RESUMO
The high attrition rate of oncology drug candidates can be in part explained by the disconnect between the standard preclinical models (e.g., 2D culture, xenograft tumors) commonly employed for drug discovery and the complex multicellular microenvironment of human cancers. As such, significant focus has recently shifted to the establishment of preclinical models that more closely recapitulate human tumors, such as patient-derived xenografts, 3D spheroids, humanized mice, and mixed-culture models. For these models to be suited to drug discovery, they should optimally exhibit reproducibility, high-throughput, and robust and simple assay readouts. In this article, we describe a protocol for the generation of an in vitro 3D co-culture spheroid model that recapitulates the interaction of tumor cells with stromal fibroblasts in pancreatic adenocarcinoma. We additionally describe protocols relevant to the analysis of these spheroids in high-throughput drug discovery campaigns such as the assessment of spheroid proliferation, immunofluorescence and immunohistochemistry staining of spheroids, live-cell and confocal imaging and analysis of cell surface markers.
