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. 2019 Aug 12;10:3637. doi: 10.1038/s41467-019-10968-6

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© The Author(s) 2019

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 — Isolation and characterization of CAFs from KPflC and KPC primary tumors. a Representation of isolation of cancer cells and CAFs from poorly metastatic KPflC (top panel) and highly metastatic KPC (lower panel) primary tumors using the previously generated LSL-E-cadherin-GFP mouse. b Brightfield and GFP imaging of cancer cells and CAFs isolated from E-Cadherin-GFP-KPflC tumors and from E-Cadherin-GFP-KPC tumors. Scale bar: 100 μm. c Immunocytochemistry staining of markers of CAF activation (ACTA2 α-smooth muscle actin; FAP fibroblast activated protein) in fl-e-CAFs and mt-e-CAFs. Scale bar: 100 μm. d Immunofluorescence staining of CAFs and cancer cells for markers of pancreatic epithelial cells (E-cadherin, keratin 19). Scale bar: 100 μm. e Representation of CAF-driven contraction assay. f Representative images of CAF-collagen matrices and quantification of matrix area following a 12-day contraction assay. Scale bar: 1 cm, n = 3 biological repeats with three technical replicates per biological repeat. g Shear rheology measurements of storage modulus of matrices remodeled by fl-e-CAFs or by mt-e-CAFs and normalized to values found in fl-e-CAFs matrices. n = 3 biological repeats with three technical replicates per biological repeat. h Polarized light imaging and quantification of picrosirius red-stained collagen matrices remodeled by fl-e-CAFs or by mt-e-CAFs. Scale bar: 100 μm, n = 3 biological repeats with three technical replicates per biological repeat. i Representative maximum projections of Second Harmonic Generation (SHG) signal in collagen matrices remodeled by fl-e-CAFs and by mt-e-CAFs and quantification of SHG signal intensity through a 60 μm z-stack and at intensity peak (bar graph inset). Scale bar: 100 μm, n = 3 biological repeats with three technical replicates per biological repeat. j Representative single-plane SHG images of collagen matrices generated by fl-e-CAFs or by mt-e-CAFs and quantification of GLCM correlation (bar graph inset depicts GLCM mean correlation normalized to mean correlation found in fl-e-CAFs matrices). Scale bar: 100 μm, n = 3 biological repeats with three technical replicates per biological repeat. k Representative images of picrosirius red staining imaged with brightfield or polarized light and quantification of collagen birefringence signal in primary KPflC and KPC tumors. n = 7 for KPflC tumors and n = 9 for KPC tumors. Data are presented as mean values with SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001