Molecular signatures suggest a major role for stromal cells in development of invasive breast cancer.

BACKGROUND: Breast cancer invasion and metastasis involves both epithelial and stromal changes. Our objective was to delineate the pivotal role stroma plays in invasion by comparing transcriptomes among stromal and epithelial cells in normal tissue and invasive breast cancer. METHODS: Total RNA was isolated from epithelial and stromal cells that were laser captured from normal breast tissue (n = 5) and invasive breast cancer (n = 28). Gene expression was measured using Affymetrix U133A 2.0 GeneChips. Differential gene expression was evaluated and compared within a model that accounted for cell type (epithelial [E] versus stromal [S]), diagnosis (cancer [C] versus normal [N]) as well as cell type-diagnosis interactions. RESULTS: Compared to NE, the CE transcriptome was highly enriched with genes in proliferative, motility and ECM ontologies. Differences in CS and NS transcriptomes suggested that the ECM was being remodeled in invasive breast cancer, as genes were over-represented in ECM and proteolytic ontologies. Genes more highly expressed in CS compared to CE were primarily ECM components or were involved in the remodeling of ECM, suggesting that ECM biosynthesis and remodeling were initiated in the tumor stroma. CONCLUSION: Based on identified molecular cross-talk between the two contiguous cell populations, a mechanistic model that spurs invasion is proposed, that shows breast cancer invasion proceeds through the acquisition of a motile phenotype in tumor epithelial cells and a reactive phenotype in cancer associated fibroblasts.

Differential expression of Oct3/4 in human breast cancer and normal tissues.

Oct3/4, a transcription factor specifically expressed in mammalian totipotent embryonic stem and germ cells, has a critical role in the regulation and maintenance of pluripotency and self-renewal. However, reactivation of Oct3/4 expression is observed in several human breast cancer cell lines, but not in non­malignant cells. To examine Oct3/4 expression in human primary breast carcinomas and normal breast tissues, we obtained breast tumor tissues from 28 patients and normal breast tissues from 9 women. According to quantitative polymerase chain reaction, all of the tumor tissues, irrespective of tumor type or clinicopathological status, expressed Oct3/4 mRNA at 10- to 100- fold higher levels than that in the normal breast tissues. Expression of the Oct3/4 protein in tumors was confirmed by western blot analysis and immunofluorescent staining. Additionally, rapid amplification of cDNA ends and DNA sequencing revealed expression of multiple Oct4 gene transcripts from chromosome 6 (POU5F1) in normal breast tissues and the non­malignant breast epithelial cell line MCF­10A; by contrast, the breast tumors and malignant breast cancer cell line MCF­7 predominantly expressed transcripts of an Oct4-like gene (POU5F1B) from chromosome 8, which was termed Oct3 in the current study. The deduced amino acid sequences of full-length Oct3 and Oct4 are 96% identical. The findings of the current study indicated that Oct3, rather than Oct4, may serve as a novel clinical marker and a potential target for gene-specific therapy of breast cancer.

The effect of tissue fixation and processing on breast cancer size.

Precise measurement of an invasive breast cancer is crucial for pathological staging and subsequent patient management. Formalin fixation and histological processing may change tissue size, but there is no agreement on which state of the specimen, fresh or fixed, should be used for final tumor measurement. To determine the influence of fixation and processing on breast tumor size, a specific 1-dimensional measurement from 50 invasive breast tumors was recorded in fresh, fixed, and processed/mounted states. Tumors varied in maximum measured dimension from 4 to 20 mm and contained 10% to 90% estimated fibrous tissue (mean, 52.8%). In 96% of cases, there was no difference in measured size between fresh and fixed states. After final processing and mounting, a decrease in size from initial fresh measurement was noted in 40% of cases (mean difference, 2.4 mm; maximum difference, 7 mm). In 9 cases (18%), the measured size increased by a maximum of 3 mm (mean, 1.7 mm) after processing/mounting. Twenty-one cases (42%) showed no change in measurement during the entire fixation and processing protocol. Increases in measured size were attributed largely to tissue expansion during histological sectioning/mounting. One can arguably measure the size of an invasive breast cancer from either the fresh or fixed state without affecting accuracy, but caution should be exercised in relying solely on the microscopic measurements.

Cancer associated fibroblasts stimulated by transforming growth factor beta1 (TGF-beta 1) increase invasion rate of tumor cells: a population study.

Cancer associated fibroblasts (CAFs) are believed to promote tumor growth and progression. Our objective was to measure the effect of TGF-beta1 on fibroblasts isolated from invasive breast cancer patients. Fibroblasts were isolated from tissue obtained at surgery from patients with invasive breast cancer (CAF; n = 28) or normal reduction mammoplasty patients (normal; n = 10). Myofibroblast activation was measured by counting cells immunostained for smooth muscle alpha actin (ACTA2) in cultures +/- TGF-beta 1. Conditioned media (CM) was collected for invasion assays and RNA was isolated from cultures incubated in media +/- TGF-beta1 for 24 h. Q-PCR was used to measure expression of cyclin D1, fibronectin, laminin, collagen I, urokinase, stromelysin-1, and ACTA2 genes. Invasion rate was measured in chambers plated with MDA-MB-231 cells and exposed to CM in the bottom chamber; the number of cells that invaded into the bottom chamber was counted. Wilcox Rank Sum tests were used to evaluate differences in CAFs and normal fibroblasts and the effect of TGF-beta 1. There was no difference in percent myofibroblasts or invasion rate between normal and CAF cultures. However, TGF-beta1 significantly increased the percent of myofibroblasts (P < 0.01) and invasion rate (P = 0.02) in CAF cultures. Stromelysin-1 expression was significantly higher in normal versus CAF cultures (P < 0.01). TGF-beta 1 significantly increased ACTA2 expression in both normal and CAF cultures (P < 0.01). Expression of fibronectin and laminin was significantly increased by TGF-beta in CAF cultures (P < 0.01). CAFs were measurably different from normal fibroblasts in response to TGF-beta 1, suggesting that TGF-beta stimulates changes in CAFs that foster tumor invasion.