Heterogeneity of gene expression in stromal fibroblasts of human breast carcinomas and normal breast

Abstract

Breast carcinoma invasion is associated with prominent alterations in stromal fibroblasts. Carcinoma-associated fibroblasts (CAF) support and promote tumorigenesis, whereas normal mammary fibroblasts (NF) are thought to suppress tumor progression. Little is known about the difference in gene expression between CAF and NF or the patient-to-patient variability in gene expression. Paired CAF and NF were isolated from six primary human breast carcinoma specimens. RNA was extracted from low-passage cultures of CAF and NF and analyzed with Affymetrix Human Genome U133 Plus 2.0 arrays. The array data were examined with an empirical Bayes model and filtered according to the posterior probability of equivalent expression and fold difference in expression. Twenty-one genes (27 probe sets) were up-regulated in CAF, as compared with NF. Known functions of these genes relate to paracrine or intracellular signaling, transcriptional regulation, extracellular matrix and cell adhesion/migration. Ten genes (14 probe sets) were down-regulated in CAF, including the pluripotency transcription factor KLF4. Quantitative RT-PCR analysis of 10 genes validated the array results. Immunohistochemical staining for three gene products confirmed stromal expression in terms of location and relative quantity. Surprisingly, the variability of gene expression was slightly higher in NF than in CAF, suggesting inter-individual heterogeneity of normal stroma.

Figure 1

Characterization of fibroblasts isolated from human breast tissue samples: A) Hematoxylin-eosin-stained frozen section of tissue piece taken from infiltrating ductal carcinoma sample prior to tissue mincing. B) Hematoxylin-eosin-stained frozen section of tissue piece taken from adjacent normal mammary tissue sample prior to mincing. C) Carcinoma-associated fibroblasts (CAF) from human breast carcinoma sample at passage 1 (p1). The cells were immunofluorescence-labeled for epithelial marker cytokeratin (red signal) and the mesenchymal marker vimentin (green signal). Note a few epithelial cells and epithelial cell fragments among the fibroblasts. D) Carcinoma-associated fibroblasts (CAF) from the same human breast carcinoma sample as shown in panel “C” at passage 3 (p3). The cells were immunofluorescently labeled for epithelial marker cytokeratin (red signal) and mesenchymal marker vimentin (green signal). At this stage, no epithelial cells are left. E) CAF labeled for FSP1 (red signal) and αSMA (green signal). F) Normal fibroblasts (NF) labeled for FSP1 (red signal) and αSMA (green signal). Abbreviations: Ca = carcinoma; S = stroma). Original magnification: 200× for all images. Blue signal in panels C – F: nuclear label DAPI

Figure 2

Cluster analysis of gene expression: Gene expression data obtained with Affymetrix arrays from CAF and NF isolated from all six cases (BCa 1, 2, 5, 6, 7, 8) were subjected to unsupervised cluster analysis. Also included were results from a human mammary fibroblast cell line (HMF) grown in 3D co-culture with S1 normal mammary epithelial cells or with malignant T4-2 cells.

Figure 3

Validation of mRNA levels: The mRNA levels of ten genes (7 overexpressed in CAF, 3 overexpressed in NF) were analyzed by qRT-PCR in CAF and NF from five breast cancer patients. The results are expressed as fold difference between CAF and NF. In all but one case (BCa 8), the results were congruent with the cumulative Affymetrix data.

Figure 4

Validation of protein levels by immunohistochemistry: Paraffin sections from breast carcinoma and adjacent normal mammary gland tissue were immuno-labeled for the protein products of genes that had been found to be overexpressed in CAF (WISP1, TGFb2) or in NF (KLF4). Blue arrowheads: stromal fibroblasts. Scale bar indicates 100 μm. The labeling intensity in fibroblasts of tumor or adjacent normal stroma was scored manually on a scale from 0 (no detectable labeling) to 3 (strong labeling). The horizontal lines indicate the means.