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. 2021 Oct 29;6:366. doi: 10.1038/s41392-021-00769-z

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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 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. 2 — DNA methylation of the metabolism genes in macrophages is induced by direct interaction with PDA cells through GARP/TGF-βRII-integrin αV/β8. a, b Nqo-1 and Aldh1a3 methylation in mouse BMDMs in a transwell system separated from KPC cells by an 8-μm or 1-μm pore membrane that, respectively, allows or not allows tumor cells to migrate through and direct contract with macrophages and in BMDMs cultured with TCM. Nqo-1 and Aldh1a3 methylation quantified as described in Supplementary Methods. *P < 0.05 (Mann–Whitney U test). c Lucifer Yellow labeled-KPC cells were co-cultured with unlabeled BMDMs. Thick arrows indicate macrophages that contain Lucifer Yellow spread from KPC cells (thin arrow) around them. Scale bar: 20 μm. d GARP expression on M0, M1-like, and M2-like macrophages measured by immunofluorescent staining with FITC-conjugated anti-GARP antibody. Arrow indicates macrophages that have the highest fluorescence within each image. Scale bar: 20 μm. *P < 0.05 (ANOVA). Histogram (right panel) shows quantification of fluorescence intensity. e Multiplex immunohistochemistry (IHC) was performed on a single slide of human PDA tissues for GARP (in green), CD68 (in red) and CD163 (in purple). A representative among 20 human PDAs tested is shown. Arrows (both panels) indicate GARP-expressing CD68⁺CD163⁺ (M2-like) macrophages^; and arrowheads (left panel) indicate GARP-expressing CD68⁺CD163^- (M1-like) macrophages^. Notched arrowheads (right panel) indicate CD68⁺CD163⁺ (M2-like) macrophages with little GARP expression. Scale bar: 50 μm. f Multiplex IHC staining of GARP (in green) on F4/80⁺ (in red) macrophages in PDAs from KPC mice. Scale bar: 50 μm. g TGF-βRII and GARP on cell surface of M0, M1-like, and M2-like macrophages co-stained and analyzed by flow cytometry. h Quantification of the percentages of TGF-βRII on cell surface of M0, M1-like, and M2-like macrophages by flow cytometry. *P < 0.05 (ANOVA). i Integrin subunits ɑV and β8 cell-surface expression was measured by flow cytometry. j IHC staining of PDA and normal pancreas tissues from KPC mice with anti-integrin ɑV and β8 antibodies. Scale bar: 100 μm. Data were from technical triplicates and representative of two experiments