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. 2020 Sep 9;5:183. doi: 10.1038/s41392-020-00230-7

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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. 1 — VDR is negatively correlated with the acidic microenvironment-associated CRC malignancy. a, b Schematic representation of the method for isolation of primary CRC cells and CRC stem cells. Representative images (a) and quantified data for tumor spheres (with diameters larger than 50 µm) formed by CC tissue CSCs cultured under pH 7.4 and 6.8 (b). Student’s t-test. c Flow cytometric analysis of CD133-positive CRC cells (CC tissue adherent and RKO cells) cultured under pH 7.4 and 6.8. Student’s t-test. d Enriched signaling pathways from the gene ontology (GO) database in RKO cells cultured under pH 7.4 and 6.8. e Schematic diagram illustrating the key factors (CYP27A1, CYP27B1, VDR, and CYP24A1) involved in anabolism and catabolism of 1α,25(OH)₂D₃ and the regulation of target genes. CYP27A1 cytochrome P450 family 27 subfamily A member 1, CYP27B1 cytochrome P450 family 27 subfamily B member 1, VDR vitamin D receptor, CYP24A1 cytochrome P450 family 24 subfamily A member 1. f, g qPCR (f) and immunoblotting (g) of CYP24A1, CYP27A1, CYP27B1, and VDR in CRC cells (HCT8, SW620, RKO, and CC tissue-adherent cells) cultured under pH 7.4 and 6.8. h, i qPCR of VDR in 86 CRC samples and paired adjacent normal samples (h) and in 48 primary CRC tissue and 48 recurrent CRC tissue samples from our hospital (SYSUCC; i). Student’s t-test. j qPCR data for VDR in normal tissue samples and CRC tissue samples from TCGA and other databases (obtained through Oncomine; https://www.oncomine.com). k, l Representative immunohistochemical images (left) and quantified data (right) for VDR (k) in 419 CRC tissue and adjacent normal tissue samples from representative patients with different stages of CRC at our hospital (SYSUCC). The overall survival of patients with low and high VDR expression (l) are shown. Student’s t-test, Kaplan–Meier method and the log-rank test were used. m Correlation between LAMP2 expression and VDR expression in 86 CRC samples, as described in Fig. 1h. The Pearson correlation coefficient (r) and P value are shown. Three independent experiments were performed to obtain the data in b, c and f, g. The data are shown as the mean ± SD; *P < 0.05, **P < 0.01, and ***P < 0.001