Novel cell culture technique for primary ductal carcinoma in situ: Role of notch and epidermal growth factor receptor signaling pathways

被引:233
作者
Farnie, Gillian
Clarke, Robert B.
Spence, Katherine
Pinnock, Natasha
Brennan, Keith
Anderson, Neil G.
Bundred, Nigel J.
机构
[1] Univ Manchester, Christie Hosp NHS Trust, Fac Med & Human Sci, Div Canc Studies,Dept Surg, Manchester M20 9BX, Lancs, England
[2] Univ Manchester, Christie Hosp NHS Trust, Fac Med & Human Sci, Div Canc Studies,Breast Biol Grp, Manchester M20 9BX, Lancs, England
[3] Univ Manchester, Fac Life Sci, Wellcome Trust Ctr Cell Matrix Res, Manchester, Lancs, England
来源
JNCI-JOURNAL OF THE NATIONAL CANCER INSTITUTE | 2007年 / 99卷 / 08期
关键词
D O I
10.1093/jnci/djk133
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
Background The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. Methods Samples of normal breast tissue (n = 15), pure DCIS tissue of varying grades (n = 35), and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e., mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor), or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 pm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least 60 months of follow-up. All statistical tests were two-sided. Results DCIS had a greater MFE than normal breast tissue (1.5% versus 0.5%, difference = 1%, 95% confidence interval [Cl] = 0.62% to 1.25%, P <.001). High-grade DCIS had a greater MFE than low-grade DCIS (1.6% versus 1.09%, difference = 0.51%, 95% Cl = 0.07% to 0.94%, P=.01). The VIFE of high-grade DCIS treated with gefitinib in the absence of exogenous EGF was lower than that of high-grade DCIS treated with mammosphere medium lacking gefitinib and exogenous EGIF (0.56% versus 1.36%, difference 0.8%, 95% Cl = 0.33% to 1.4%, P=.004). Increased Notch signaling as detected by NICD staining was associated with recurrence at 5 years (P=.012). DCIS MFE was reduced when Notch signaling was inhibited using either DAPT (0.89% versus 0.51%, difference = 0.38%, 95% Cl = 0.2% to 0.6%, P <.001) or a Notch 4-neutralizing antibody (0.97% versus 0.2%, difference = 0.77%, 95% Cl = 0.52% to 1.0%, P <.001). Conclusion We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE.
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页码:616 / 627
页数:12
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