Determining molecular mechanisms of damage and repair in an oral mucosa chip - 7. Project Summary/Abstract Oral mucositis is a painful, treatment-modifying side effect of cancer therapy that affects roughly 50% of cancer patients annually in the United States. Currently, the only FDA-approved drug to treat oral mucositis, palifermin, is used 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 pro-inflammatory cytokines, and the timing of cell- specific events, and also to facilitate the discovery and translation of new anti-mucositis drug candidates. These discoveries will be achieved by experiments on a novel mucosa chip platform representing the epithelial and subepithelial layers of the oral mucosa. This project is expected to directly support high-throughput on-chip testing of single and combination oral mucositis therapies and also a future, personalized medicine approach for treating oral mucositis in cancer patients, using information from the patients’ own cells and saliva. This proposal aims to (1) determine the effects of cisplatin and 5-fluorouracil treatments mirroring the pre-clinical and clinical administration schedule of three separate infusions, with evaluations of cell layer morphology, reactive oxygen, pro-inflammatory cytokines, cell proliferation, and cell death. Effects of fractionated radiotherapy will also be evaluated on the chip, extending previous findings that used single-dose therapy. A second aim (2) will determine single and synergistic effects of putative anti-mucositis drugs: a superoxide dismutase mimetic, a defensin-mimetic, and two interleukin inhibitors. These reagents are applied clinically via intravenous infusion or by topical rinse, respectively, so they will be applied to both peripheral channels of the mucosa chip, with the same cell and molecular endpoints. Achievement of Aims 1 and 2 will involve performing microscopy assays, ELISAs, antibody arrays, fluorescence wellplate and imaging assays, and RNA sequencing. Specific molecular endpoints will include activation, expression and nuclear translocation of transcription factors Nrf-2 and NF-κB; expression of numerous pro-inflammatory cytokines including TNF-α, IL-1 α,β, IL-2, -3, -6, and -13, among others; and intracellular plus live-cell reactive oxygen species assays. Dose-response studies will be conducted for chemotherapy and anti-mucositis agents applied singly before combination treatments on the mucosa chip. 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 in vitro. The proposed systems 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. This approach is particularly effective as anti-mucositis drugs with poor performance or with unacceptable side effects can be identified and abandoned before costly pre-clinical and clinical trials.
