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Cancer\u2010associated fibroblasts (CAFs) is a critical part of the tumor microenvironment, heavily influenced TIC function and fate. However, deeply investigations have been hindered by the lack of accurate preclinical models to investigate the interaction between CAFs and TIC. Organoids model have achieved major advancements as a precious research model for recapitulating the morphological aspects of organs, and thus also serving as a candidate model to investigate the mutual interaction between different cell types. Consequently, this study aimed to construct a three\u2010dimensional (3D) co\u2010culture organoid model of primary LGR5\u2010expressing tumor stem cells from primary murine liver tumors with CAFs to investigate the impact of CAFs on LGR5 marked TICs in liver cancer.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Materials and Methods<\/jats:title>\n                    <jats:p>First, both of the transgenic LGR5\u2010diphtheria toxin receptor (DTR)\u2010GFP knock\u2010in mice and transgenic Rosa26\u2010mT mice developed primary liver tumors by diethylnitrosamine (DEN) administration. Tumor organoids and CAFs were generated from those primary liver cancer separately. Second, LGR5\u2010expressing TICs organoid with CAFs were established ex vivo based on cell\u2013cell contact or trans\u2010well co\u2010culture system, and the mutual influence between those two types of cells was further investigated. Subsequently, immunodeficient mouse\u2010based xenograft model was further adopted to evaluate the influence of CAFs to LGR5 tumor stem cell, tumor formation, and metastasis.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>\n                      The co\u2010culture organoid model composed of murine liver tumor LGR5+ tumor\u2010initiating cells and CAFs in 3D co\u2010culture was successfully established, with the intention to investigate their mutual interaction. The existence of CAFs upon engrafting tumor organoids resulted in dramatic higher number of LGR5+ cells in the neoplasia when compared with engrafting tumor organoids alone. Furthermore, ex vivo culture of isolated LGR5+ cells from tumors of co\u2010engrafted mice formed significantly larger size of organoids than mono\u2010engrafted. Our results also indicated significantly larger size and number of formed organoids, when LGR5+ cells co\u2010cultured with CAF in both cell\u2013cell contact and paracrine signaling in vitro\n                      <jats:italic>,<\/jats:italic>\n                      comparing to LGR5+ cells alone. Furthermore, we found that specific knockout of LGR5 expressing cells suppressed CAF\u2010mediated promotion of tumor formation, growth, and metastasis in the experimental mice model.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>Altogether, in a 3D co\u2010culture type of murine liver LGR5+ cells and cancer\u2010associated fibroblasts, we have demonstrated robust effects of CAFs in the promotion of LGR5 marked liver TICs. We also further revealed the influence of tumor microenvironment on stem cell\u2010related therapy, suggesting the possibility of combing CAF\u2010targeted and tumor stem cell targeted therapy in treating liver cancer.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/cam4.6408","type":"journal-article","created":{"date-parts":[[2023,8,14]],"date-time":"2023-08-14T10:21:21Z","timestamp":1692008481000},"page":"18032-18049","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["Cancer\u2010associated fibroblasts nurture\n                    <scp>LGR5<\/scp>\n                    marked liver tumor\u2010initiating cells and promote their tumor formation, growth, and metastasis"],"prefix":"10.1002","volume":"12","author":[{"given":"Mingna","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Oncology Postgraduate Training Base of Jinzhou Medical University, Shanghai East Hospital  Shanghai 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Molecular Network, West China Hospital, West China School of Medicine Sichuan University  Chengdu Sichuan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Menglin","family":"Yao","sequence":"additional","affiliation":[{"name":"Department of Respiratory and Critical Care Medicine, National Clinic al Research Center for Geriatrics, Center of Precision Medicine, Precision Medicine Key Laboratory of Sichuan Province, Frontiers Science Center for Disease\u2010related Molecular Network, West China Hospital, West China School of Medicine Sichuan University  Chengdu Sichuan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qiuwei","family":"Pan","sequence":"additional","affiliation":[{"name":"Department of Gastroenterology and Hepatology Erasmus Medical Center  Rotterdam the Netherlands"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wanlu","family":"Cao","sequence":"additional","affiliation":[{"name":"Department of Oncology Shanghai 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