Oral cancer induces severe pain that disrupts talking, eating and drinking. Patients develop tolerance to the opioids used to treat this pain (mechanical allodynia); progressively larger doses are required. Unfortunately, opioids produce debilitating side effects including profound sedation. A non-opioid pharmacologic strategy to alleviate oral cancer pain is imperative; patients wait days or weeks before surgical resection, suffer from recurrence or cannot be cured. In previous work we found that protease-activated receptor-2 (PAR2) contributes to oral cancer mechanical allodynia. In more recent work we demonstrated that oral cancer and associated macrophages secrete proteases into the oral cancer microenvironment. We found that two poorly characterized proteases, legumain (Lgmn) and cathepsin-S (Cat-S), exhibit highly upregulated expression and activity in the oral cancer microenvironment and potentially mediate cancer pain. These proteases cleave PAR2 on sensory neurons. Cleavage at specific sites on PAR2 stabilizes receptor conformations which in turn promote activation of receptor signaling and trafficking pathways. These signaling pathways lead to TRPV4 activation and hyperexcitability of nociceptors. The work we now propose will define mechanisms of Lgmn and Cat-S in oral cancer pain. We propose to identify and localize activated proteases in oral cancers using our fluorescently-quenched activity-based probes (qABPs) that covalently interact with activated proteases. We will unequivocally identify and determine the cellular origin of activated proteases present in tumors from patients with oral cancer. We will then analyze the correlation between protease activity and pain scores, and determine whether tumor proteases induce PAR2-dependent activation of nociceptors. Using mice that lack PAR2 or TRPV4 in Nav1.8 nociceptors, and our validated models of oral cancer pain, we will determine whether tumor cell and macrophage proteases cause pain by activating PAR2 and TRPV4 on nociceptors. Furthermore, we will use our new Par2-muGFP mice for PAR2 localization and trafficking with high specificity and spatiotemporal fidelity. Lastly we will define the mechanisms by which tumor cell and macrophage proteases activate PAR2 and TRPV4, induce hyperexcitability of nociceptors, and cause oral nociception. We assert that protease inhibitors as well as PAR2 and TRPV4 antagonists hold therapeutic potential for oral cancer pain; we ultimately seek to exploit these mechanisms to develop cancer pain therapies.