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© The Author 2007. Published by Oxford University Press.
ARTICLES |
Novel Cell Culture Technique for Primary Ductal Carcinoma In Situ: Role of Notch and Epidermal Growth Factor Receptor Signaling Pathways
Affiliations of authors: Department of Surgery (GF, NP, NGA, NJB) and Breast Biology Group (GF, RBC, KS), Division of Cancer Studies, Faculty of Medicine and Human Sciences, University of Manchester, Christie Hospital NHS Trust, Manchester, UK; Wellcome Trust Centre for Cell Matrix Research, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, UK (KB)
Correspondence to: Nigel J. Bundred, MD, Department of Surgery and Breast Biology Group, Division of Cancer Studies, Faculty of Medicine and Human Sciences, University of Manchester, Christie Hospital NHS Trust, Wilmslow Road, M20 9BX, Manchester, UK (e-mail: bundredn{at}manchester.ac.uk).
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 
-secretase inhibitor), or Notch 4–neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 µm 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 [CI] = 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% CI = 0.07% to 0.94%, P = .01). The MFE 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 EGF (0.56% versus 1.36%, difference 0.8%, 95% CI = 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% CI = 0.2% to 0.6%, P<.001) or a Notch 4–neutralizing antibody (0.97% versus 0.2%, difference = 0.77%, 95% CI = 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.
| CONTEXT AND CAVEATS Prior knowledge The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells, but their involvement in ductal carcinoma in situ (DCIS) of the breast is unclear. Study design In vitro study using DCIS-derived epithelial cells. Contribution The authors used a novel method to culture DCIS cells and showed that EGFR is necessary for DCIS growth and self-renewal and that Notch signaling is important for cell survival and/or self-renewal in nonadherent cultures. Implications This nonadherent cell culture method may facilitate discovery of pathways that are important for DCIS propagation and testing of novel inhibitors to prevent DCIS recurrence and progression to invasive disease. Limitations It is not known whether DCIS mammospheres will recapitulate the DCIS lesion from which they are derived when implanted into immunodeficient mice.
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Manuscript received September 26, 2006; revised January 31, 2007; accepted February 27, 2007.
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J Natl Cancer Inst 2007 99: 577.
J Natl Cancer Inst 2007 99: 1284-1285.
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