Low invasive breast cancer cells and fibroblasts reciprocally influenced their plasma membrane fluidity.

<p>Pseudocoloured GP images of living tumor (MCF-7) and fibroblast (NFs or CAFs) cells (A), and reciprocal effects on membrane fluidity (B) are shown. MCF-7 cells were cultured for 6 days, alone or in presence of NFs or CAFs, in 35 mm glass-bottom Petri dishes and labelled with Laurdan (2 µM). Tumor cell/fibroblast interaction determined an enhancement of cancer cell membrane fluidity. On the other hand, MCF-7 cells promoted an increase in fibroblast membrane packing density. Data are mean±SD (error bars) of three independent experiments. Statistical significance was determined using Student's t-test, *p<0.05 vs respective homotypic culture.</p

Cancer-associated fibroblasts and their normal counterpart affected E-cadherin expression in breast cancer cells.

<p>Frozen nodule sections from MCF-7 cell homotypic cultures (A) or MCF-7 cell/fibroblast mixed cultures (B and D) were processed for E-cadherin immunostaining (Cy3: red). DAPI was used for nuclear counterstaining (blue) (C). Confocal images show the up-regulation of E-cadherin in MCF-7 cell population co-cultured with NFs (B) as compared to the MCF-7 homotypic culture (A). On the contrary, interaction with CAFs promoted a strong reduction in E-cadherin expression (D). A representative experiment of three is shown. (E) <i>F (a.u.)</i>, fluorescence intensity (in arbitrary units). Data are mean±SD (error bars) of three independent experiments. Statistical significance was determined using Student's t-test, *p<0.05.</p

Spatial distribution of breast tumor cells and fibroblasts in nodule co-cultures.

<p>Sections (10 µm) of paraformaldehyde-fixed frozen nodule co-cultures of either MCF-7 or MDA-MB-231 breast tumor cells with fibroblasts (NFs or CAFs) at day 6 of culture are shown. Sections were stained immunohistochemically using an anti-cytokeratin (large spectrum, pankeratin) monoclonal antibody to identify the reciprocal location of the two cell types (tumor cells and fibroblasts) in the nodule co-cultures. The more invasive and less differentiated MDA-MB-231 cells showed a looser association as compared to MCF-7/fibroblast co-cultures, with various cells infiltrating the fibroblast nodule core (arrows). DAB for detection; hematoxylin counterstaining; original magnification for upper panels (MCF-7/NFs, MCF-7/CAFs) and lower insets, ×100; original magnification for lower panels (MDA-MB-231/NFs and MDA-MB-231/CAFs), ×200.</p

Breast tumor cell induction of α-smooth muscle actin (α-SMA) in tumor stroma-derived fibroblasts.

<p>Sections (10 µm) of paraformaldehyde-fixed frozen nodule culture (CAFs) and co-cultures (MCF-7 or MDA-MB-231 breast tumor cells with CAFs) at day 6 of culture are shown. Sections were stained immunohistochemically using an anti-α-SMA monoclonal antibody to verify the effect of breast cancer cells on fibroblast-to-myofibroblast transdifferentiation. α-SMA expression was induced in the tumor-associated fibroblast population as a result of the interaction with the more aggressive and less differentiated MDA-MB-231 cells, demonstrating the ability of these cells to promote stroma activation. DAB for detection; hematoxylin counterstaining; original magnification, ×200.</p

Highly invasive and metastatic breast cancer cells underwent a plasma membrane fluidity increase when co-cultured with normal or cancer-associated fibroblasts.

<p>Pseudocoloured GP images of living tumor (MDA-MB-231) and fibroblast (NFs or CAFs) cells (A), and reciprocal effects on plasma membrane fluidity (B) are shown. MDA-MB-231 cells were cultured for 6 days, alone or in presence of NFs or CAFs, in 35 mm glass-bottom Petri dishes and labelled with Laurdan (2 µM). Tumor cell/fibroblast interaction determined an enhancement of cancer cell membrane fluidity, while MDA-MB-231 cells did not affect fibroblast membrane packing density. Data are mean±SD (error bars) of three independent experiments. Statistical significance was determined using Student's t-test, *p<0.05 vs respective homotypic culture.</p

Morphology of isolated normal and tumor-derived fibroblasts.

<p>Light microscopic view showing the different morphology of fibroblasts isolated from human healthy mammary skin (A) or human breast cancer (B) samples after 6 days of culture. Original magnification, ×200.</p

Primer sequences.

<p>Primer sequences.</p

Consistent anomalies observed in ET and BAT by a-CGH.

*<p>further amplification in ET, with respect to BAT.</p><p>In bold: anomalies limited to ETs.</p><p>In square bracket: genomic positions (according to NCBI 36 build) of the observed anomalies.</p><p>In round brackets: mosaicism degree of the observed anomaly. The percentage of abnormal cells was inferred using the formula proposed by Valli et al. That formula cannot be applied to the amplified segments (EGFR, CDK4, MDM2 and 15q24.1) since their ploidy status is unknown.</p

Selection of the genes significantly different between ET and BAT with a 10<i>-</i>fold difference in expression levels.

<p>Selection of the genes significantly different between ET and BAT with a 10<i>-</i>fold difference in expression levels.</p