Figure 3

Association of TWIST1 mRNA expression with stromal content of the tumor tissue. A: TWIST1 mRNA expression levels in laser capture microdissected breast tumor tissues. The stromal and epithelial tumor cell areas were separately analyzed after microdissecting 10 breast tumor tissues (n = 9 with high ESR1expression, n = 1, (T6), with low ESR1 expression). After LCM, RNA isolation and cDNA synthesis were done as described in the Patients and Methods sections. TWIST1 mRNA levels were measured by qRT-PCR in RNA isolated from both the stromal (S) and the epithelial tumor cell (T) compartments of these tissues, and levels were normalized to our set of reference genes by the delta (Δ) Ct method. To ensure our set of reference genes did not cause a bias in these analyses, and our LCM material was indeed enriched for stromal and epithelial cells, we also measured VIM and EpCAM in these fractions. A two-sided paired t-test was performed to evaluate the differences. B: TWIST1 mRNA expression in SR and SP ER-positive tumors and C: TWIST1 mRNA expression in SR- and SP ER-negative tumors. For figure sections B and C, the mRNA expression of TWIST1 was log-transformed and the expression relative to reference genes is presented on the y-axis of the box-and-whisker plots. The box-plot shows the five statistics (box bottom-line is the 25^th percentile, solid line in the middle of the box presents the median, upper line of the box is the 75^th percentile and the whiskers extend to 1.5 times the inter-quartile range, observations beyond these values are plotted, separately). Mann-Whitney test was performed to evaluate the difference between the SR and the SP groups in both ER-positive and ER-negative patients (B, C).