Supplementary Figure 1: Pericytic spreading of metastatic cells.

(a) Representative images of CellTracker⁺ cells (green) co-cultured with endothelial tubes (bright field) on matrigel (scale bar, 20 μm). (b) Representative images of GFP⁺ H2030-BrM (white), CD31⁺ endothelial cells (red) and CSPG4⁺ pericytes (green) in cultured brain tissue. Right panels are insets. Yellow arrows indicate pericytes 1) displaced, 2) localized across from the cancer cells and 3) stomped on (scale bars, 20 μm). (c) Representative image of a GFP⁺ MDA231-BrM cell (white) as in b (scale bars, 10 μm). (d-e) Representative immunofluorescence images H2030-BrM cells (GFP, white) and pericytes co-stained with pericyte markers (d) (PDGFRβ, red and CSPG4, green) and oligodendrocyte markers (e) (O4 sulfatide, red and CSPG4, green). Arrowheads: double positive cells (scale bars, 5μm). Figures (a-e) are representative of 2 independent experiments. (f) Representative time lapse confocal images of H2030-BrM control (Ca: white) displacing CSPG4-DsRed⁺ pericytes (p1, p2 and p3: green) along the endothelium (DiD staining, red) in cultured brain tissue. Right panels, H2030-BrM L1CAM knockdown cells interacting with pericytes (p1-p3, DsRed: green). Arrows: direction of cancer cell movement in the next frame; solid circles: stalling. L1CAM knockdown cells eventually round up. Images are representative of 4 (Control) and 3 (shL1CAM) independent experiments (scale bars, 10 μm). (g) Mean pericyte coverage in cancer cell invested versus adjacent uninvolved vasculature depicted in Fig. 1d (n = 62, 55, 83, 96 vessels per H2030-BrM “+”, “-”, MDA231-BrM “+”, “-” cancer cell groups pooled from 2 independent experiments). P values are calculated using Mann-Whitney test. Error bars, S.E.M. (h) Immunohistochemistry images of metastatic cells (white arrowheads and L1CAM staining) interacting with pericytes and vascular smooth muscle cells (alpha-smooth muscle actin staining, brown) along the vascular wall (red dotted line) in clinically silent micrometastases in brain tissue obtained during autopsy of a treatment-naïve ER+ breast cancer patient (scale bars, 50 μm). (i) Immunofluorescence staining of brain tissue in (h) for pericytes (aSMA staining, green), endothelial cells (CD31 staining, red) and cancer cells (panKeratin staining, white) (scale bar, 5μm). Data in h-i is from one biological replicate. See Supplementary Table 1 for statistics source data.