Functional Screen of Paracrine Signals in Breast Carcinoma Fibroblasts

<div><p>Stromal fibroblasts actively participate in normal mammary gland homeostasis and in breast carcinoma growth and progression by secreting paracrine factors; however, little is known about the identity of paracrine mediators in individual patients. The purpose of this study was to characterize paracrine signaling pathways between breast carcinoma cells and breast carcinoma-associated fibroblasts (CAF) or normal mammary fibroblasts (NF), respectively. CAF and NF were isolated from breast carcinoma tissue samples and adjacent normal mammary gland tissue of 28 patients. The fibroblasts were grown in 3D collagen gel co-culture with T47D human breast carcinoma cells and T47D cell growth was measured. CAF stimulated T47D cell growth to a significantly greater degree than NF. We detected a considerable inter-individual heterogeneity of paracrine interactions but identified FGF2, HB-EGF, heparanase-1 and SDF1 as factors that were consistently responsible for the activity of carcinoma-associated fibroblasts. CAF from low-grade but not high-grade carcinomas required insulin-like growth factor 1 and transforming growth factor beta 1 to stimulate carcinoma growth. Paradoxically, blocking of membrane-type 1 matrix metalloprotease stimulated T47D cell growth in co-culture with NF. The results were largely mirrored by treating the fibroblasts with siRNA oligonucleotides prior to co-culture, implicating the fibroblasts as principal production site for the secreted mediators. In summary, we identify a paracrine signaling network with inter-individual commonalities and differences. These findings have significant implications for the design of stroma-targeted therapies.</p> </div

Functional screen of paracrine factors in co-culture of NF and T47D cells. A.

<p>Red-green heat map representation of NF co-culture-induced T47D cell growth in the presence or absence of neutralizing antibodies. Antibody treatment and calculation of Co-culture-induced T47D cell growth were performed as described in the legend of <b>Fig. 3 and 4</b>. Color changes from green to red as value increases. Each row depicts data for NF from an individual patient (Pt number on left). The tumor grade is indicated on the left-hand side of Pt number. Cases are stratified into low grade (G1 and G2) and high grade (G3). <b>B.</b> Scatter plot representation of the data shown in panel “<b>A</b>”. Student t-test was applied to compare specific treatment vs. no antibody control. Each data point represents data of one NF sample.</p

siRNA screen of T47D cell growth in co-culture with CAF or NF and inter-subject heterogeneity of paracrine interactions. A & B

<p>. Red-green heat map representation of T47D cell growth stimulation in co-culture with CAF (<b>A</b>) or NF (<b>B</b>). SiRNA oligonuleotide transfection was performed as described in Materials and Methods to knock down expression of specific mediators. Co-culture-induced T47D cell growth was calculated as described for Fig. 3. Each data point represents the mean of 3–6 replicates. <b>C</b>. Inter-subject heterogeneity of T47D cell growth response to neutralizing antibody is highest in co-culture with NF and lowest in co-culture with CAF from high-grade tumors. Co-culture-induced T47D cell growth in the presence of antibody was normalized to the no-treatment control for each patient. The sample variance for the group of NF, CAF low-grade, or CAF high-grade were then calculated. F test was applied to compare variances between the groups. * P<0.05, CAF of low grade tumor vs. NF, # P<0.05, CAF of high grade tumor vs. CAF of low grade tumor.</p

Functional screen of paracrine factors in co-culture of CAF with T47D cells. A.

<p>Red-green heat map representation of CAF co-culture-induced T47D cell growth in the presence or absence of neutralizing antibodies. Antibody treatment and calculation of Co-culture-induced T47D cell growth were performed as described in the legend of <b>Fig. 3</b>. Color changes from green to red as value increases. Each row depicts data for CAF from an individual patient (Pt number on left). The tumor grade is indicated on the left-hand side of Pt number. Cases are stratified into low grade (G1 and G2) and high grade (G3). Asterisk denotes neutralizing antibodies, where significant differences were detected between low-grade and high-grade cancer group. Each data point represents the mean of 3–6 replicates. <b>B.</b> Scatter plot representation of the data shown in panel “<b>A</b>”. Student t-test was applied to compare specific treatment vs. no antibody control. * P = 0.0006, ** P<0.0001.</p

Characterization of T47D and HMF growth in micro-channel collagen gel co-culture system. A.

<p>T47D cell growth is significantly induced in co-culture with HMF compared to mono-culture. Co-culture or mono-culture gels were fixed and stained at day 1 to 6. The cytokeratin-positive area was measured with Image J. <b>B.</b> HMF proliferate at a significantly higher rate in mono-culture than in co-culture with T47D cells.</p

Identification of paracrine signaling factors regulating T47D cell growth in co-culture with HMF. A.

<p>In co-culture with HMF, T47D cell growth is significantly reduced by neutralizing antibodies against FGF-2, Heparanase-1, or MT1-MMP. Neutralizing antibodies (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046685#pone.0046685.s010" target="_blank">Table S1</a></b> for details) were added to collagen gel and culture media. Co-cultures were fixed and stained after 3–4 days of treatment. Co-culture-induced T47D cell growth was calculated as described in Materials and Methods. <b>B.</b> Neutralizing antibodies do not affect HMF growth in co-culture. <b>C.</b> T47D cell growth was significantly decreased in co-culture with HMF transfected with siRNA targeting FGF-2, HB-EGF, Heparanase-1, MT1-MMP, SDF-1, or TGF-β1. HMF were transfected with siRNA oligonuleotides (100 nM) 3 days before co-culture with T47D cells. Co-cultures were incubated for 3 days, then fixed and stained. Data shown represent the mean of 3 independent experiments. In each experiment, 3–6 micro-channels were used as technical replicates for every group. Student’s t-test was applied to compare specific treatment vs. no antibody control. The asterisk denotes P<0.05.</p

Immunocytochemical staining of array-based micro-channel 3D collagen gel co-culture of T47D cells and HMF. A.

<p>Image of micro-channel device. Inset: Cartoon of single channel design. <b>B.</b> Top view of the image of single channel taken with fluorescent microscope. T47D cells were specifically labeled with anti-pancytokeratin antibody and Alexa 594-conjugated secondary antibody (red). HMF were specifically labeled with anti-vimentin antibody and Alexa 488-conjugated secondary antibody (green) (4x objective). <b>C.</b> Side view of the integrated Z-series image stack images taken with confocal microscope (20x objective).</p