“Interferon response genes regulated by STAT1 signature” and “IL-6 gene signature” in co-culture.

<p>(<b>A</b>) Biologically independent replicates of the monocultured normal human osteoblasts (NHOst), the breast cancer cell line MDA-MB-231, and the mixed co-culture of NHOst and MDA-MB-231 cells were incubated for 48 hours under low serum conditions and characterized by DNA microarray hybridization. The figure shows the heat map of the hierarchical clustering of a total of 1461 elements that display a greater than 1.5-fold variance in expression of at least 2 different experimental samples. The co-culture of NHOst and MDA-MB231 induced two prominent sets of genes: An “interferon–response genes regulated by STAT1” signature and an “IL-6 gene signature” (zoomed image). (<b>B</b>) Real time PCR confirms a significantly higher expression of IL-6 mRNA in the co-culture than in either of the two monocultures (p = 4e-7; un-paired, two-tailed <i>t</i>-test). (<b>C</b>) As implied by the higher expression levels of IL-6 mRNA, the IL-6 concentration in the co-culture supernatants, as determined by ELISA, were significantly higher than the average concentration of the two monocultures. (p = 0.0046; un-paired, two-tailed <i>t</i>-test). (<b>D</b>) Also IL-6 was significantly more highly induced in NHOst cells stimulated with conditioned medium from MDA-MB-231 cells than in MDA-MB-231 cells stimulated with conditioned medium from NHOst (p = 0.045; un-paired, two-tailed <i>t</i>-test).</p

Effects of the “interferon response genes regulated by STAT1 signature” and “IL-6 gene signature” on bone metastasis formation.

<p>Kaplan-Meier plots for overall survival (OS) (A,D), distant metastasis-free survival (DMFS) (B,E) and bone metastasis-free survival (BMFS) (C, F) estimates are shown for the “interferon response genes regulated by STAT1 signature” and the “IL-6 gene signature”. P-values are given for the Cox regression analysis. The “IL-6 gene signature” segregated tumors with a significant difference in BMFS. The colors of the curves correspond to the colors of the bars below the heatmaps of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029743#pone-0029743-g004" target="_blank">figure 4 A and B</a>.</p

Effects of a heterotypic interaction between normal human osteoblasts and breast cancer cells.

<p>(<b>A</b>) Biologically independent replicates of NHOst, Hs578T, and the mixed co-culture of NHOst and Hs578T were kept for 48 hours at low serum conditions and characterized by DNA microarray profiling. We performed hierarchical clustering of 1923 elements that display a greater than 3-fold variance in expression in more than 2 different experimental samples. Genes are represented in rows and experiments in columns. Unsupervised hierarchical clustering of the experiments grouped the biological replicates together. The vertical black bar marks a cluster of genes that were higher expressed in all co-cultures compared to both monocultures, which indicated that they were induced by the heterotypic interaction. Further analysis of these genes revealed that they were specific for proliferation and mitosis. (<b>B</b>) The proliferation rate of NHOst and Hs578T cell monocultures and of their 1∶1 co-culture was determined by measuring increases in cell number by direct cell counting. Quadruplicates of pre-starved cells were plated at a density of 8500 cells/cm² and after 48 hours the cell number was determined using a cell counter All figures represent averages from four replicates, and error bars denote standard deviation. The increase in cell number of the co-culture is significantly higher than the increase in both of the monocultures (p = 0.0084, un-paired, two-tailed <i>t</i>-test). (<b>C</b>) Hs578T cells and NHOst cells were incubated for 24 hours with conditioned media (CM) from NHOst cells or Hs578T cells, respectively, and compared to a negative control of the same cells incubated with autologous medium. All experiments were performed in triplicates. After 24 hours cell numbers were measured by the cell counting with FACS. (<b>D</b>) The expression values of the genes in the “tumor-osteoblast cell-induced M phase/cell cycle” gene signature were extracted from a published expression study of 295 early stage breast cancers from the Netherlands Cancer Institute. Genes and samples were organized by hierarchical clustering. The tumors were segregated into two groups that were defined by high or low expression levels of the 36 genes matching the proliferation gene cluster. The histogram below the heat map represents the differences in the sums of log2 ratios among groups. Based on the distributions, the sums of the log2 ratios for the “proliferation” transcripts were over-expressed in the majority of the cases in the right branch of the cluster compared to the left branch of the cluster (32/113 versus 105/45 scores below/above reference zero value, respectively). (<b>E+F</b>) Correlation of the “proliferation” gene signature with distant metastasis-free survival (DMFS) (E) and overall survival (OS) (F). Kaplan-Meier curves for the clinical outcomes of the indicated tumors that exhibited high (red curve) and low (black curve) expression of the “proliferation” signature are shown.</p

