The development and characterization of a humanmesothelioma in vitro 3Dmodel to investigate immunotoxin therapy

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