Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways.
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 microm 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).


CONCLUSIONS
We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE.
Farnie
Gillian
G
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.
Clarke
Robert B
RB
Spence
Katherine
K
Pinnock
Natasha
N
Brennan
Keith
K
Anderson
Neil G
NG
Bundred
Nigel J
NJ
D001940:Breast / Q000166:cytology / Q000502:physiology; D001943:Breast Neoplasms / Q000473:pathology* / Q000503:physiopathology / Q000601:surgery; D002278:Carcinoma in Situ / Q000473:pathology / Q000503:physiopathology / Q000601:surgery; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000473:pathology* / Q000503:physiopathology / Q000601:surgery; D002448:Cell Adhesion; D018929:Cell Culture Techniques / Q000379:methods; D066246:ErbB Receptors / Q000502:physiology*; D005260:Female; D006801:Humans; D009361:Neoplasm Invasiveness; D051880:Receptors, Notch / Q000502:physiology*; D012008:Recurrence; D014407:Tumor Cells, Cultured
D051880:Receptors, Notch; D066246:ErbB Receptors