Generation and Characterization of Patient‐Derived Head and Neck, Oral, and Esophageal Cancer Organoids

Abstract Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient‐derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head‐and‐neck and anal origin. Formed rapidly (<14 days) from a single‐cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate‐to‐high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1 : Generation of esophageal cancer PDO Basic Protocol 2 : Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3 : Imaged‐based monitoring of organoid size and growth kinetics Basic Protocol 4 : Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5 : PDO content analysis by flow cytometry Basic Protocol 6 : Evaluation of drug response with determination of the half‐inhibitory concentration (IC 50 ) Support Protocol : Production of RN in HEK293T cell conditioned medium