Abstract This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. Fluctuations in the active biomechanical properties of cells are understudied, but evidence suggest they play a critical role in both core physiological processes and in disease. For example, tissue phase transitions, from elastic- to fluid-like (or jammed to unjammed) are thought to arise in part from increased noise in cell junctional mechanics. These forces can also result in shedding of cellular material like exosomes. Both of these processes play a central role in cancer progression, most notably in invasion and metastasis. However, a major challenge is identifying the specific subcellular origin of these forces and the machinery responsible for them. For example, studies of tissue fluidization using vertex modeling approaches have defined the necessary and sufficient geometric changes for tissue fluidization in epithelia. Specifically, active fluctuations in cell junction length are required for the T1 transitions that change junctional topology and allow cells to diffuse like a fluid. Actomyosin dynamics can drive these transition in the absence of cell division and apoptosis, but these models focus on actomyosin activity at apical/lateral interfaces (between cells), but largely ignore more indirect sources of activity, for example deriving from tractions generated at the basal surface which acts to sheer lateral and apical cell junctions. By tracking the dynamics of single cells in both normal and transformed primary human mammary epithelial organoids we see evidence that the activity necessary to fluidize a tissue derives from interactions between cells and their basal interface at the ECM. At the single cell level, we reason that this activity manifests as fluctuations in cell tractions, specifically at basal cell-ECM interfaces, and at the tens of minutes timescale. In this supplemental proposal, we will first develop a platform allowing the measurement of dynamic cell tractions at the cell-ECM interface which we will apply to single normal and transformed mammary epithelial cells. Second, we will develop a parallel assay for measuring cell tractions at the cell-cell interface. We hypothesize that the magnitude of fluctuations at the cell-ECM interface will be several fold higher than at lateral interfaces, and that the largest fluctuations will occur at the tens of minutes timescale, consistent with that of junctional fluctuations in intact tissues. Successful development of this assay will allow us to investigate the impact of mechanical fluctuations in processes spanning tissue fluidization, cancer cell invasions, and exosome shedding.