Gene expression changes in multiple co-cultures of breast cancer cell lines with osteoblasts.

<p>Overview of collapsed data from repeat co-culture experiments of six benign and malignant epithelial cell lines with NHOst cells. In a gene expression profiling experiment 7 monocultures and 6 co-cultures with NHOst were analyzed independently in duplicates. Genes with missing data in more than 20% of the arrays were removed, leaving 10774 gene IDs. Based on this dataset, the calculation of the interaction factors were performed separately for all co-cultures, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029743#s2" target="_blank">Methods</a> section. The interaction factors of the 6 co-cultures were further analyzed for their distribution, and factors with a standard deviation of <2 in at least two co-cultures were eliminated, leaving interaction factors for 635 genes, which are shown as a heat map (unsupervised hierarchical clustering). Red and green denote induction and repression due toheterotypic interaction. The magnitude of induction or repression is represented by color intensity. Specific clusters involved in IFN signaling, a “tumor-osteoblast cell-induced M phase/cell cycle” signature and response to TGF-β are marked.</p

Correlations of the “IL-6 gene signature” with other published prognostic gene signatures in early stage breast cancer.

<p>Scatter plots showing the relationship between the value of the centroids of the “70-gene signature” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029743#pone.0029743-vandeVijver1" target="_blank">[24]</a>, the “wound signature” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029743#pone.0029743-Chang2" target="_blank">[53]</a>, the “luminal type B signature” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029743#pone.0029743-Sorlie2" target="_blank">[26]</a>, the “proliferation” signature and the “IL-6 gene signature” in the NKI study. Each point in the scatter plots represents a single one of the 295 tumors analyzed in the NKI dataset. A 5×5 pair-wise scatter plot matrix of the five gene signatures is shown. Columns and rows are labeled in the diagonal panels, i.e. the first top right panel represents data for the centroids of the “Wound” signature plotted against the centroids of the “IL-6 gene signature”. The overall correlation between each pair of expression signatures across this set of 295 samples is indicated in each panel.</p

Induction of interferon response in two types of breast cancer cell lines

MDA-MB231 cells were incubated in conditioned media from CCL-171 monoculture, MDA-MB231 monoculture, T47D monoculture, CCL-171/MDA-MB231 co-culture and CCL-171/T47D co-culture. gene expression was determined by quantitative RT-PCR. The gene expression level of GAPDH was used for normalization between the samples. A strong induction of by the supernatant from the CCL-171/MDA-MB231 co-culture can be seen in MDA-MB231. Incubation of T47D cells with conditioned media from CCL-171 monoculture, MDA-MB231 monoculture, T47D monoculture, CCL-171/MDA-MB231 co-culture and CCL-171/T47D co-culture showed that only the CCL-171/MDA-MB231 co-culture supernatant induced gene expression, although to a much lesser extent than in MDA-MB231 cells. Gene expression levels of were determined by quantitative RT-PCR. CCL-171 cells show much higher expression levels when isolated by FACS after co-culture with MDA-MB231 than with T47D cells. Expression levels in tumor cells are shown as controls. The error bars show the standard deviation from the normalized mean.<p><b>Copyright information:</b></p><p>Taken from "Characterization of heterotypic interaction effects to deconvolute global gene expression profiles in cancer"</p><p>http://genomebiology.com/2007/8/9/R191</p><p>Genome Biology 2007;8(9):R191-R191.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2375029.</p><p></p

