Assessing the Role of L. brevis and S. salivarius Bacterial-mammalian Cell Interactions in Methotrexate Cytotoxicity in an Imageable in vitro Co-Culture Model of the Oral Mucosa Using an Aqueous Two-Phase System

Abstract

Chemotherapy-induced oral mucositis (CIOM) is characterized by the ulceration of the oral mucosal tissues caused by the systemic cytotoxic effects of chemotherapy. It is considered a major side-effect that may lead to the interruption of cancer treatment, which often compromises the treatment prognosis for patients. Recently, there has been interest in exploring the potential of bacteria to treat CIOM, primarily driven by the promising effects of probiotics in reducing the severity of intestinal mucositis. The application of probiotics for treating CIOM has not been properly explored due to the lack of adequate study models and clear mechanisms through which probiotics exert their benefits. We fabricated an imageable in vitro model using stained immortalized human oral keratinocytes (OKF6/OKF6-TERT2) and then treated it with Methotrexate aiming to recapitulate the cellular damages that are typically observed in CIOM. The establishment of the microbe-mammalian co-culture was achieved using an aqueous-two phase system (ATPS), a liquid-based scaffold of polyethylene glycol and dextran. An ATPS was optimized for each bacteria to maintain them contained and viable for 48 hours within the DEX-phase without damaging a monolayer of OKF6 cells. This research aimed to provide a tool to fill the knowledge gap in our understanding of the mechanistic effects of specific probiotics in CIOM. The effect of applying L. brevis or S. salivarius to a monolayer of OKF6 before exposure to MTX was tested. The results suggested that the application of S. salivarius to the OKF6 cells before MTX exposure maintained cell viability in comparison to the bacteria-free control. In this study L. brevis had a high susceptibility to the tested MTX dosage. The establishment of an in vitro model capable of allowing mammalian-microbial interactions in a system with representative damage of CIOM provided the possibility of elucidating the mechanism behind the benefits of probiotics that past studies have demonstrated in clinical trials and using in vivo models. To our understanding, this is the first study to test in vitro the effects of probiotics in an oral mucositis model, furthermore, allowing mammalian-microbial interactions.