PROJECT SUMMARY/ABSTRACT Radiation-induced xerostomia (RIX) is a condition of subjective dry mouth caused by radiation therapy to the head and neck and manifested as hyposalivation and altered sialochemistry. RIX is the most common chronic side effect observed in HNC patients receiving radiation therapy and despite improvements in radiation delivery remains a critical issue for patients. Currently available treatments provide temporary palliation and in some cases (e.g. pilocarpine) can be accompanied by side effects that are as bad or worse than xerostomia. There is a critical need for a treatment that will safely and effectively alleviate RIX without compromising patient quality of life. A small phase I study suggested that MSC therapy could improve the perception of dry mouth and salivary gland function in patients with RIX. However, the mechanisms by which MSCs elicit this effect remain unknown and none of the successful studies investigated to use of cryopreserved MSCs. We aim to fill this important knowledge gap by identifying an optimal source of MSCs for treatment and understanding how MSCs improve salivary function to optimize treatment to maximize patient benefit. This proposal intends to fill this knowledge gap which will potentially lead to the development of novel and effective therapies for RIX. We hypothesize that MSCs provide a reparative effect to the salivary gland through paracrine signaling. We will investigate the short-term and long-term functions of MSCs following injection into the submandibular gland and identify whether there is an ideal potential tissue source for MSCs in terms of cryo-recovery and RIX treatment. We seek to address three questions critical to understand and treat RIX: 1) identify an ideal tissue source for MSC to treat RIX; 2) investigate the effects of MSC treatment on the salivary gland in acute and late-phase radiation damage; and 3) determine if MSC-based products elicit the same effects as MSCs alone. In Aim 1, we will evaluate the ability of MSCs derived from marrow, adipose, and submandibular gland tissue to recover after cryopreservation following IFN-ɣ pre-licensing and evaluate the effects of these MSCs on salivary tissue in vivo and in vitro. We will characterize the secretome of MSCs from different tissue origins alone and investigate bi-directional effects of salivary gland tissue on this secretome using a salivary organoid model. Finally, we will confirm in vivo, differences in reparative effects based on MSC source. In Aim 2, we will define the effects of IFN-ɣ pre-licensed, cryopreserved MSCs in RIX by studying both acute and long-term effects on saliva production and salivary gland architecture in-vivo. We will also investigate the effects of MSC- based products like MSC conditioned media and MSC-derived exosomes on salivary function in vivo and in vitro. Together this work will provide an improved understanding of how MSCs ameliorate radiation damage, support the development of novel...