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. 2020 Jan 24;11:515. doi: 10.1038/s41467-019-14060-x

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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 — a Histological H&E examination (left) and multiplex immunohistochemistry (IHC) staining of CD73 (red), a-smooth muscle actin (a-SMA, green), CD11b (cyan), and CD3 (gray) were performed with de-identified FFPE CRC-specimen. Nuclei were counterstained with DAPI (blue). Representative images illustrate the level of CD73 expression and its bio-distribution on CAFs (α-SMA⁺), CD11b⁺ myeloid, and CD3⁺ T cells in CAF-rich, moderate, and poor specimens. Scale bars, 100 μm. b, c Randomly selected and evenly distributed areas (~5 × 10⁵ mm²) from each multiplex IHC-stained CRC specimen were analyzed for the percentage of CD73⁺ signal that distributed on α-SMA⁺ b, CD11b⁺, and CD3⁺ cells c among the total CD73⁺ region in each area, which was set as 100%, was calculated and plotted. d Multiplex IHC staining was performed to determine tumor (EpCAM, magenta), CD73 ⁺ (red) cells, and CAFs (α-SMA, green) distribution and their potential co-localization. Scale bars, 50 μm. e The percentage of CD73⁺ signal on EpCAM⁺ tumors against total CD73⁺ region was presented. f CD73 (NT5E) gene expression in paired purified CRC-CAFs (red box), TILs, and tumors from published dataset (GSE39396) was compared. g Principle component analysis was performed with a published CRC cohort dataset (GSE39582) of 557 CRC specimens. The NT5E^high (red) and NT5E^low (blue) specimens were first defined by their CD73 expression in the top 20% and bottom 20% level, respectively, and clustered against a group of 18 fibroblast-specific (FB) genes that shows a positive correlation and against a group of 15 pre-defined group immune effector function associated genes as Immune response genes that reveals negative association. h The Kaplan–Meier survival curve demonstrates clinical correlation of NT5E^high (red) and NT5E^low (blue) expression in CRC patients (GSE 39582) with event-free survival. Data depict mean ± SEM. Unpaired Student’s t-test for b, c, e and paired Student’s t-test for f. Source data are provided in the Source Data file.