Reduction of Int6 in the fibroblasts in human breast tumors. (A) Left: gene expression data from the breast cancer TCGA project were directly exported from Oncomine, in which mRNA levels of INT6 in normal breast tissue and invasive ductal carcinomas were compared. INT6 mRNA levels were reported to be 50% lower in the latter. Right: stroma gene expression data in the Finak study available in Oncomine were analyzed to show that INT6 mRNA levels were approximately 42 times higher in the tissue surrounding the normal adjacent ducts than in the stroma in the tumor. (B) Control or INT6-repressed MCF7 cells were analyzed by Western blot (left) or IHC (right) by an anti-Int6 antibody. We note that agreeing with our previous finding using GFP-tagging , Int6 is mainly cytoplasmic. (C) This is a typical IHC experiment examining fibroblasts in the adjacent normal and tumor region from the same human tumor sample. The top pictures were captured using a 10× objective, and one area in each was then examined by a 40× objective to reveal more details. Closed and open arrowheads mark Int6-positive vs. Int6-negative fibroblasts. (D) The histoscore differences between the normal and tumor regions were compared by the Wilcoxon signed rank test. While there was no difference in Int6 intensity between the normal and tumor regions (p = 0.66), a much lower percentage of Int6-positive fibroblasts was found in the tumor. As a result, all but two samples (marked red) show lower values for tumor fibroblasts. The mean normal and tumor histoscores are marked orange. On the right is a boxplot of the differences between normal and tumor histoscores (individual values shown as circles, mean difference shown in orange). Mean difference ± SEM = 27.0 ± 7.2 (p < 10−3, Wilcoxon signed rank test).