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This work presents a versatile protocol for establishing and maintaining oral and esophageal organoid cultures from murine and human samples, including normal and cancer states. It also develops an ex vivo co-culture model using patient tumor-derived organoids and autologous CD8+ T lymphocytes to evaluate CD8+ T cell–mediated tumor killing. 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Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2024) 17, dmm050319 . doi:10 . 1242/dmm.050319  \nRESOURCES & METHODS  \nEstablishing mouse and human oral esophageal organoids to investigate the tumor immune response  \nYuan Jiang1,2,*, Hua Zhao3,4,*, Shuai Kong1,2, Dan Zhou1,5, Jinxiu Dong1, Yulan Cheng4, Shuo Zhang1,2, Fei Wang1,2, Andrew Kalra4, Nina Yang4, Dan-Dan Wei2,6, Jian Chen1, Yuan-Wei Zhang1, De-Chen Lin3,‡, Stephen J. Meltzer4,‡ and Yan-Yi Jiang1,2,‡  \nABSTRACT  \nOrganoid culture systems are very powerful models that recapitulate in vivo organ development and disease pathogenesis, offering great promise in basic research, drug screening and precision medicine. However, the application of organoids derived from patients with cancer to immunotherapeutic research is a relatively untapped area. Esophageal cancer is one of the most lethal malignancies worldwide, including two major pathological subtypes: esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC shares many biological and genomic features with oral squamous cell cancers. Herein, we provide a versatile protocol for the establishment and maintenance of oral and esophageal organoid cultures derived from both murine and human samples. We describe culture conditions for organoids derived from normal tongue, esophagus and gastroesophageal junction, esophageal cancer and Barrett’s esophagus. In addition, we establish an ex vivo model by co-culturing patient tumor-derived organoids and autologous CD8+ T lymphocytes to assess CD8+ T cell-mediated tumor killing. Our protocol can also be modified for organoid establishment from othersquamous epithelia and carcinomas. The co-culture model can serve as a template for studies of other tumor-immune cell interactions and the efficacy of immune checkpoint blockade therapy.  \nKEY WORDS: Organoids, Immune response, Oral and esophageal cancer  \nINTRODUCTION  \nOrganoids are three-dimensional (3D) cell cultures based on the principles of stem cell differentiation. Compared with traditional  \n1Institute of Health and Medical Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China. 2University of Science and Technology of China, Hefei 230026, China. 3Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry, and Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA. 4Division of Gastroenterology and Hepatology, Department of Medicine and Oncology, Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA. 5Institutes of Physical Science and Technology, Anhui University, Hefei 230601, China. 6Hefei Cancer Hospital, Chinese Academy of Sciences, Hefei 230031, China.  \n*These authors contributed equally to this work.  \n‡Authors for correspondence ([dechenli@usc.edu](dechenli@usc.edu); [smeltzer@jhmi.edu](smeltzer@jhmi.edu);  \n[yanyij@cmpt.ac.cn](yanyij@cmpt.ac.cn))  \n D.-C. L. , 0000-0002-1951-367X; S.J. M. , 0000-0002-9131-1052; Y.-Y.J. , 0000- 0002-7432-8661  \nThis is an Open Access article distributed under the terms of the Creative Commons Attribution License ([https://creativecommons.org/licenses/by/4.0](https://creativecommons.org/licenses/by/4.0)), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.  \nHandling Editor: Owen Sansom  \nReceived 24 May 2023; Accepted 24 November 2023  \ntwo-dimensional (2D) cell cultures, organoids exhibit complex structures and functions that more closely recapitulate the tissues and organs from which they are derived, providing a superior model for studies of development and disease pathogenesis (Drost and Clevers, 2018; Lancaster and Knoblich, 2014; Tuveson and Clevers, 2019) . In addition, relative to animal models, it is more convenient and feasible to establish organoids, while avoiding the potential ri","cbCaie7DQNy4YEng","https://ap.wps.com/l/cbCaie7DQNy4YEng","pdf",4674825,16,"English","# RESOURCES & METHODS\n## Establishing mouse and human oral esophageal organoids to investigate the tumor immune response\n# ABSTRACT\n# KEY WORDS\n# INTRODUCTION","[{\"question\":\"What is the main goal of establishing mouse and human oral esophageal organoids in this protocol?\",\"answer\":\"To investigate the tumor immune response using organoid cultures derived from normal and cancer samples, including an ex vivo co-culture system with CD8+ T lymphocytes.\"},{\"question\":\"Which esophageal cancer subtypes and related tissue sources are covered?\",\"answer\":\"The protocol addresses oral and esophageal organoids derived from normal tongue, esophagus, and gastroesophageal junction, esophageal cancer, and Barrett’s esophagus, and it frames cancer biology around ESCC and EAC.\"},{\"question\":\"How is the ex vivo tumor-immune model designed to assess immune function?\",\"answer\":\"By co-culturing patient tumor-derived organoids with autologous CD8+ T lymphocytes to evaluate CD8+ T cell–mediated tumor killing.\"}]","Establishing mouse and human oral esophageal organoids to investigate the tumor immune response | PDF",1790000916]