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 dehydrogenase, 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.

No abstract