The mechanics, 3D structure, and biochemical composition of the extracellular matrix (ECM) significantly influences diverse biological outcomes, including cellular polarization, differentiation, apoptosis, migration, and proliferation For example, ECM alignment triggers polarized cell morphologies and facilitates migration away from the primary tumor. However, it remains poorly understood how cells spatiotemporally integrate 3D ECM cues in order to selectively orchestrate downstream signaling. The objective of the proposed research here aims to further our understanding this process. Specifically, I will investigate how mesenchymal cells spontaneously establish anteroposterior cell polarity in 3D ECM environments. Towards these means, I will evaluate the spatiotemporal evolution of cell-collagen interfaces, integrin activation, Rho family GTPase signaling, and cytoskeletal dynamics, in mesenchymal cells undergoing 3D polarization. I hypothesize that ECM fibers oriented normal to the cell surface will trigger integrin clustering and the formation of mature matrix adhesions (Aim 1). Furthermore, I hypothesize that Rho family GTPase activation will be bifurcated into focal adhesion-dependent and independent regimes (Aim 2), and that this will result in ECM context-dependent modulation of actin polymerization, bundling, dendritic branching, actomyosin contraction, and depolymerization (Aim 3). This will be accomplished by light- sheet imaging of mesenchymal cells embedded within reconstituted ECM-like environments, acute pharmacological perturbations, and computer vision analysis of ECM fiber orientation, cell shape, signal transduction, and cytoskeletal dynamics. This research will serve as the foundation from which further studies can evaluate 3D spatiotemporal cell signaling in physiological and pathophysiological ECM states.