PMC

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 .

Scatter plots showing the relationship between the value of the centroids of the “70-gene signature” , the “wound signature” , the “luminal type B signature” , 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.

(A) The expression values of genes in the “interferon response genes regulated by STAT1 signature” were extracted from a published gene expression study of 295 early stage breast cancers from the Netherlands Cancer Institute . Genes and samples are organized by unsupervised hierarchical clustering. The tumors segregated into two groups defined by high (blue bar below the heatmap) or low (golden bar below the heatmap) expression levels of 26 genes matching the “interferon response genes regulated by STAT1 signature”. (B) For the “IL-6 gene signature” an analogous analysis was performed, resulting in tumors segregated into two groups based on the expression levels of 28 genes. The two main clusters of tumors with high and low expression levels are marked with colored bars below the heatmap (blue and golden respectively).

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 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.

(A) 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) 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 t-test). (C) 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 t-test). (D) 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 t-test).

(A) 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) 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 t-test). (C) 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. (D) 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). (E+F) 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.