Correlation of the 70 genes signature 38, the wound signature 24, the hypoxia signature 25 and the interferon response score in the NKI dataset

Pairwise scatterplot-matrix of four gene signatures. Pearson correlations are shown in the lower part of each plot.<p><b>Copyright information:</b></p><p>Taken from "Characterization of heterotypic interaction effects to deconvolute global gene expression profiles in cancer"</p><p>http://genomebiology.com/2007/8/9/R191</p><p>Genome Biology 2007;8(9):R191-R191.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2375029.</p><p></p

Overview of gene expression changes over multiple co-cultures of breast cancer cell lines and normal breast epithelial cells with fibroblasts

Correlation of the measured co-culture gene expression levels and their estimated expression levels based on the proportional contribution of each cell type determined by a linear regression fit of the monoculture to the co-culture data. Fold change of each gene associated with co-culturing of MDA-MB231 and CCL-171. Genes of the interferon response gene set (Additional data file 1) as determined by SAM are indicated in red. Fold change in expression of the interferon response gene set (Additional data file 1) in co-culture of MCF-7, HMECs and MDA-MB-231 with either the CCL-171 lung fibroblast or the HTB-125 breast fibroblast, showing that CCL-171 and HTB-125 induce a distinct, but very similar response in co-culture with different epithelial cells. Overview of collapsed data from repeat co-culture experiments of eight benign and malignant epithelial cells with three different fibroblasts. Hierarchical clustering of the interaction effects of 3,000 genes in co-cultures of 7 breast cancer cell lines and normal breast epithelial cells with fibroblasts. Red and green denote relative changes in expression associated heterotypic interaction. The magnitude of the relative change is given by color intensity.<p><b>Copyright information:</b></p><p>Taken from "Characterization of heterotypic interaction effects to deconvolute global gene expression profiles in cancer"</p><p>http://genomebiology.com/2007/8/9/R191</p><p>Genome Biology 2007;8(9):R191-R191.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2375029.</p><p></p

Immunohistochemical staining of STAT1 in a cohort of primary breast cancers: Kaplan-Meier disease-specific survival curve for 353 primary tumors assessed for STAT1

The red curve shows 102 patients bearing tumors with high STAT1 expression whereas the blue curve represents 251 patients with low or absent STAT1 expression. X = censored data.<p><b>Copyright information:</b></p><p>Taken from "Characterization of heterotypic interaction effects to deconvolute global gene expression profiles in cancer"</p><p>http://genomebiology.com/2007/8/9/R191</p><p>Genome Biology 2007;8(9):R191-R191.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2375029.</p><p></p

Effect of heterotypic interaction between breast cancer cell line MDA-MB231 and CCL-171 fibroblasts

Biologically independent replicates of the monocultured fibroblast CCL-171, the breast cancer cell line MDA-MB231 and the mixed co-culture of CCL-171 and MDA-MB231 were grown for 48 h at low serum conditions and characterized by DNA microarray hybridization. Hierarchical clustering of a total of 4,333 elements that display a greater than 3-fold variance in expression in more than 3 different experimental samples. Data from individual elements or genes are represented as single rows, and different experiments are shown as columns. Red and green denote expression levels of the samples. The intensity of the color reflects the magnitude of the deviation from baseline. Unsupervised hierarchical clustering of the experiments grouped the biological replicates together. Gene expression varied considerably between fibroblast and MDA-MB231 cultures, as expected for cells of mesenchymal or epithelial origin, respectively. The co-culture profile showed mainly intermediate expression levels. However, the vertical black bar marks a cluster of genes induced in all co-cultures compared to both monocultures, indicating that they are induced by heterotypic interaction. Zooming in on the genes up-regulated in co-culture compared to monocultures reveals that they are associated with the response to interferon.<p><b>Copyright information:</b></p><p>Taken from "Characterization of heterotypic interaction effects to deconvolute global gene expression profiles in cancer"</p><p>http://genomebiology.com/2007/8/9/R191</p><p>Genome Biology 2007;8(9):R191-R191.</p><p>Published online 14 Sep 2007</p><p>PMCID:PMC2375029.</p><p></p