Oral mucositis is a painful, treatment-modifying side effect of cancer therapy that affects roughly 250,000 cancer patients annually in the United States. Currently, the only FDA-approved drug to treat oral mucositis, palifermin, is in only about 4% of clinical cases. The long-term objectives of this project are to elucidate molecular mechanisms of damage initiation in mucositis, including RNA expression signatures, and the timing of cell- specific events, and also to facilitate the discovery and translation of new anti-mucositis drug candidates. This project is expected to directly support a future, personalized medicine approach for treating oral mucositis in cancer patients. The initiation of mucositis is thought to involve the innate immune and damage response of structural gingival cells, beginning with sub-epithelial endothelial and mesenchymal cells, and evolving toward damage to gingival keratinocytes and epithelial ulceration. Therefore, development of a gingiva-on-a-chip, involving culture of the cells implicated in the initiation of mucositis, is expected to support these objectives. This proposal aims to (1) determine initiation of gingival disruption on a chip exposed to cancer therapies, and (2) determine responses of the gingiva-on-a-chip to putative drug treatments for oral mucositis. Achievement of Aim 1 will involve establishing a baseline of cell function and molecular expression in a tri-culture chip featuring human gingival keratinocyte, fibroblast, and microvascular endothelial cell lines. Viability, apoptosis, proliferation, transcriptional programming, and cytokine secretion of each cell type on the chip will be determined using (immuno)fluorescence assays on cells, tetrazolium salt and cytokine antibody array assays on conditioned nutrient media, and whole transcriptome RNA sequencing. Responses to four doses of four chemotherapy agents implicated in oral mucositis will be assessed and compared to baseline in the chip and in wellplate monoculture using the same assays. Achievement of Aim 2 will involve testing of four anti-mucositis therapies on the chip, for cultures exposed to one chemotherapy agent at a dose found to elicit hallmarks of mucositis, using the same assays as in Aim 1. Gingival barrier function will be assessed in all experiments by tracking fluorescently labeled oral bacterium and carbohydrate molecules of known size across the cell layers. The health-relatedness of this proposal lies in directly impacting oral health and supporting development of precision medicine approaches through knowledge of oral mucositis and the effects of treatments on a chip. The proposed system would serve as a model of the initiation of mucositis in human gingiva and could test novel anti-mucositis drug candidates in parallel with pre-clinical tests. Such knowledge would potentially lead to more successes at the level of clinical trials targeting oral mucositis interventions. Translated to primary culture of gingival biopsies of cancer patients, the chip-based platform would support personalized modeling and mitigation of mucositis by pairing cancer therapies with the best anti-mucositis therapy, determined on the chip.
The specific effects of potential anti-mucositis drugs on gingival cells exposed to cancer chemotherapy agents is unclear, in part because of the difficulty in simultaneously visualizing and measuring rapid (seconds to hours) responses of epithelial and subepithelial cell layers. A novel gingiva on-a-chip platform would enable the determination of the spatiotemporal pattern of molecular and cellular alterations in multiple gingival cell layers exposed to chemotherapy agents and anti-mucositis drug candidates. Fundamental knowledge gained from such an approach would guide mechanistic studies of mucositis, and test novel mucositis therapies, leading to mitigation of mucositis symptoms in a broad swathe of cancer patients undergoing chemotherapy.