Sircar Kanishka, Gaboury Louis, Ouadi Lydia, Mecteau Melanie, Scarlata Eleonora, Saad Fred, Aprikian Armen, Tanguay Simon, Lapointe Steven, Lussier Christian, Miletti Tina, Lanoix Joel
Department of Pathology, McGill University Health Center, Canada.
Clin Cancer Res. 2006 Jul 15;12(14 Pt 1):4178-84. doi: 10.1158/1078-0432.CCR-06-0328.
To isolate human prostatic epithelial plasma membranes for the identification of cell surface proteins in the therapeutic targeting of cancer cells while permitting the retrieval of banked samples for clinical purposes.
Radical prostatectomies from 84 patients (median, 61 years; prostate-specific antigen, 5.9; 66% nonpalpable) were processed with alternate, mirror image slices submitted for histology and tissue banking. Benign and malignant foci were macrodissected from the banked sections using the pathologically mapped, mirror image histology sections as a guide. Epithelial plasma membranes were isolated using novel immunomagnetic purification and their purity was assessed. Tissue homogenates were probed by Western blot for malignant (AMACR) and benign (p63) markers to test the accuracy of this protocol. Selected banked tissue slices were retrieved, thawed, and compared pathologically to their corresponding routinely processed alternate slices.
Plasma membrane preparations showed the enrichment of epithelial plasma membrane markers (prostate-specific membrane antigen and epithelial-specific antigen) with minimal marker expression from nonepithelial cells or intracellular organelles. Cancer homogenates showed up-regulated AMACR and down-regulated p63, whereas benign homogenates showed up-regulated p63 and down-regulated AMACR. There was 30% benign (p63+) contamination in cancer slices and <6% cancer (AMACR+) contamination in benign slices. Retrieved tissues showed the retention of immunoreactivity while their histology was always adequate for diagnosis.
We have successfully isolated purified epithelial plasma membranes from benign and malignant human prostates and provided validation data for the accuracy of our protocol in a prostate-specific antigen-screened cohort. Our method also enabled the retrieval of banked tissues for clinical purposes with the retention of good histologic and immunohistochemical quality.
分离人前列腺上皮细胞膜，用于鉴定癌细胞治疗靶向中的细胞表面蛋白，同时允许为临床目的获取储存样本。
对84例患者（中位年龄61岁；前列腺特异性抗原5.9；66%不可触及）的根治性前列腺切除术标本进行处理，将交替的镜像切片分别用于组织学检查和组织储存。以病理映射的镜像组织学切片为指导，从储存切片中宏观解剖出良性和恶性病灶。使用新型免疫磁珠纯化法分离上皮细胞膜，并评估其纯度。通过蛋白质免疫印迹法检测组织匀浆中的恶性（α-甲基酰基辅酶A消旋酶）和良性（p63）标志物，以测试该方案的准确性。取出选定的储存组织切片，解冻，并与相应的常规处理的交替切片进行病理比较。
质膜制备物显示上皮细胞膜标志物（前列腺特异性膜抗原和上皮特异性抗原）富集，非上皮细胞或细胞内细胞器的标志物表达极少。癌组织匀浆显示α-甲基酰基辅酶A消旋酶上调，p63下调，而良性组织匀浆显示p63上调，α-甲基酰基辅酶A消旋酶下调。癌组织切片中有30%的良性（p63阳性）污染，良性组织切片中有<6%的癌（α-甲基酰基辅酶A消旋酶阳性）污染。取出的组织显示免疫反应性得以保留，同时其组织学始终足以用于诊断。
我们已成功从良性和恶性人前列腺中分离出纯化的上皮细胞膜，并为我们的方案在前列腺特异性抗原筛查队列中的准确性提供了验证数据。我们的方法还能够为临床目的获取储存组织，同时保留良好的组织学和免疫组织化学质量。
Zotero 插件