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. Author manuscript; available in PMC: 2016 Nov 18.
Published in final edited form as: Nature. 2016 May 18;533(7604):493–498. doi: 10.1038/nature18268

Figure 1. Cx43 and PCDH7 are associated with brain metastasis.

Figure 1

a, Upper Left: Contrast-enhanced MRI of representative patient with brain metastasis. Tumor (white) is surrounded by parenchymal reaction (dark grey). Upper Right: Hematoxallin-Eosin staining (H&E) of resected brain metastasis (T) and parenchyma (P). Lower Panels: Immunohistochemistry of adjacent sections for GFAP (Lower Left) and Cx43 (Lower Right). Scale bar, 10 μm. (n = 6 patient samples) b, Cx43 expression is increased in brain metastases compared with primary and normal tissue. Representative images of Cx43 staining in clinical samples from triple-negative breast cancer (TNBC) and non-small cell lung carcinoma (NSCLC). Proportion of CX43-positive samples was quantified in primary (1ry) tumours (TNBC n = 98, NSCLC n = 138), brain metastases (Mets) (TNBC n= 117; NSCLC n = 91) and normal lung tissues (n = 75) Scale bar, 100 μm. c, Upper: GFP+ H2030-BrM3 cells (green) are surrounded by GFAP+ activated astrocytes (red) in the brain parenchyma at early (day 7) and later (day 21) time points following intracardiac inoculation in mice. Blue, collagen IV (ColIV) staining in vessels. Scale bar, 10 μm. Lower: Cx43 staining (arrowhead) at the interface of GFP+ H2030-BrM3 (green) and GFAP+ astrocytes (blue). Scale bar, 10 μm. d-e, Gap junction communication between astrocytes and BrM cells. d, Time-lapse images of dye transfer from MDA231-BrM2 cells to astrocytes. See also Supplementary Information Video S1. Scale bars, 100 μm. e, Quantification of dye transfer from astrocytes to cancer cells. Histograms show red fluorescent signal in parental (Par) and BrM cells. Values are mean ± S.E.M. (Data are from n=3 biological replicates over 3 independent experiments). f-i, Cx43 and PCDH7 western immunoblotting in the indicated parental and brain metastatic derivatives (f, n=3 independent experiments), in brain metastatic cells compared to brain cell types (g, n=2 independent experiments), and in MDA231 derivatives metastatic to brain, lung (LM) or bone (BoM) (h, n=2 independent experiments). Full blots are shown in Supplementary Data. i-j, Kaplan-Meier plots of brain metastasis-free survival in 189 cases of triple-negative breast cancer (i) and 129 cases (MSKCC set2) and 58 cases (GSE3141) of lung adenocarcinoma (j), based on Cx43/PCDH7 expression in the primary tumour.