Differential isolation of normal luminal mammary epithelial cells and breast cancer cells from primary and metastatic sites using selective media.
Differential isolation of normal luminal mammary epithelial cells and breast cancer cells from primary and metastatic sites using selective media.
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发表时间：
1993-02
期刊：
影响因子：
11.2
通讯作者：
S. Ethier;Michael L. Mahacek;W. Gullick;T. S. Frank;B. Weber
中科院分区：
文献类型：
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作者：
S. Ethier;Michael L. Mahacek;W. Gullick;T. S. Frank;B. Weber
本研究旨在确定使用支持管腔谱系人乳腺上皮细胞增殖的细胞培养基是否允许从原发性和转移性肿瘤标本中常规分离乳腺癌细胞。来自乳房缩小术标本和原发性乳腺癌的乳腺上皮细胞获得的结果表明，细胞在I型胶原蛋白包被的培养皿上在补充有胰岛素、氢化可的松、表皮生长因子、霍乱毒素和5%胎牛血清的Ham's F-12培养基中生长，导致腔细胞标记物细胞角蛋白19染色呈阳性的细胞生长和连续传代。相比之下，乳腺上皮细胞在生长因子补充的无血清培养基中的生长导致出现角蛋白19一致阴性的乳腺上皮细胞集落。使用过滤分离方法从源自乳腺癌标本的原代培养物中分离单个角蛋白-19阳性菌落。所有从乳腺癌组织中分离的乳腺腔上皮细胞均表达正常细胞的特征。角蛋白-19阳性克隆从几个不同的肿瘤中分离出来，在培养中快速生长30至60天，然后衰老。从一个已知在p53基因的特定位点发生杂合性丢失的肿瘤中分离细胞。从该标本中分离的所有菌落均含有两种p53等位基因，这与其来源于正常管腔细胞一致。还从一个肿瘤中分离细胞，其中c-erbB 2蛋白在肿瘤细胞中显著过表达。再次，角蛋白-19阳性克隆从这个肿瘤中分离没有过度表达c-erbB-2蛋白。然后用源自胸腔积液和转移性淋巴结的细胞进行实验。这些标本获得的结果表明，支持正常管腔乳腺上皮细胞生长的生长条件不支持肿瘤细胞的生长。然而，省略霍乱毒素，表皮生长因子，和I型胶原基质导致两个长期的细胞系的分离。这两种细胞系的群体倍增时间约为100小时，是超二倍体，染色阳性细胞角蛋白19。因此，支持正常腔乳腺上皮细胞生长的培养条件通常不支持乳腺癌细胞的生长。
The present studies were aimed at determining if the use of a cell culture medium that supports proliferation of human mammary epithelial cells of the luminal lineage would allow routine isolation of breast cancer cells from primary and metastatic tumor specimens. Results obtained with mammary epithelial cells derived from reduction mammoplasty specimens and primary breast carcinomas indicated that growth of cells on type I collagen-coated dishes in Ham's F-12 medium supplemented with insulin, hydrocortisone, epidermal growth factor, cholera toxin, and 5% fetal bovine serum resulted in the growth and serial passage of cells that stained positively for the luminal cell marker cytokeratin 19. By contrast, growth of mammary epithelial cells in a growth factor-supplemented serum-free medium resulted in the emergence of mammary epithelial cell colonies that were uniformly negative for keratin 19. Filter isolation methods were used to isolate individual keratin-19-positive colonies from primary cultures derived from breast cancer specimens. All of the luminal mammary epithelial cells isolated from breast cancer tissues expressed characteristics of normal cells. Keratin-19-positive colonies isolated from several different tumors all grew rapidly for 30 to 60 days in culture and then senesced. Cells were isolated from one tumor that was known to have undergone a loss of heterozygosity at a specific locus in the p53 gene. All colonies isolated from this specimen contained both p53 alleles, which was consistent with their origin from normal luminal cells. Cells were also isolated from one tumor in which the c-erbB2 protein was drastically overexpressed in the neoplastic cells. Once again, keratin-19-positive colonies isolated from this tumor did not overexpress the c-erbB-2 protein. Experiments were then performed with cells derived from pleural effusions and metastatic lymph nodes. Results obtained with these specimens indicated that the growth conditions that support the growth of normal luminal mammary epithelial cells do not support the growth of neoplastic cells. However, the omission of cholera toxin, epidermal growth factor, and type I collagen substratum resulted in the isolation of two long-term cell lines. Both cell lines have population doubling times of approximately 100 h, are hyperdiploid, and stain positively for cytokeratin 19. Thus, culture conditions that support the growth of normal luminal mammary epithelial cells do not, in general, support the growth of breast cancer cells.