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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. 2 — mt-cells further potentiate fl-e-CAFs and mt-e-CAFs to induce fl-CCs invasion. a Representation of (i) local crosstalk between cancer cells and CAFs, (ii) long-range CC driven interactions, and (iii) long-range CAF-driven interactions between cancer cells and CAFs across a primary pancreatic tumor. b Top panel: representation of media conditioning driven by cancer cells or by CAFs and treatment of fl-e-CAFs with conditioned media (CM) during remodeling of a collagen matrix over 12 days. Lower panel: representative images and quantification of collagen birefringence in fl-e-CAFs matrices treated with CM generated by fl-e-CAFs, mt-e-CAFs, mt-CCs or fl-CCs, and normalized to treatment with CM from fl-e-CAFs. Scale bar: 100 μm. c Top panel: representation of media conditioning driven by cancer cells or by CAFs and treatment of mt-e-CAFs with CM during remodeling of a collagen matrix over 12 days. Lower panel: representative images and quantification of collagen birefringence in mt-e-CAFs matrices treated with CM generated by mt-e-CAFs, fl-e-CAFs, mt-CCs or fl-CCs, and normalized to treatment with CM from mt-e-CAFs. Scale bar: 100 μm. d Schematic representation of 3D CAF-based organotypic invasion assay. e Representative H&E staining of organotypic invasion assay and quantification of invasive index for fl-CCs or mt-CCs invading into fl-e-CAF matrices (blue bars) or into mt-e-CAFs matrices (purple bars). Scale bar: 200 μm. f Schematic representation of 3D organotypic invasion assay including pharmaceutical removal of CAFs following matrix establishment and continuous media conditioning driven by CAFs during cancer cell invasion. g Quantification of invasive index and representative images of H&E stained organotypic matrices with fl-CCs and mt-CCs invading into matrices generated by fl-e-CAFs and with continuous media conditioning driven by fl-e-CAFs or by mt-e-CAFs during invasion. Scale bar: 200 μm. h Schematic representation, i quantification of invasive index and representative images of H&E stained organotypic matrices with fl-CCs and mt-CCs invading into matrices generated by mt-e-CAFs and with continuous media conditioning driven by fl-e-CAFs or by mt-e-CAFs during cancer cell invasion. Scale bar: 200 μm. Contraction and invasion assays were conducted in three biological repeats with three technical replicates per biological repeat. Individual data points are presented with mean values and SEM. *p < 0.05