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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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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.
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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.

Mentions: As previously stated, several reports illustrate a difference in protein expression between the intralobular and interlobular stroma. Atherton et al. (1998) reported that immuno-localization of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma [25]. Fibroblasts isolated from the interlobular stroma synthesized 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesized type I and type IV collagens in similar amounts. The authors suggest this protein is a way to separate the two types of distinct stroma for analysis. Collagen fibers have also been reported to be more abundant and densely packed throughout interlobular stroma compared to intralobular stroma in the bovine mammary gland [23]. Thus, we examined the collagen fiber deposition in the tissue samples from the patients used in the microarray data. Using Sirius Red, a pan stain for collagen fibers, there was a clear visible difference in the deposition of collagen fibers between the two types of stroma (Fig. 4A). In our microarray data, the fold change and p-value for type XIV collagen/undulin were -0.199 and 0.785, respectively. Undulin had the best p-value compared to all other types of collagen, but again, no values for any of the collagen genes were significant. RT-PCR analysis of the patients samples used in the microarray revealed an inconsistent expression of undulin between samples, resulting in no significant change between intralobular and interlobular expression (Fig. 4B&4C).


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)

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

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

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.
Mentions: As previously stated, several reports illustrate a difference in protein expression between the intralobular and interlobular stroma. Atherton et al. (1998) reported that immuno-localization of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma [25]. Fibroblasts isolated from the interlobular stroma synthesized 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesized type I and type IV collagens in similar amounts. The authors suggest this protein is a way to separate the two types of distinct stroma for analysis. Collagen fibers have also been reported to be more abundant and densely packed throughout interlobular stroma compared to intralobular stroma in the bovine mammary gland [23]. Thus, we examined the collagen fiber deposition in the tissue samples from the patients used in the microarray data. Using Sirius Red, a pan stain for collagen fibers, there was a clear visible difference in the deposition of collagen fibers between the two types of stroma (Fig. 4A). In our microarray data, the fold change and p-value for type XIV collagen/undulin were -0.199 and 0.785, respectively. Undulin had the best p-value compared to all other types of collagen, but again, no values for any of the collagen genes were significant. RT-PCR analysis of the patients samples used in the microarray revealed an inconsistent expression of undulin between samples, resulting in no significant change between intralobular and interlobular expression (Fig. 4B&4C).

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