Three-dimensional single cell patterning for mid- to high-throughput studies of tumor cell and extracellular matrix heterogeneity

Abstract

Cancer heterogeneity includes cancer cell genetic heterogeneity and heterogeneity in the tumor microenvironment (TME)--both stromal cells and extracellular matrix (ECM). Determining which combinations of the vast array of possibly interacting heterogeneities drive tumor progression presents a major multi-disciplinary challenge in cancer research. To make effective treatment decisions this challenge must be addressed. The practical challenge is assessing cellular heterogeneity in a statistically powerful way with respect to targeted TME characteristics. Here we present a simple, but extensible, and low-cost method for conducting mid- to high-throughput and long-term studies of heterogeneous cell responses to various biomechanical stimuli in 3D models mimicking the biomechanical properties of the ECM. Using a platform we term \"the drop-patterning chip\" thousands of cells are simultaneously transferred from microwells and fully embedded, only using the force of gravity, in precise patterns in 3D. This method allows for throughputs approaching flow-through methods, which lack phenotypic information on cell-matrix interactions, and does not rely on expensive or harsh patterning forces, which often times also result in a proximal stiff surface.