The development and characterization of a @entity1 XXXX in vitro 3D model to investigate immunotoxin therapy.

@entity1 @entity137 @entity5 @entity5962 @entity2615 @entity3620
BACKGROUND: @entity5 microenvironments present significant barriers to penetration by antibodies and immunoconjugates. @entity5 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 @entity5 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 @entity2615 . METHODOLOGY/PRINCIPAL FINDINGS: Here, we examined how the @entity5 microenvironment affects the penetration and killing activity of SS1P in a new three-dimensional (3D) spheroid model cultured in vitro using the @entity1 @entity2615 cell line ( @entity1 ) and two primary cell lines isolated from the ascites of @entity3620 @entity1 . 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 @entity137 , 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 @entity5962 , a protein involved in the assembly and sealing of tight junctions and highly expressed in @entity3620 , was found significantly increased in spheroids as compared to monolayers. Moreover, we found that siRNA silencing and antibody inhibition targeting @entity5962 could enhance SS1P immunotoxin therapy in vitro. CONCLUSION/SIGNIFICANCE: This work is one of the first to investigate immunotoxins in 3D @entity5 spheroids in vitro. This initial description of an in vitro @entity5 model may offer a simple and more representative model of in vivo @entity5 and will allow for further investigations of the microenvironmental effects on drug penetration and @entity5 cell killing. We believe that the methods developed here may apply to the studies of other @entity5 -targeting antibodies and immunoconjugates in vitro.
