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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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5-Bromo-2'-deoxyuridine (BrdU) labeling, assessed by flow cytometry, of MCF10AT monocultures and co-cultures with normal breast-associated fibroblasts (NAF) and carcinoma-associated fibroblasts (CAF). These data are representative of replicate experiments indicating that NAF suppress proliferation of MCF10AT cells to a greater extent than do CAF. Again some variability in extent of suppression is present among individual NAF cultures and individual CAF cultures.
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Figure 5: 5-Bromo-2'-deoxyuridine (BrdU) labeling, assessed by flow cytometry, of MCF10AT monocultures and co-cultures with normal breast-associated fibroblasts (NAF) and carcinoma-associated fibroblasts (CAF). These data are representative of replicate experiments indicating that NAF suppress proliferation of MCF10AT cells to a greater extent than do CAF. Again some variability in extent of suppression is present among individual NAF cultures and individual CAF cultures.

Mentions: In replicate co-cultures of MCF10A cells with three different NAF and CAF grown in an E:F of 2:1, both types of fibroblasts significantly reduced proliferation of MCF10A cells. The mean BrdU-labeling index of MCF10A cells, when measured by immunocytochemistry, was decreased by 47% in co-culture with NAF (n = 19, P = 0.009) and by 39% in co-culture with CAF (n = 19, P = 0.024) relative to the MCF10A monoculture (Table 1 and Fig. 4). The BrdU-labeling index of MCF10AT cells was reduced by 49% in the presence of NAF (n = 20, P = 0.013), relative to the MCF10AT monoculture, whereas co-culture with CAF failed to significantly lower the MCF10AT BrdU-labeling index (n = 22, P = 0.935) (Table 2 and Fig. 4). The effect of NAF versus CAF on the rate of proliferation of MCF10AT cells was significantly different (P < 0.001). The effect was further confirmed by repeating the co-cultures to measure the BrdU-labeling index by flow cytometry, rather than by immunocytochemistry (Fig. 5).


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)

5-Bromo-2'-deoxyuridine (BrdU) labeling, assessed by flow cytometry, of MCF10AT monocultures and co-cultures with normal breast-associated fibroblasts (NAF) and carcinoma-associated fibroblasts (CAF). These data are representative of replicate experiments indicating that NAF suppress proliferation of MCF10AT cells to a greater extent than do CAF. Again some variability in extent of suppression is present among individual NAF cultures and individual CAF cultures.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064098&req=5

Figure 5: 5-Bromo-2'-deoxyuridine (BrdU) labeling, assessed by flow cytometry, of MCF10AT monocultures and co-cultures with normal breast-associated fibroblasts (NAF) and carcinoma-associated fibroblasts (CAF). These data are representative of replicate experiments indicating that NAF suppress proliferation of MCF10AT cells to a greater extent than do CAF. Again some variability in extent of suppression is present among individual NAF cultures and individual CAF cultures.
Mentions: In replicate co-cultures of MCF10A cells with three different NAF and CAF grown in an E:F of 2:1, both types of fibroblasts significantly reduced proliferation of MCF10A cells. The mean BrdU-labeling index of MCF10A cells, when measured by immunocytochemistry, was decreased by 47% in co-culture with NAF (n = 19, P = 0.009) and by 39% in co-culture with CAF (n = 19, P = 0.024) relative to the MCF10A monoculture (Table 1 and Fig. 4). The BrdU-labeling index of MCF10AT cells was reduced by 49% in the presence of NAF (n = 20, P = 0.013), relative to the MCF10AT monoculture, whereas co-culture with CAF failed to significantly lower the MCF10AT BrdU-labeling index (n = 22, P = 0.935) (Table 2 and Fig. 4). The effect of NAF versus CAF on the rate of proliferation of MCF10AT cells was significantly different (P < 0.001). The effect was further confirmed by repeating the co-cultures to measure the BrdU-labeling index by flow cytometry, rather than by immunocytochemistry (Fig. 5).

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