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. 2020 Feb 17;10:2778. doi: 10.1038/s41598-020-59368-7

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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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Confocal images of immunofluorescence staining images of ALDH1A1, CXCR4, EpCAM, and PDX1 (red) scale bars 40 μm in (A) PT291 2D primary cell line (B) PT291 organoids (C) PT291 CLO (D) PT127 2D primary cell line and (E) PT127 CLO. DAPI counterstain (blue). Images are representative of three independent experiments. (F–I) Analysis comparing the fluorescence intensity between PT291 CLOs and 2D cell lines in (F) ALDH1A1 (G) CXCR5 (H) EpCAM and (I) PDX1. (J,K) Relative quantification of stem cell markers NANOG, OCT4 and SOX2 assessed using qRT-PCR in (J) PT291 PDX derived cell line, organoids and CLOs, and (K) PT127 PDX derived cell line, CLOs and PDX tumour sample. Expression is shown relative to the expression in matched cell line. 18 S rRNA used as endogenous control n = 3.