Fig. 2

The impact of FN-EDA on macrophage functions in breast cancer. A FN-EDA and CD68 immunofluorescence staining in HR- and HER2-positive breast cancer and in triple-negative breast cancer (TNBC) tissue (scale bar, 20 µm). B–D Freshly isolated monocytes were incubated for 48 h with CM from HR⁺ breast cancer cell lines and from TNBC cell lines. The data from high-level fibronectin-expressing TNBC cancer cell lines MDA-MB-231 and HCC38 are shown in red. Expression of CD163, CD206, CD80, and CD86 macrophage differentiation and activation markers was assessed by flow cytometry. B Representative flow cytometry dot plots show the gating strategy for the monocytes treated with CM from HR⁺ MCF-7 and TNBC MDA-MB-231 cells. C Percentage of CD163⁺CD206⁺ macrophages and D median fluorescence intensity (MFI) value of CD80 and CD86 on CD163⁺CD206⁺ cells. E Increasing amounts of monocytes treated with MCF-7 or MDA-MB-231 CM were co-cultured with T cells. T cell proliferation was assessed by flow cytometric CFSE dilution assay. F Phagocytosis, G production of reactive oxygen species, and H migration capacity of monocytes following the treatment with MCF-7 or MDA-MB-231 CM. I Polarization of monocytes on the surfaces coated with the plasma fibronectin (pFN), the recombinant FN lacking the EDA domain (rFN-EDA⁻) and the recombinant FN with the EDA domain (rFN-EDA⁺). The cells were pre-treated with CM from the TNBC cell lines. MDA-MB-468 cell line represents a TNBC cell line with low-level fibronectin expression (A.U, arbitrary units). Fluorescence microscopy images of actin staining for monocyte polarization analysis on different surfaces are presented on the right-hand side (scale bar, 40 µm). Each experiment was repeated at least three times with the monocytes from different donors. (Mean ± SEM, Student’s t test; *, P < 0.05; **, P < 0.01)