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. 2017 Sep 6;7:10636. doi: 10.1038/s41598-017-09928-1

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© The Author(s) 2017

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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Prospective isolation of mesenchymal cell subset in NSCLC with increased expression of PD-L1. (A–E) Representative pseudocolor dot plots from a patient specimen (BE0133, squamous cell carcinoma) showing the gating strategy to identify clusters of mesenchymal cells within the NSCLC tumor specimen. Cells (R1 gate) initially displayed on a SSC/FSC color density plot (A) subgated to select single cells based on FSC-Area versus FSC-Height (R2 gate) (B), were further subgated for identification of live (7-AAD negative) cells (R3 gate) (C). Single, live cells (Gate R3) were displayed on a bivariate plot showing the presence of a cluster of Lin- cells that lacked the epithelial cell adhesion marker EpCAM (CD326) (R4 gate) and a cluster positive for EpCAM (R5 gate) (D). Lin-EpCAM- cells (Gate R4) displayed as a bivariate plot to identify CD73 and CD90 subset of cells (E). The expression of each subset for PD-L1 is shown on a histogram plot (F) (black: CD73+CD90+, red: CD73-CD90+, blue: CD73+CD90−, gray: CD73-CD90−). (G) Scatter plot showing the Lin-EpCAM-CD73+CD90+ cells (gate R6), as a percentage (%) of total counted events determined from Gate R4 (n = 13, biological replicates). (H) Scatter plot showing the mean fluorescence intensity (MFI) for PD-L1 in the Lin-EpCAM-CD73+CD90+ mesenchymal cell subset (gate R6) in tumor (T) versus matched nonadjacent uninvolved tissue (N) (n = 13, biological replicates). (I) Representative images obtained from a single lung adenocarcinoma cell using ImageStream®. Statistical analysis in G and H by Student t-test, two-tailed, for comparison of paired parametric data. All tests were two-tailed. *p < 0.05 were considered significant. See related supplementary data Figures S2–4.