Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor\u27s heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein, the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing
