Silencing integrin β3 expression diminishes migration and invasion-modulating properties of the HMF-Sdc1-derived ECM.

<p>A) Quantitative assessment of migration directionality of MDA-MB-231 and MCF10DCIS.com cells within the ECMs produced by HMF-mock or HMF-Sdc1 cells treated with control or β3 siRNA using time-lapse microscopy. Different letters above columns indicate significant difference (p<0.001). B) Invasion of MDA-MB-231 and MCF10DCIS.com cells through ECMs produced by HMF-mock or HMF-Sdc1 cells treated with control or β3 siRNA. Different letters above columns indicate significant difference (at least p<0.05). Ctrl si, control siRNA; β3 si, β3 siRNA.</p

Integrin αvβ3 activity is involved in Sdc1-induced morphological changes during 3D ECM production.

<p>A-B, representative phase contrast images of HMF mock and Sdc1 cells cultured on gelatin-coated plates under semiconfluent states (A; original magnification: 100x) and the corresponding mean elongation indices (ratio of cell length/width) of HMF mock and Sdc1 cells (B). C, representative phase contrast images of HMF mock and Sdc1 cells after 7 d of culture under conditions permissive for 3D ECM production (Original magnification: 100x). D-E, representative confocal images of the nuclei of HMF mock and Sdc1 cells cultured on gelatin-coated plates under semiconfluent states (D; original magnification: 200x) and the corresponding mean nuclear elongation indices (ratio of nuclear length/width) of HMF mock and Sdc1 cells (E). The nuclei were labeled with Hoechst 33342. F-G, representative confocal images of the nuclei of HMF mock and Sdc1 cells after 7 d of culture under conditions permissive for 3D ECM production (F; Original magnification: 200x) and the corresponding mean nuclear elongation indices of HMF mock and Sdc1 cells (G). The nuclei were labeled with Hoechst 33342. *indicates that the difference was statistically significant (p<0.0001). H-I, representative confocal images of the nuclei of control siRNA or β3 siRNA treated HMF mock and Sdc1 cells after 7 d of culture under conditions permissive for 3D ECM production (H; original magnification: 200x) and the corresponding mean nuclear elongation indices (I). SiRNA treatment was performed as described in Material and Methods. The nuclei were labeled with Hoechst 33342. Letters above the columns indicate the results of statistical comparisons by ANOVA. Columns sharing the same letter are not significantly different; columns labeled with different letters are significantly different (at least p<0.05).</p

Activation of integrin αvβ3 alone is not sufficient for generating a migration and invasion-permissive ECM.

<p>A) Quantitative assessment of migration directionality of MDA-MB-231 and MCF10DCIS.com cells within the ECMs from HMF mock and Sdc1 cells treated with control or β3 clasp peptide using time-lapse microscopy. Columns labeled with different letters are significantly different (p<0.001). B) Invasion of MDA-MB-231 and MCF10DCIS.com cells through ECMs from HMF mock and Sdc1 cells treated with control or β3 clasp peptide. Columns labeled with different letters are significantly different (at least p<0.05). Ctrl pep, control peptide; β3 pep, β3 clasp peptide.</p

Potential molecular mechanisms by which stromal Sdc1 regulates ECM fiber alignment.

<p>A) In Sdc1-negative normal fibroblasts, FN fibrillogenesis is likely initiated by integrin α5β1, which gives rise to an ECM with haphazard fiber arrangement. B) In the stromal fibroblasts of some invasive breast carcinomas, Sdc1 is aberrantly expressed. Sdc1 induces morphological changes of the fibroblasts, which may contribute to the production of aligned ECM. Sdc1 may also modulate FN fibrillogenesis by directly regulating the unfolding of dimeric FNs and the interactions between cell-associated FN dimers through its HS chains. In addition, Sdc1 may modulate the initiation of FN fibrillogenesis by activating integrin αvβ3 through its ectodomain. The combined effects of Sdc1 affect the architecture of the multimeric FN fibrils, resulting in an ECM with an organized, aligned fiber arrangement.</p

Generation of stable HMF cell lines expressing mutant forms of Sdc1.

<p>A) Schematic representation of Sdc1 mutant constructs introduced into HMF cells. B) Flow cytometry analysis of exogenous Sdc1 in stably transfected HMF reveals that wildtype Sdc1 and Sdc1 mutants are expressed at comparable levels.</p

Forced activation of integrin αvβ3 leads to partial ECM fiber alignment.

<p>A) Activation of the αvβ3 integrin can be achieved independent of Sdc1 by β3 clasp peptide. Integrin αvβ3 activities were accessed by measuring the attachment of HMF cells, including untreated HMF cells, HMF cells treated with 250μM control peptide (ctrl pep), HMF cells treated with 250μM β3 clasp peptide (β3 pep) and HMF mock cells treated with 2 mM MgCl₂, to culture dishes pre-coated with vitronectin after 15min incubation at 37°C. Columns labeled with different letters are significantly different (at least p<0.05). B) Representative confocal images of immunofluorescently labeled FN fibers of ECMs from HMF mock and Sdc1 cells treated with control or β3 clasp peptide. Original magnification: 200x. C) Mean fiber-to-fiber angles of the indicated HMF ECMs. Columns labeled with different letters are significantly different (at least p<0.05). Ctrl pep, control peptide; β3 pep, β3 clasp peptide.</p

Sdc1 ectodomain and HS chains are required for producing an ECM that promotes directional migration and invasion.

<p>A) Time-lapse migration study of MDA-MB-231 and MCF10DCIS.com cells in 3D ECMs derived from HMF-mock cells, HMF-Sdc1 cells, HMF cells expressing the HS-deficient Sdc1 (Sdc1-ΔHS) and HMF cells expressing the ectodomain-truncated Sdc1 (Sdc1-ΔEcto). Cell movements were recorded every 30 minutes for a period of 5–6 hours. The directional persistence of cell migration was determined as the ratio of the migration distance (net distance in a direct line from start to end point) to the total distance traveled. Columns labeled with different letters are significantly different (p<0.001). B) Invasion of MDA-MB-231 and MCF10DCIS.com cells through the indicated HMF-derived ECMs deposited in the inserts of Matrigel invasion chambers. Invasion is reported as the number of invading cells per field. Columns labeled with different letters are significantly different (p<0.001).</p

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 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

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