Skip to main content
Fig. 1 | Breast Cancer Research

Fig. 1

From: A positive feedback loop driven by fibronectin and IL-1β sustains the inflammatory microenvironment in breast cancer

Fig. 1

Expression of total fibronectin (tFN) and the alternatively spliced variant of cellular fibronectin harboring the extra domain A (FN-EDA) in breast cancer. A Gene expression of tFN and FN-EDA was evaluated in freshly obtained treatment-naïve breast tumor samples (n = 62) by quantitative RT-PCR. Quantitation of the target gene expression was first normalized to the housekeeping β-actin gene expression for each sample. Then, the Ct values from the tumor-adjacent histopathologically normal breast tissue were used for additional normalization of the data. Results were distributed according to hormone receptor (HR) and HER2 status. B Kaplan–Meier plot for the estimation of survival in breast cancer patients (n = 995, from METABRIC database) with low and high FN-EDA expression. C Immunofluorescence analysis of FN-EDA in the breast tumor specimens. Representative images of epithelial cellular adhesion molecule (EpCAM) and FN-EDA are demonstrated for each breast cancer subtype according to HR and HER2 expression. Large magnifications of the merged images are given as indents. The micrographs from one patient analyzed out of eight are shown for each subtype. (Scale bar, 10 µm). D Total FN and FN-EDA protein levels were determined in breast cancer cell lines by Western blot. E Change in tFN mRNA expression upon treatment with recombinant IL-1β, IL-6, or TNF-α in breast cancer cell lines was analyzed by RT-PCR. Quantitation of the target gene expression was first normalized to the housekeeping β-actin gene expression. Then, the Ct values from the control (untreated) cells were used for additional normalization. The dashed line crossing at 2.−ΔΔCt = 1 represents an equal expression level with the untreated control cells. The change in gene expression was calculated with 2e-ΔΔCt or -ΔΔCt formulas, which was previously described [23]. The data from high-level fibronectin-expressing TNBC cancer cell lines MDA-MB-231 and HCC38 are shown in red. F The total FN secretion was assessed by ELISA and G the change in cell-associated (both intracellular and surface-bound) tFN levels were assessed by flow cytometry in the breast cancer cell lines stimulated with IL-1β, IL-6, or TNF-α for 24 h. H The impact of IL-1β on FN-EDA expression and cell morphology was assessed by immunofluorescence (scale bar, 40 µm). Representative micrographs are shown for MCF-7 and MDA-MB-231 cells. I Total FN and FN-EDA levels secreted into the culture media of breast cancer cell lines upon IL-1β treatment. Western blot bands were quantified, and the proportion of tFN to FN-EDA was shown in the bar graph on the right. J A correlation analysis between IL-1β and FN-EDA gene expression levels from breast tumor samples. The cell line data are from at least three independent experiments (Mean ± SEM, Student’s t test; *, P < 0.05; **, P < 0.01)

Back to article page