Abstract 648: A 3D spheroid model of human mesothelioma to investigate antibody therapeutics
Authors: Xiang Maria , Phung Yen , Ho Mitchell 
Published: February 2011

Abstract
The formation of tumor cells traditionally cultured in vitro as monolayers frequently exhibit less drug resistance than tumors in vivo and are inadequate models for screening anti-cancer agents. Mesothelioma, a highly fatal asbestos-induced cancer of the mesothelium, is notoriously resistant to traditional chemotherapy and radiation. In this study, the development of multicellular and apoptotic resistance in human mesothelioma cell lines is examined using a 3D spheroid model cultured in vitro, which more closely depicts in vivo tumor resistance. We hypothesized that poor drug penetration of spheroid tumors is associated with the number of cellular contacts related to its 3D shape. In addition, we asked whether its resistance to cell death was directly related to the Bcl-2-regulated mitochondrial pathway. To test this, we cultured tumors cells both as monolayers and spheroids and measured the concentration of a fluorescently-labeled immunotoxin, SS1P, as a percentage of live cells. Antigen expression was determined via flow cytometry, and tumors were visualized using transmission and scanning electron microscopy, where a higher number of tight junctions were found in the core area of the spheroid. Additionally, we analyzed the expression of several proteins related to apoptosis in cell lysates for both monolayers and spheroids by western blotting. We found that some human mesothelioma cells form spheroids within 24 hours in the absence of Matrigel. The expression levels of mesothelin and CA125 - two established tumor antigens - are comparable between mesothelioma cells obtained from monolayer and spheroid; however, the cytotoxicity assay (IC50) of the anti-mesothelin immunotoxin SS1P shows that it is at least 100 times less active for spheroids than for monolayers. SS1P also shows incomplete penetration in spheroids, with limited penetration after 4 to 8 hours. Regarding cellular contacts, E-Cadherin is expressed in both monolayers and spheroids, but is significantly increased in spheroids. However, no bands are seen for prosurvival protein Bcl-2, and no difference in expression is found for prosurvival protein MCL-1. As for pro-apoptotic molecules, Bax and Bak, Bax did not show any changes, whereas Bak appeared only in monolayer and was absent for spheroid. Our results indicate that the upregulation of E-Cadherin expression in spheroids may be related to the increased number of tight junctions, thereby inhibiting the penetration of immunotoxins. Likewise, downregulation of the pro-apoptotic molecule Bak may cause resistance to immunotoxin-induced mitochondria-dependent apoptosis in spheroids. We conclude that using a biologically relevant 3D spheroid model more closely resembles similar types of resistance to that of in vivo tumors. We believe that it is a useful in vitro tumor model that will provide greater opportunity for investigating and screening antibody therapeutics.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 648.