the preservation of epithelial phenotype. This is not an irrelevant goal in the field since the prognosis for patients with OSCC still poor (Jadhav and Gupta, 2013).