原发性导管原位癌的新型细胞培养技术：Notch和表皮生长因子受体信号通路的作用

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

作者信息

Farnie Gillian, Clarke Robert B, Spence Katherine, Pinnock Natasha, Brennan Keith, Anderson Neil G, Bundred Nigel J

机构信息

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.

出版信息

J Natl Cancer Inst. 2007 Apr 18;99(8):616-27. doi: 10.1093/jnci/djk133.

DOI:10.1093/jnci/djk133

PMID:17440163

Abstract

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).

CONCLUSION

We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE.

摘要

背景

表皮生长因子受体（EGFR）和Notch信号通路与正常乳腺干细胞的自我更新有关。我们研究了这些信号通路在乳腺导管原位癌（DCIS）中的作用。

方法

取正常乳腺组织样本（n = 15）、不同分级的纯DCIS组织样本（n = 35）以及浸润性癌周围的DCIS组织样本（n = 7）进行非贴壁（即乳腺球）培养。在第0天用吉非替尼（一种EGFR抑制剂）、DAPT（N-[N-(3,5-二氟苯乙酰-L-丙氨酰)]-S-苯甘氨酸叔丁酯）（一种γ-分泌酶抑制剂）或Notch 4中和抗体处理乳腺球培养物。乳腺球形成效率（MFE）通过形成的直径60微米或更大的乳腺球数量除以接种的单细胞数量来计算，并以百分比表示。对50例随访至少60个月的石蜡包埋DCIS组织进行免疫组织化学检测Notch 1细胞内结构域（NICD）。所有统计检验均为双侧检验。

结果

DCIS的MFE高于正常乳腺组织（1.5%对0.5%，差异 = 1%，95%置信区间[CI] = 0.62%至1.25%，P<.001）。高级别DCIS的MFE高于低级别DCIS（1.6%对1.09%，差异 = 0.51%，95% CI = 0.07%至0.94%，P = .01）。在无外源性表皮生长因子（EGF）的情况下，用吉非替尼处理的高级别DCIS的MFE低于用不含吉非替尼和外源性EGF的乳腺球培养基处理的高级别DCIS（0.56%对1.36%，差异0.8%，95% CI = 0.33%至1.4%，P = .004）。通过NICD染色检测到的Notch信号增加与5年复发相关（P = .012）。当使用DAPT（0.89%对0.51%，差异 = 0.38%，95% CI = 0.2%至0.6%，P<.001）或Notch 4中和抗体（0.97%对0.2%，差异 = 0.77%，95% CI = 0.52%至1.0%，P<.001）抑制Notch信号时，DCIS的MFE降低。