In this protocol, we outline the procedure for generation of tumor spheroids within 384-well
hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a
physiologically representative microenvironment. We outline the formation of patient derived
cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough
analysis following drug treatment. Specifically, we describe collection of spheroid
morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data …