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. 2020 Apr 23;11:1961. doi: 10.1038/s41467-020-15846-0

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a An outline of the experimental strategy for studying ex vivo organoid initiation of cells derived from primary murine liver tumors. b A representative picture of organoids derived from single LGR5⁺ cells. Scale bar = 50 µm. c Representative confocal micrograph of a single LGR5⁺ cell-initiated organoid dominated by LGR5-expressing cells. LGR5-driven GFP: green. Scale bar = 20 µm. d Organoid initiation efficiency of LGR5–GFP⁺ and LGR5–GFP⁻ cells, isolated from primary tumors (LGR5⁺ cells: 25 out of 71 tissues, 35.2%; LGR5⁻ cells: 11 out of 71 tissues, 15.5%) (paired T test, 2.13 ± 0.67% vs. 0.065 ± 0.023%, n = 30, P = 0.0048). e An outline of the strategy used to study ex vivo organoid initiation of allograft tumor-derived cells. f Efficiency of organoid initiation by allograft liver tumor-derived LGR5–GFP⁺ and LGR5–GFP⁻ cells (paired T test, 40.46 ± 10.19% vs. 9.84 ± 3.93%, n = 10, P = 0.0187). g Outline of the experimental strategy used to assess in vivo tumor initiation of cells isolated from primary murine liver tumors. h Weight of tumors initiated by LGR5⁺ and LGR5⁻ cells (LGR5⁺ vs. LGR5⁻: 0.46 ± 0.046 g vs. 0.10 ± 0.10 g, n = 3) (formed tumor number: LGR5⁺ cells—3 out of 9; LGR5⁻ cells—1 out of 9). i LGR5 expression in single LGR5⁺ cell-derived allograft tumors and the corresponding primary tumors (17.42 ± 15.29% vs. 2.47 ± 1.27%, n = 3). j–n Representative pictures showing that LGR5–GFP⁺ and LGR5–GFP⁻ cells (k) were isolated from DEN-induced primary liver tumors (j). Then, LGR5–GFP⁺ cells (green arrow) initiated allograft tumors in immunodeficient mouse (l–n). The initiated allograft tumors sustained LGR5 expression (n). o An outline of the experimental strategy for in vivo tumor initiation assay of cells isolated from allograft murine liver tumors. p, q Tumor weight (p) and macroscopic aspect (q) of allografts initiated by LGR5–GFP⁺ cells and LGR5–GFP⁻ cells (isolated from allograft tumors) (0.64 ± 0.19 g vs. 0.27 ± 0.08 g, n = 11, P = 0.0418). Mean ± SEM. Source data are provided as a Source Data file.