We have launched a collaborative effort between MD Anderson Cancer Center and University of Texas at Dallas that uses dorsal root ganglion (DRG) removed from pain phenotyped patients during neurological surgery. DRGs are taken from thoracic levels during a spine stabilization surgery and then cut in thirds. One third is saved for immunohistochemistry (IHC) or in situ hybridization (ISH), one third goes for culturing and electrophysiology, and one third is used for RNA sequencing. We have developed an extensive dataset with patient pain phenotype information, DRG neuron electrophysiological characterization and RNA sequencing. In many cases we have pairs of DRGs from the same patient where the patient had pain in one dermatome and not in another, allowing for precise case-control analysis. Our electrophysiology results clearly demonstrate that chronic pain is associated with spontaneous activity (SA) in DRG nociceptors. This is the first time that this has been demonstrated. Our RNA sequencing results identify transcriptional changes associated with chronic pain and SA in the DRG that show indications of sexual dimorphism. In males we find clear signs of immune infiltration and neuro-immune interactions as well as an increase in expression for some members of the FOS/JUN transcription factor family. In females we see an upregulation of some G-protein coupled receptors (GPCRs) and other signs of intrinsic changes in neuronal excitability. These findings give unique insights into drivers of chronic pain in the DRG in a diverse cohort of patients with important implications for chronic pain therapeutic development, including the potential need for sex-specific treatment. Our overarching hypothesis is that SA in human DRG neurons, which is a critical factor for pain generation in patients, is driven by fundamentally different mechanisms in male and female patients. We will test this hypothesis using human DRG samples and a combination of electrophysiology (Aim 1) and RNA-seq (Aim 2). In Aim 3 IHC and ISH along with pharmacological interventions guided by preliminary findings and data generated during this project will be used to define new potential therapeutic avenues. In sum, the experiments in this project will give fundamental new insight into mechanisms of chronic pain that will enable therapeutic and biomarker discovery with the opportunity for an almost immediate impact on clinical care.