Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways
G Farnie, RB Clarke, K Spence… - Journal of the …, 2007 - academic.oup.com
G Farnie, RB Clarke, K Spence, N Pinnock, K Brennan, NG Anderson, NJ Bundred
Journal of the National Cancer Institute, 2007•academic.oup.comBackground 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 (ie,
mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an …
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 (ie,
mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an …
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 γ-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.
Oxford University Press