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. 2023 Sep 4;21:336. doi: 10.1186/s12916-023-03034-y

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Metformin inhibited HRCA growth by restricting stemness maintenance in vitro and in vivo. A Clustered heatmap of log2-transformed RPKMs showed the differentially expressed genes (DEGs) after metformin treatment (5 mM, 24 h). The organoids treated with metformin were cultured from P4 and P8. c-Myc was marked. B, C Immunofluorescent staining of β-catenin and Axin2 on organoids in metformin-treated (48h) and mock groups. Scale bar, 50 μm. (right panel). D Quantification of fluorescence intensity in organoids was performed and analyzed for β-catenin and Axin2. E After metformin treatment for 24 and 48 h, HRCA-PDOs were harvested to perform qRT-PCR for evaluation of the mRNA expression of LGR5, OLFM4 and c-Myc. F Expression of OLFM4 and c-Myc in HRCA-PDOs after metformin treatment for 48 h were detected by immunohistochemical staining and immunofluorescent staining, respectively. Scale bar, 50 μm. (right panel). G HRCA-PDOs were harvested to examine the expression of proliferation markers including Ki67 and PCNA by immunofluorescence staining after metformin treatment for 48 h. A representative result of three independent experiments was shown. Scale bar, 50 μm. H FACs analysis revealed an obvious cell cycle change of adenoma cells after metformin treatment (5 mM, 48 h). FACs, fluorescence-activated cell sorting. I, J Bright-field image showed the colorectal tumor formation in colon between mock and metformin-treated groups (left panel) and quantification of colorectal tumors in mice (n = 5 in control group and n = 4 in metformin-treated group) were counted and statistically analyzed (right panel). K–L qRT-PCR examination of expression of Lgr5 and c-Myc mRNA levels and immunohistochemical staining of proliferative marker Ki67 in colorectal tumors between mock and metformin-treated groups. All data were presented as mean ± SD (n = 4-5) and subjected to one-way ANOVA by the Bonferroni post hoc test in E, Two-way ANOVA followed by the Bonferroni post hoc test in H and J and Student’s t-test in D and F. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 versus mock group