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Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture.

Sadlonova A, Novak Z, Johnson MR, Bowe DB, Gault SR, Page GP, Thottassery JV, Welch DR, Frost AR - Breast Cancer Res. (2004)

Bottom Line: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells.However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells.The degree of growth inhibition varied among NAF or CAF from different individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of Alabama at Birmingham, Alabama, USA. asadlono@path.uab.edu

ABSTRACT

Background: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed.

Methods: NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR.

Results: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation.

Conclusion: Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF, suggesting that the ability of fibroblasts to inhibit epithelial cell proliferation is lost during breast carcinogenesis. Furthermore, as the degree of transformation of the epithelial cells increased they became resistant to the growth-inhibitory effects of CAF. Insulin-like growth factor II could not be implicated as a contributor to this differential effect of NAF and CAF on epithelial cell growth.

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Relative 5-bromo-2'-deoxyuridine (BrdU) indices of MCF10A cells in co-culture with varying quantities of normal breast-associated fibroblast NAF-2 and carcinoma-associated fibroblast CAF-1. With increasing numbers of NAF-2, the mean rate of proliferation of co-cultured MCF10A cells increased, with a significant difference in BrdU-labeling index observed between a ratio of epithelial cells to fibroblasts (E:F) of 2:1 versus an E:F of 1:3 (P < 0.05). With increasing numbers of CAF-1, the mean rate of proliferation was highest at an E:F of 1:1. The rate of proliferation at an E:F of 1:1 was significantly higher than that at an E:F of 2:1 (P < 0.05). At an E:F of 1:3, CAF-1 caused a decreased proliferation of and enhanced cell death of MCF10A cells.
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Figure 6: Relative 5-bromo-2'-deoxyuridine (BrdU) indices of MCF10A cells in co-culture with varying quantities of normal breast-associated fibroblast NAF-2 and carcinoma-associated fibroblast CAF-1. With increasing numbers of NAF-2, the mean rate of proliferation of co-cultured MCF10A cells increased, with a significant difference in BrdU-labeling index observed between a ratio of epithelial cells to fibroblasts (E:F) of 2:1 versus an E:F of 1:3 (P < 0.05). With increasing numbers of CAF-1, the mean rate of proliferation was highest at an E:F of 1:1. The rate of proliferation at an E:F of 1:1 was significantly higher than that at an E:F of 2:1 (P < 0.05). At an E:F of 1:3, CAF-1 caused a decreased proliferation of and enhanced cell death of MCF10A cells.

Mentions: In a prior report, CAF was found to promote, rather than inhibit, the growth of MCF10A cells in a similar 3D co-culture system [22]. One of several possible explanations for this discrepancy between the prior result and the present result is a difference in E:F. Shekhar and colleagues used an E:F of 1:1 rather than the E:F of 2:1 we initially used [22]. The number of fibroblasts has been shown to have an effect on the response of epithelial cells [7,9,14]. We therefore repeated the 3D co-cultures of MCF10A cells using NAF-2 and CAF-1 with increasing numbers of fibroblasts (i.e. a decreasing E:F) (Fig. 6). BrdU labeling was assessed by immunocytochemistry of histologic sections of 3D cultures.


Breast fibroblasts modulate epithelial cell proliferation in three-dimensional in vitro co-culture.

Sadlonova A, Novak Z, Johnson MR, Bowe DB, Gault SR, Page GP, Thottassery JV, Welch DR, Frost AR - Breast Cancer Res. (2004)

Relative 5-bromo-2'-deoxyuridine (BrdU) indices of MCF10A cells in co-culture with varying quantities of normal breast-associated fibroblast NAF-2 and carcinoma-associated fibroblast CAF-1. With increasing numbers of NAF-2, the mean rate of proliferation of co-cultured MCF10A cells increased, with a significant difference in BrdU-labeling index observed between a ratio of epithelial cells to fibroblasts (E:F) of 2:1 versus an E:F of 1:3 (P < 0.05). With increasing numbers of CAF-1, the mean rate of proliferation was highest at an E:F of 1:1. The rate of proliferation at an E:F of 1:1 was significantly higher than that at an E:F of 2:1 (P < 0.05). At an E:F of 1:3, CAF-1 caused a decreased proliferation of and enhanced cell death of MCF10A cells.
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Related In: Results  -  Collection

