Fig. 2: Rab11b is up-regulated during metastatic adaptation to the brain microenvironment.

a Representative images of H&E (top) or Rab11 immunohistochemical staining of human primary breast cancer or breast cancer brain metastases (arrowheads). Left, Rab11 IHC scoring. Analysis of contingency, Fisher’s exact test. Scale bar 100 μm. b qPCR for Rab11 isoforms in MDA-231 cells grown in culture, primary tumors (21 dpi), or brain metastases (21 dpi). Values for each isoform normalized to cells in culture. n = 3 independent cell samples, 7 animals/group. Boxes, first to third interquartile range, line, mean, whiskers, minimum and maximum values. ANOVA, Dunnett’s multiple comparison. c qPCR for Rab11b in cells in culture versus brain metastases. Values are normalized to MDA-231 cells in culture. n = 3 independent cell samples, 4 animals/group Line, mean. Student’s t test. d Representative H&E and Rab11b immunohistochemical staining of brain metastases, and MDA-231 primary tumor or murine brain. Scale bar 100 μm. e Top, qPCR for Rab11b in cell lines co-cultured with primary murine glia for 2 days. All values normalized to single culture. Bars, mean ± s.d. ANOVA, Dunnett’s multiple comparison. Bottom, immunostaining for Rab11b (green, red) and nuclei (DAPI, blue) in cell lines cultured alone or co-cultured with primary murine glia for 5 days. n = 3. Scale bar 50 μm. f qPCR for Rab11b in MDA-231 primary tumors or metastases as indicated, collected at time points indicated. All values are normalized to MDA-231 cells in culture. n = 3. Boxes, first to third interquartile range, line, mean, whiskers, minimum and maximum values. Two-way ANOVA with Tukey’s multiple comparison test. g Representative H&E, cytokeratin 8 (K8), and Rab11b immunohistochemical staining of MDA-231 primary tumors, brain or lung metastases, at 7 dpi. Scale bar 20 μm. h MDA-231 cells were intracranially injected and brain metastases dissected at 7 dpi using fluorescence signal as a guide. Metastases dissociated and brain parenchymal cells were removed using magnetic bead-based stromal cell depletion. Naive brain (brain), or stroma depleted brain met (7 dpi brain met) samples were lysed and subjected to Rab11b activation assay, followed by immunoblotting. For all panels, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.