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. 2021 Feb 25;11:4690. doi: 10.1038/s41598-021-84058-3

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© The Author(s) 2021

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/.

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Single-cell RNA-seq (scRNA-seq) of Sr-CDX CAFs revealed the differentiation of AD-MSCs into CAF subpopulations. (a) Schematic illustration of the scRNA-seq analysis platform. The transcript profiles of Sr-CDX CAFs were compared with those of the original AD-MSCs. Sr-CDX CAFs labeled with GFP were obtained by tumor digestion, followed by FACS, and viable CAFs were analyzed with droplet-based scRNA-seq. (b and c) Original AD-MSCs; (b) clustering of represented t-distributed stochastic neighbor embedding (t-SNE) plot formed three clusters. (c) Violin plot of marker genes for myCAFs (ACTA2 and TPM1), iCAFs (IL-6 and LIF), apCAFs (CD74 and HLA-DRA), and common CAFs (COL1A1 and FAP). Red plots indicate median expression level. The myCAF marker expression was observed mainly in clusters 1 and 2. Clusters 1 and 2 were defined as “myCAF-like cluster.” (d–f) Sr-CDX CAFs; (d) clustering of t-SNE plots formed seven clusters. (e) Violin plot of various marker genes for myCAFs, iCAFs, apCAFs, and common CAFs. Red plots indicate median expression level. (f) Heatmap showing different expression levels of CAF markers among the seven clusters. The iCAF and apCAF markers were expressed mainly in clusters 1–4 and 7, whereas myCAF markers were expressed mainly in clusters 5–7. iCAF, apCAF, myCAF, and common CAF marker expression was identified in cluster 7. Clusters 1–4 were defined as iCAF/apCAF-like clusters, clusters 5 and 6 as myCAF-like clusters, and cluster 7 as myCAF/iCAF/apCAF-like cluster. (g and h) Representative IHC image of human PDAC and Sr-CDX tumors. (g) Each plot is stained for cluster markers IL-6 (cluster 1), NCAPG (cluster 2), PTX3 (cluster 5), and COMP (cluster 6). (h) Each plot is stained for cluster 7 marker NCAM1 (upper image) and stained for αSMA (red) and IL-6 (brown, lower image). The small plots on the right show a magnification of the indicated area (black rectangle). Hematoxylin (blue) was used as a nuclear counterstain. Arrowheads indicate each cluster marker positive CAF. Arrows indicate αSMA/IL-6 double-positive CAFs. D, cancer duct structure. Scale bar, 100 µm.