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Figure 6: Relative 5-bromo-2'-deoxyuridine (BrdU) indices of MCF10A cells in co-culture with varying quantities of normal breast-associated fibroblast NAF-2 and carcinoma-associated fibroblast CAF-1. With increasing numbers of NAF-2, the mean rate of proliferation of co-cultured MCF10A cells increased, with a significant difference in BrdU-labeling index observed between a ratio of epithelial cells to fibroblasts (E:F) of 2:1 versus an E:F of 1:3 (P < 0.05). With increasing numbers of CAF-1, the mean rate of proliferation was highest at an E:F of 1:1. The rate of proliferation at an E:F of 1:1 was significantly higher than that at an E:F of 2:1 (P < 0.05). At an E:F of 1:3, CAF-1 caused a decreased proliferation of and enhanced cell death of MCF10A cells.
Mentions: In a prior report, CAF was found to promote, rather than inhibit, the growth of MCF10A cells in a similar 3D co-culture system [22]. One of several possible explanations for this discrepancy between the prior result and the present result is a difference in E:F. Shekhar and colleagues used an E:F of 1:1 rather than the E:F of 2:1 we initially used [22]. The number of fibroblasts has been shown to have an effect on the response of epithelial cells [7,9,14]. We therefore repeated the 3D co-cultures of MCF10A cells using NAF-2 and CAF-1 with increasing numbers of fibroblasts (i.e. a decreasing E:F) (Fig. 6). BrdU labeling was assessed by immunocytochemistry of histologic sections of 3D cultures.

Bottom Line: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells.However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells.The degree of growth inhibition varied among NAF or CAF from different individuals.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, The University of Alabama at Birmingham, Alabama, USA. asadlono@path.uab.edu

ABSTRACT

Background: Stromal fibroblasts associated with in situ and invasive breast carcinoma differ phenotypically from fibroblasts associated with normal breast epithelium, and these alterations in carcinoma-associated fibroblasts (CAF) may promote breast carcinogenesis and cancer progression. A better understanding of the changes that occur in fibroblasts during carcinogenesis and their influence on epithelial cell growth and behavior could lead to novel strategies for the prevention and treatment of breast cancer. To this end, the effect of CAF and normal breast-associated fibroblasts (NAF) on the growth of epithelial cells representative of pre-neoplastic breast disease was assessed.

Methods: NAF and CAF were grown with the nontumorigenic MCF10A epithelial cells and their more transformed, tumorigenic derivative, MCF10AT cells, in direct three-dimensional co-cultures on basement membrane material. The proliferation and apoptosis of MCF10A cells and MCF10AT cells were assessed by 5-bromo-2'-deoxyuridine labeling and TUNEL assay, respectively. Additionally, NAF and CAF were compared for expression of insulin-like growth factor II as a potential mediator of their effects on epithelial cell growth, by ELISA and by quantitative, real-time PCR.

Results: In relatively low numbers, both NAF and CAF suppressed proliferation of MCF10A cells. However, only NAF and not CAF significantly inhibited proliferation of the more transformed MCF10AT cells. The degree of growth inhibition varied among NAF or CAF from different individuals. In greater numbers, NAF and CAF have less inhibitory effect on epithelial cell growth. The rate of epithelial cell apoptosis was not affected by NAF or CAF. Mean insulin-like growth factor II levels were not significantly different in NAF versus CAF and did not correlate with the fibroblast effect on epithelial cell proliferation.

Conclusion: Both NAF and CAF have the ability to inhibit the growth of pre-cancerous breast epithelial cells. NAF have greater inhibitory capacity than CAF, suggesting that the ability of fibroblasts to inhibit epithelial cell proliferation is lost during breast carcinogenesis. Furthermore, as the degree of transformation of the epithelial cells increased they became resistant to the growth-inhibitory effects of CAF. Insulin-like growth factor II could not be implicated as a contributor to this differential effect of NAF and CAF on epithelial cell growth.

Show MeSH
Related in: MedlinePlus