Adrenocortical carcinoma (ACC) is an aggressive malignancy with a poor 5-year survival rate of 6% for patients with metastatic disease. There are no targeted therapies for these patients. Unfortunately, current treatments only slow disease progression for an average of 5 months, after which drug resistance quickly develops. As such, there is a critical need to identify new targets of intervention and develop new treatments for ACC. ACC can be characterized by significant heterogeneity both intertumorally between patients and intratumorally within an individual tumor, making choosing an effective therapeutic strategy challenging. Several mechanistic pathways have been identified that may correlate with disease progression: IGF2, Wnt/β-catenin (Wnt), and cell cycle regulating pathways such as p53/retinoblastoma protein (Rb) are dysregulated in 90% of ACC, and correlate with metastatic disease. Unfortunately, the intratumoral heterogeneity of IGF2, Wnt, and p53/Rb dysregulation and its contributions to ACC tumor progression is unknown, although each of these pathways has been implicated or correlated with tumor invasion, matrix metalloproteinase (MMP) expression, or metastasis in other cancers. This lack of understanding can partially be attributed to the lack of appropriate preclinical research models. Most attempts to generate ACC models have been unsuccessful. The small number of existing models do not adequately reflect the oncogenic signaling pathways and intratumoral heterogeneity of human ACC. A human-based ACC model system permitting functional and transcriptomic characterization of tumor subpopulations following metastasis does not exist. To address these critical research gaps, we developed the first patient-derived organoids (PTOs) from patient samples as well as an ACC metastasis-on-a-chip (MOC) platform. Our MOC platform is an in vitro microfluidic system that incorporates tumor organoids, recirculating fluid flow, and downstream tissue organoids, which can recapitulate aspects of metastasis from a primary tumor site to metastatic sites. In addition, we have developed MMP peptide biosensor technology that integrates into our organoids, enabling near-real time observation of MMP-mediated tumor cell invasion. We will deploy this platform to sort tumor cells into metastatic/motile versus non-metastatic/less motile subpopulations for analysis of expression of dysregulated pathways in ACC (IGF2, Wnt, and p53/Rb) and subpopulation heterogeneity determined by single cell RNA sequencing. We hypothesize that IGF2, Wnt, and cell cycle dysregulation correlates with increased metastasis kinetics and MMP activity in ACC PTOs deployed in our MOC platform. Towards this hypothesis: Aim 1 will delineate metastasis kinetics, MMP activity, and proliferation of ACC PTO cells; Aim 2 will define ACC intratumoral heterogeneity of critical oncogenic pathway dysregulation using single cell-RNA-sequencing; Aim 3 will determine the relative importance of each dri...