Fig. 1 | Oncogene

Fig. 1

From: Estradiol induces BDNF/TrkB signaling in triple-negative breast cancer to promote brain metastases

Fig. 1

E2 promotes metastases of TN EGFR¯ 4T1BR5 cells. a EGFR expression in brain-metastatic TNBC cells (α-tubulin was used as loading control). b OVX BALB/c female mice supplemented with E2 (n = 14), placebo pellets (OVX, n = 15), or placebo pellets + letrozole (OVX + Letrozole, n = 15) were injected IC with 4T1BR5 cells. Left: Each dot represents the median number of micrometastases (<300 µm) per mouse and the line designates the group median. Representative H&E of a micrometastasis is shown. Right: Total number of metastatic clusters per mouse in the same experiment. Representative H&E stain of a metastatic cluster is shown. The data were analyzed using Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test. c 4T1BR5 cells were injected IC in OVX female BALB/c mice supplemented with placebo or E2 pellets as in b (n = 11/group) and survival to humane end point was recorded. Log rank Mantel-Cox test was used to compare survival curves. d E0771-GFP-luc cells were IC injected in C57Bl/6 female mice treated as in b, (E2, n = 14; OVX, n = 11; OVX + let, n = 13). Left: Total number of brain metastatic clusters per mouse quantified as in b. Right: Metastatic burden quantified as total flux (photons/sec) in excised brains with representative images of luminescence signal in brains from each group (line marks group median). The data were analyzed using Kruskal–Wallis ANOVA followed by Dunn’s multiple comparisons test. e Invasion of 4T1BR5 and 231BR cells on organotypic brain slices from E2 or OVX BALB/c female mice was quantified as the number of intersections between cancer cells and concentric circles drawn from the edge of the sphere (Sholl analysis, see supplementary Fig 2 for details). Top shows representative image of a sphere at 0, 24, and 48 h after plating. Left, plots show the median number of new intersections/sphere in 5 µm increments from initial sphere edge 48 h after plating. Right graphs show total number of new intersections per sphere, 48 h after plating. n = 16 spheres/group for 4T1BR5 and n = 6/group for 231BR. Two-tailed Mann–Whitney test. Line represents median. f Double IF staining of ERα (green) and reactive astrocytes (GFAP, red) associated with BM in brain sections of OVX (n = 4) or E2-treated mice (n = 4). Left: representative image shows percentage of GFAP+ /ERα+ cells surrounding BM in OVX (n = 3) and E2-treated (n = 3) mice. Scale bar is 100 µm. Right, dots show the mean percentage of GFAP+/ERα+cells in 15 × 0.134 mm2 fields per mouse brain, two-tailed Mann–Whitney test. Line shows the group median

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