The Development and Characterization of a Human Mesothelioma In Vitro 3D Model to Investigate Immunotoxin Therapy
The Development and Characterization of a Human Mesothelioma In Vitro 3D Model to Investigate Immunotoxin Therapy
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DOI：
10.1371/journal.pone.0014640
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发表时间：
2011-01-31
期刊：
影响因子：
3.7
通讯作者：
Ho, Mitchell
中科院分区：
文献类型：
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作者：
Xiang, Xinran;Phung, Yen;Ho, Mitchell
背景：肿瘤微环境是抗体和免疫结合物穿透的重要障碍。然而，肿瘤微环境很难在体外进行研究。与体内培养的细胞相比，培养成单层的细胞对治疗的抵抗力更低，更能代表体内肿瘤的替代研究模型更可取。SS1P是一种免疫毒素，由间皮瘤特异性抗体的Fv部分与细菌毒素融合而成，目前正在进行间皮瘤的临床试验。方法/主要研究结果：在这里，我们研究了肿瘤微环境如何影响SS1P的渗透和杀伤活性，在一个新的三维(3D)球体模型中，使用人间皮瘤细胞系(NCI-H226)和从恶性间皮瘤患者腹水分离的两个原代细胞系，体外培养了SS1P。生长为单层或球形的间皮瘤细胞表达类似水平的间硫蛋白；然而，球形细胞受SS1P的影响至少低100倍。为了了解这种细胞毒性的差异，我们制作了荧光标记的SS1P分子，并使用共聚焦显微镜检查SS1P在球体内渗透的时间进程。4小时后透皮吸收受限。有趣的是，我们在电子显微镜下发现，球体核心区的紧密连接数量显著增加。E-钙粘附素是一种参与紧密连接组装和封闭的蛋白，在恶性间皮瘤中高表达，与单层相比，球体中的表达显著增加。此外，我们还发现针对E-钙粘附素的siRNA沉默和抗体抑制可以在体外加强SS1P免疫毒素的治疗。结论/意义：本工作是首次在体外研究3D肿瘤球体中的免疫毒素。这种体外肿瘤模型的初步描述可能提供一个简单和更具代表性的体内肿瘤模型，并将允许进一步研究微环境对药物渗透和肿瘤细胞杀伤的影响。我们相信，本文开发的方法也可应用于其他肿瘤靶向抗体和免疫结合物的体外研究。
Background: Tumor microenvironments present significant barriers to penetration by antibodies and immunoconjugates. Tumor microenvironments, however, are difficult to study in vitro. Cells cultured as monolayers exhibit less resistance to therapy than those grown in vivo and an alternative research model more representative of the in vivo tumor is more desirable. SS1P is an immunotoxin composed of the Fv portion of a mesothelin-specific antibody fused to a bacterial toxin that is presently undergoing clinical trials in mesothelioma.Methodology/Principal Findings: Here, we examined how the tumor microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the human mesothelioma cell line (NCI-H226) and two primary cell lines isolated from the ascites of malignant mesothelioma patients. Mesothelioma cells grown as monolayers or as spheroids expressed comparable levels of mesothelin; however, spheroids were at least 100 times less affected by SS1P. To understand this disparity in cytotoxicity, we made fluorescence-labeled SS1P molecules and used confocal microscopy to examine the time course of SS1P penetration within spheroids. The penetration was limited after 4 hours. Interestingly, we found a significant increase in the number of tight junctions in the core area of spheroids by electron microscopy. Expression of E-Cadherin, a protein involved in the assembly and sealing of tight junctions and highly expressed in malignant mesothelioma, was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting E-Cadherin could enhance SS1P immunotoxin therapy in vitro.Conclusion/Significance: This work is one of the first to investigate immunotoxins in 3D tumor spheroids in vitro. This initial description of an in vitro tumor model may offer a simple and more representative model of in vivo tumors and will allow for further investigations of the microenvironmental effects on drug penetration and tumor cell killing. We believe that the methods developed here may apply to the studies of other tumor-targeting antibodies and immunoconjugates in vitro.