Developing in vitro models of human ductal carcinoma in situ from primary tissue explants
Developing in vitro models of human ductal carcinoma in situ from primary tissue explants
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DOI：
10.1007/s10549-015-3551-8
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发表时间：
2015-09-01
影响因子：
3.8
通讯作者：
McAuliffe, Priscilla F.
中科院分区：
文献类型：
--
作者：
Brown, Daniel D.;Dabbs, David J.;McAuliffe, Priscilla F.
由于目前没有可靠的预测导管原位癌（DCIS）进展为浸润性疾病，几乎所有患者都接受综合治疗，导致许多情况下过度治疗。很少有研究DCIS进展的体外模型已经开发出来。我们在这里报告的成功文化和扩大原发性DCIS手术标本使用条件重编程协议。经皮芯针活检证实DCIS的患者在获得知情同意后被纳入组织库方案。新鲜组织取自乳房肿瘤切除术或乳房切除术标本，机械和酶促解离，培养在条件培养基中的照射小鼠成纤维细胞和rho相关蛋白激酶（ROCK）抑制剂补充，并通过免疫细胞化学。在33例DCIS病例中，58%（19例）在培养中扩增长达2个月，42%（14例）在机械分离后立即冷冻以备将来生长。培养物几乎完全由细胞角蛋白8-和EpCAM阳性管腔和细胞角蛋白14-，细胞角蛋白5-和p63阳性基底乳腺上皮细胞组成，表明在体外维持异质性。此外，如通过管腔和基底标志物表达所评估的，这些细胞在“条件性重编程”增殖状态下以及在条件培养基和ROCK抑制剂撤回后都保留了它们的细胞身份。当生长至100%汇合时，培养物组织成腔和基底层以及由基底细胞包围的腔室。可以产生直接来源于患者组织的DCIS的原代培养物，并且可以用作DCIS研究的体外模型。
Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive comprehensive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. Patients with percutaneous core-needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Fresh tissue was taken from lumpectomy or mastectomy specimens, mechanically and enzymatically dissociated, cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and characterized by immunocytochemistry. Out of 33 DCIS cases, 58 % (19) were expanded for up to 2 months in culture, and 42 % (14) were frozen immediately after mechanical dissociation for future growth. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the "conditionally reprogrammed" proliferative state and after conditioned media and ROCK inhibitor withdrawal. When grown to 100 % confluency, the cultures organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. Primary cultures of DCIS derived directly from patient tissues can be generated and may serve as in vitro models for the study of DCIS.