Hassan Raffit, Lerner Megan R, Benbrook Doris, Lightfoot Stan A, Brackett Daniel J, Wang Qing-Cheng, Pastan Ira
Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA.
Clin Cancer Res. 2002 Nov;8(11):3520-6.
Mesothelin, a cell surface glycoprotein overexpressed in ovarian cancer, mesotheliomas, and some squamous cell carcinomas, is an attractive candidate for targeted therapy because it is not shed in significant amounts into the bloodstream and is not present in significant amounts on normal human tissues except for mesothelial cells. The objective of this study was to determine the antitumor activity of SS1(dsFv)PE38, a recombinant antimesothelin immunotoxin, against human gynecologic tumors grown in short-term culture in vitro.
Tumor cells obtained from primary cultures of five ovarian and one cervical tumor were mixed with an equal proportion of NIH-3T3 fibroblasts and plated inside collagen gels in tissue culture plates. After 4-7 days of growth, these organotypic cultures were treated with media alone, SS1(dsFv)PE38, and a control immunotoxin RFB4(dsFv)PE38, which targets the CD22 antigen not present on gynecologic tumors, every other day x 3. The organotypic culture gels were then formalin fixed, paraffin embedded, and evaluated for immunotoxin sensitivity using light microscopic examination of H&E-stained slides and also evaluated for apoptosis using the terminal deoxynucleotidyl transferase-mediated nick end labeling assay.
Tumors expressing mesothelin showed a significant dose-dependent sensitivity to SS1(dsFv)PE38 even at concentrations as low as 1 ng/ml, whereas no antitumor activity was seen at 100 ng/ml in tumors that did not express mesothelin. This activity was specifically attributable to mesothelin targeting because RFB4 (dsFv)-PE38 had no activity against mesothelin-expressing tumors.
These results demonstrate that ovarian and cervical tumor cells obtained from patients can be grown in short-term culture using an organotypic culture model. Our results also show low concentrations of an immunotoxin targeting mesothelin is cytotoxic to mesothelin-expressing human tumors by inducing apoptosis.
间皮素是一种在卵巢癌、间皮瘤和一些鳞状细胞癌中过表达的细胞表面糖蛋白，是靶向治疗的一个有吸引力的候选对象，因为它不会大量释放到血液中，除间皮细胞外，在正常人体组织中也不存在大量间皮素。本研究的目的是确定重组抗间皮素免疫毒素SS1(dsFv)PE38对体外短期培养的人妇科肿瘤的抗肿瘤活性。
从5例卵巢肿瘤和1例宫颈肿瘤的原代培养物中获得的肿瘤细胞与等量的NIH-3T3成纤维细胞混合，并接种于组织培养板中的胶原凝胶内。生长4-7天后，每隔一天用单独的培养基、SS1(dsFv)PE38和对照免疫毒素RFB4(dsFv)PE38处理这些器官型培养物，共处理3次。然后将器官型培养凝胶用福尔马林固定、石蜡包埋，通过苏木精和伊红染色切片的光学显微镜检查评估免疫毒素敏感性，并使用末端脱氧核苷酸转移酶介导的缺口末端标记法评估细胞凋亡情况。
即使在低至1 ng/ml的浓度下，表达间皮素的肿瘤对SS1(dsFv)PE38也表现出显著的剂量依赖性敏感性，而在不表达间皮素的肿瘤中，100 ng/ml时未观察到抗肿瘤活性。这种活性特别归因于间皮素靶向，因为RFB4(dsFv)-PE38对表达间皮素的肿瘤没有活性。
这些结果表明，使用器官型培养模型可以在短期培养中培养从患者获得的卵巢和宫颈肿瘤细胞。我们的结果还表明，低浓度的靶向间皮素的免疫毒素通过诱导细胞凋亡对表达间皮素的人类肿瘤具有细胞毒性。
