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Interlobular and intralobular mammary stroma: genotype may not reflect phenotype.

Fleming JM, Long EL, Ginsburg E, Gerscovich D, Meltzer PS, Vonderhaar BK - BMC Cell Biol. (2008)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. flemingjo@mail.nih.gov

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.

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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.
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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.

Mentions: Microarray analysis was employed to identify genes that were differentially regulated between the intralobular and interlobular stromal subtypes. Normal human mammary tissue was obtained from healthy, pre-menopausal, reduction mammoplasty patients with no incidence of neoplasia. For each sample collected, tissue sections with distinctive intralobular and interlobular regions were selected for laser capture and microarray analysis (Fig. 1). RNA was extracted from the samples, checked for quality, and then hybridized with the Affymetrix Human Genome133A GeneChips, containing over 22,000 oligonucleotide probes. Surprisingly, no significant difference in gene expression was found between the two stroma subtypes. The microarray data demonstrated a wide range of expression values and a 45-degree straight line in each pair of samples, indicating the microarray assay and the normalization procedure were valid. Despite the small sample number, the scatter plots showed limited spread of the off-diagonal lines, suggesting that any differential expression between samples is subtle, and not significant (Fig. 2A). A heatmap with dendrograms was also generated from the data (Fig. 2B). At first glance, genes from sample numbers 13L, 27L, and 29R appeared to separate as different clusters with respect to interlobular and intralobular samples. However, further in-depth analysis using hierarchical clustering of the samples, based on the 1,115 most variable genes, did not reveal a distinct expression pattern between the intralobular and interlobular stromal tissues, further indicating there was no significant difference in terms of gene expression at the transcriptional level. Six of the genes with the largest difference in expression levels between intralobular in interlobular stroma are listed in Table 2, along with the fold-change and p-value. The lowest p-value was found to be 0.4726, which is far from statistically significant. In order to validate the microarray data, RT-PCR was performed on three of the top six genes listed in Table 2. The RT-PCR products from three patient samples as well as the quantitation of the products are shown in Figure (3A&3B). The expression levels of c-Met, SOS2, and CD44 reflect the findings of the microarray data; there was no significant difference between the intralobular and interlobular stroma.


Interlobular and intralobular mammary stroma: genotype may not reflect phenotype.

Fleming JM, Long EL, Ginsburg E, Gerscovich D, Meltzer PS, Vonderhaar BK - BMC Cell Biol. (2008)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2529294&req=5

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.
Mentions: Microarray analysis was employed to identify genes that were differentially regulated between the intralobular and interlobular stromal subtypes. Normal human mammary tissue was obtained from healthy, pre-menopausal, reduction mammoplasty patients with no incidence of neoplasia. For each sample collected, tissue sections with distinctive intralobular and interlobular regions were selected for laser capture and microarray analysis (Fig. 1). RNA was extracted from the samples, checked for quality, and then hybridized with the Affymetrix Human Genome133A GeneChips, containing over 22,000 oligonucleotide probes. Surprisingly, no significant difference in gene expression was found between the two stroma subtypes. The microarray data demonstrated a wide range of expression values and a 45-degree straight line in each pair of samples, indicating the microarray assay and the normalization procedure were valid. Despite the small sample number, the scatter plots showed limited spread of the off-diagonal lines, suggesting that any differential expression between samples is subtle, and not significant (Fig. 2A). A heatmap with dendrograms was also generated from the data (Fig. 2B). At first glance, genes from sample numbers 13L, 27L, and 29R appeared to separate as different clusters with respect to interlobular and intralobular samples. However, further in-depth analysis using hierarchical clustering of the samples, based on the 1,115 most variable genes, did not reveal a distinct expression pattern between the intralobular and interlobular stromal tissues, further indicating there was no significant difference in terms of gene expression at the transcriptional level. Six of the genes with the largest difference in expression levels between intralobular in interlobular stroma are listed in Table 2, along with the fold-change and p-value. The lowest p-value was found to be 0.4726, which is far from statistically significant. In order to validate the microarray data, RT-PCR was performed on three of the top six genes listed in Table 2. The RT-PCR products from three patient samples as well as the quantitation of the products are shown in Figure (3A&3B). The expression levels of c-Met, SOS2, and CD44 reflect the findings of the microarray data; there was no significant difference between the intralobular and interlobular stroma.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Mammary Biology and Tumorigenesis Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. flemingjo@mail.nih.gov

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.

Show MeSH
Related in: MedlinePlus