Figure 7.
Figure 7. FGFR1 localizes to the nucleus in invading cells in 3D culture and in vivo. (A) A breast cancer organotypic model was designed using a collagen/Matrigel mix containing human foreskin fibroblasts as the stromal equivalent, overlaid with MDA-MB-231 cells. OCs were raised into an air–liquid interface and cultured for 10 d before fixation in 4% PFA. (B) Hematoxylin and eosin staining revealed invading particles (group of cells) in the stroma. (C) FGF-10 was highly expressed in both invading and noninvading cells. FGFR1 was localized mainly to the nucleus (DAPI stain) of invading cells and remained mainly in the plasma membrane and cytoplasm of noninvading cells. (D) Still optical projection tomography image of an OC stained with NESO antibody, a marker for metastatic breast cancer cells (Chioni et al., 2005), highlighted that the invading cells formed distinct groups within the stroma (Video 1). (E) Anti-Ki67 staining of transverse sections from OCs revealed that cell proliferation was restricted to invading cells and not those cells that remained at the air–liquid interface. (F and G) Breast cancer OCs growing for 10 d in the presence of PD173074 showed significantly less cancer cell invasion compared with control. The invasion index comprises the combined measurements of (a) depth of invasion (mean of several measurements from each OC, taken from the top layer of the noninvading cells to the middle of the invading particles). (b) number of invading particles, and (c) mean area of the invading particles (n = 4 OCs for each condition). Error bars show means ± SEM. (H) Immunohistochemistry of human tissues from patients with an anti-FGFR1 antibody revealed that, although the IgG negative control remained clear for brown DAB staining (left), specific FGFR1 staining was detected in the myoepithelial compartment of ductal carcinoma in situ (arrows in middle image) and even stronger staining was detected in invasive lobular carcinoma (arrows in right image), including nuclear staining in some invading cells (B). This is shown by confocal sectioning of immunofluorescently stained invasive carcinoma, in which FGFR1 (green) can be seen in the nuclei (red) as labeled by open arrows (colocalization in yellow). **, P ≤ 0.01 (ANOVA). Bars: (B, F, and H) 100 µm; (C, E, and I) 50 µm; (D) 500 µm.

FGFR1 localizes to the nucleus in invading cells in 3D culture and in vivo. (A) A breast cancer organotypic model was designed using a collagen/Matrigel mix containing human foreskin fibroblasts as the stromal equivalent, overlaid with MDA-MB-231 cells. OCs were raised into an air–liquid interface and cultured for 10 d before fixation in 4% PFA. (B) Hematoxylin and eosin staining revealed invading particles (group of cells) in the stroma. (C) FGF-10 was highly expressed in both invading and noninvading cells. FGFR1 was localized mainly to the nucleus (DAPI stain) of invading cells and remained mainly in the plasma membrane and cytoplasm of noninvading cells. (D) Still optical projection tomography image of an OC stained with NESO antibody, a marker for metastatic breast cancer cells (Chioni et al., 2005), highlighted that the invading cells formed distinct groups within the stroma (Video 1). (E) Anti-Ki67 staining of transverse sections from OCs revealed that cell proliferation was restricted to invading cells and not those cells that remained at the air–liquid interface. (F and G) Breast cancer OCs growing for 10 d in the presence of PD173074 showed significantly less cancer cell invasion compared with control. The invasion index comprises the combined measurements of (a) depth of invasion (mean of several measurements from each OC, taken from the top layer of the noninvading cells to the middle of the invading particles). (b) number of invading particles, and (c) mean area of the invading particles (n = 4 OCs for each condition). Error bars show means ± SEM. (H) Immunohistochemistry of human tissues from patients with an anti-FGFR1 antibody revealed that, although the IgG negative control remained clear for brown DAB staining (left), specific FGFR1 staining was detected in the myoepithelial compartment of ductal carcinoma in situ (arrows in middle image) and even stronger staining was detected in invasive lobular carcinoma (arrows in right image), including nuclear staining in some invading cells (B). This is shown by confocal sectioning of immunofluorescently stained invasive carcinoma, in which FGFR1 (green) can be seen in the nuclei (red) as labeled by open arrows (colocalization in yellow). **, P ≤ 0.01 (ANOVA). Bars: (B, F, and H) 100 µm; (C, E, and I) 50 µm; (D) 500 µm.

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