Interlobular and intralobular mammary stroma: genotype may not reflect phenotype

Abstract

Background: The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. In this report, we compared the gene expression profiles between the two stromal subtypes.

Methods: Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry.

Results: No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different.

Conclusion: This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. This report also highlights the importance of studying gene regulation at both the transcriptional and post-translational level.

Figure 1

Identification of intralobular and interlobular stroma in normal human breast tissue. Hemotoxylin and eosin staining of 8–10 micron sections of normal mammary tissue. The intralobular stroma isolated for laser capture microscopy is outlined in green while the interlobular stroma is outlined in black. Scale bar = 200 μM.

Figure 2

Scattered plots of normalized data and unsupervised hierarchical clustering of the samples and genes. A. Raw intensity data was background-corrected and normalized as described in Materials and Methods. The normalized data from seven samples were plotted against one sample (SB13L-Intra). B. 1,115 most variable genes were used for hierarchical clustering among samples. The gene expression values were scaled by row and shown in the heat map.

Figure 3

c-Met, SOS2, and CD44 expression levels in intralobular and interlobular normal human breast stroma. A. RT-PCR analysis of the indicated genes expression. B. Mean ± SD gained by densitometric examination of RT-PCR product from three independent samples. C. Tissues were subjected to immunohistochemical analysis with the specific antibody indicated (left panels) or corresponding negative controls (right panels). Scale bar = 200 μM.

Figure 4

Localization and expression levels of collagen fibrils, CD13, Tenascin-C, and CD26 in intralobular and interlobular normal human breast stroma. A. Tissues were stained with Sirius Red alone (top panel) or with Fast Green counterstain (bottom panel). B. RT-PCR analysis of the indicated genes expression. C. Mean ± SD gained by densitometric examination of RT-PCR product from three independent samples. D. Tissues were subjected to immunohistochemical analysis with the specific antibody indicated (left panels) or corresponding negative controls (right panels). Note large quantities of intensely stained interlobular stroma (asterisks) compared to the paler-staining intralobular stroma (arrow). Scale bar = 200 μM.