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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 .

The Development and Characterization of a Human Mesothelioma In Vitro 3D Model to Investigate Immunotoxin Therapy