Proteomic Analysis Reveals Differences in Protein Expression in Spheroid versus Monolayer Cultures of Low-Passage Colon Carcinoma Cells Lars Gaedtke, †,‡ Lilja Thoenes, †,‡ Carsten Culmsee, † Barbara Mayer, § and Ernst Wagner* ,† Pharmaceutical Biology-Biotechnology, Department of Pharmacy, Center of Drug Research, and Department of Surgery, University Clinic Center Grosshadern, Ludwig Maximilians University, Munich, Germany Received January 30, 2007 Spheroid cultures of cancer cells may better reflect characteristics of tumors than traditional monolayer cultures. Furthermore, low-passage cancer cell lines recapitulate the properties of the original tumor cells more closely than commonly used standard cell lines that experience artificial selection processes and mutations over years of passaging. Here we established spheroid cultures of the low-passage colon cancer cell line COGA-5 and stable COGA-12 aggregates with local areas of compaction. The proteomes of both three-dimensional cultures were analyzed versus their corresponding two- dimensional cultures. 2-D gel electrophoresis followed by peptide mass fingerprinting identified three differently expressed proteins in COGA-5 spheroids (acidic calponin, hydroxyprostaglandin dehydro- genase, and lamin A/C) and two in COGA-12 partly compact aggregates (two isoelectric variants of the acidic ribosomal protein P0) compared to the respective monolayer cultures. The lamin A/C spot showed a lower molecular weight in the 2-D gel (30 kDa) than expected for full-length lamin. Further Western blot analysis and immunocytochemistry identified the lamin protein as a caspase-6-cleavage product in apoptotic cells of the spheroid. Similar caspase-dependent lamin cleavage was observed in monolayer cultures after serum withdrawal and further increased under hypoxic conditions, suggesting cleaved lamin as an indicator for apoptotic stress. In conclusion, proteome analysis of multicellular spheroids versus monolayers cultures identifies differential protein expression relevant to tumor cell proliferation, survival, and chemoresistance and thus may reveal novel targets for cancer therapy. Keywords: acidic calponin • acidic ribosomal protein P0 • colon cancer • hydroxyprostaglandin dehydrogenase • lamin A/C • mass spectrometry • spheroid culture • two-dimensional gel electrophoresis Introduction The lack of suitable cancer model systems, which sustain the properties of the original tumor in vitro, is a general flaw in cancer research. For example, obtaining primary cultures of colon carcinoma cells from patients is very laborious, can hardly be standardized, and the success rate is far below 10%. In addition, most cancer cell lines accumulate genetic alter- ations or undergo artificial selection processes during several in vitro passages, thereby loosing their original phenotype. A promising prospect has been provided by recently developed low-passage colon cancer cell lines that closely resemble the phenotypes of the corresponding original tumors. 1 Extensive investigation of these low-passage cell lines demonstrated, for example, a pronounced diversity of the individual tumor cells regarding oncogenic mutations. However, traditional monolayer cultures of these low- passage cell lines still hardly recapitulate characteristics that result from the three-dimensional growth in tumors in vivo. Multicellular spheroid cultures are intermediates between monolayer cultures and tumors 2,3 since they resemble the in vivo situation more closely with regard to cell proliferation, differentiation, and cell environment, i.e., cell-cell contacts and different growth areas. A major feature of multicellular sphe- roids is that they consist of proliferating cell populations as well as areas with post-mitotic, yet viable, cells and also compact structures, often in the spheroid core, which may contain necrotic or apoptotic cells. 4 This structure reflects the assembly of a tumor better than monolayer cultures and may therefore provide an advanced model system for in vivo tumors. For example, murine EMT6 mamma carcinoma cells exhibiting chemoresistance after treatment of the tumor-bearing mice recapitulated this resistant phenotype in multicellular spheroids but not in monolayer cultures. 5 Similarly, spheroids of the low-passage colon carcinoma cells may better reflect the original patient phenotype than mono- layer cultures and, therefore, provide adequate model systems for analysis of colon cancers and development of novel * To whom correspondence should be addressed. Ernst Wagner, Phar- maceutical Biology-Biotechnology, Department of Pharmacy, Center of Drug Research, Butenandtstr. 5-13, Building D, 81377 Munich, Germany. Phone: +49 89 2180 77841. Fax: +49 89 2180 77791. E-mail: ernst.wagner@ cup.uni-muenchen.de. † Department of Pharmacy. ‡ The first two authors have equally contributed to this work. § Department of Surgery. 10.1021/pr0700596 CCC: $37.00  2007 American Chemical Society Journal of Proteome Research 2007, 6, 4111-4118 4111 Published on Web 10/06/2007