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Proteomics shows 3D cell cultures are better than 2D

By Dr Matt Wilkinson , 09-Oct-2007

German researchers have shown 3D cell structures better resemble tumour phenotypes than traditional cultures, suggesting they will make better models for testing new anticancer agents.

One of the major stumbling blocks to designing new anticancer agents is the lack of adequate tumour models on which to test new drug candidates on as traditional monolayer cell cultures do not mimic the in vivo 3D environment closely enough. This latest research, published in an early view article in the Journal of Proteome Research by researchers from the Ludwig Maximilans University in Munich, Germany, used a combination of proteomics techniques to show that multicellular spheroids more closely resemble in vivo tumours than monolayer cell cultures. Multicellular spheroids consist of proliferating cell populations as well as areas with viable post-mitotic cells and compact structures in the spheroid core that can contain necrotic or apoptotic cells and therefore more closely resemble in vivo tumours. "The lack of suitable cancer model systems, which sustain the properties of the original tumour in vitro, is a general flaw in cancer research," write the authors. They continue by saying that obtaining primary cultures of colon carcinoma from patients is very laborious process that is impossible to standardise and leads to success rates of less than 10 per cent. In addition, they claim that most cancer cell lines accumulate genetic alterations and undergo artificial selection processes during multiple growth and passaging cycles. The researchers showed that the spheroids derived from the COGA-5 cell line expressed different levels of proteins to the monolayer cultures and they state that many of these alterations have been previously observed in in vivo tumours. 2D electrophoresis combined with MALDI-TOF (matrix assisted laser desorption ionisation)-(time of flight) mass spectral (MS) analysis of the spheroids showed that five proteins were up-regulated and one down regulated. Acidic calponin was found to be down-regulated in the spheroids and has been implicated in cytoskeletal organisation, suggesting that the spheroids exhibit a different architecture than the monolayer cultures, which was shown to be the case by fluorescent confocal laser scanning microscopy. The up-regulated proteins included 15-hydroxyprostaglandin dehydrogenase (15-PGDH), lamin A/C fragments and two isoelectric variants of acidic ribosomal proteins P0. 15-PGDH is often considered to expose tumour suppressor activity and may contribute to proliferation inhibition by degrading the prostaglandins that promote growth factor signalling. Lamin A/C has been linked with DNA replication and transcription as well as being cleaved during caspase-dependant cell apoptosis. The presence of the lamin A/C fragments was only observed in the spheroids and not in monolayer cultures, suggesting that that apoptosis occurs in the spheroids due to hypoxia at the core. The researchers showed that cell apoptosis could be induced using anticancer agents such as 5-fluorouracil or camptothecin and this increased the presence of the lamin A/C fragments further still. Interestingly, the authors note that this drug-induced apoptosis was accompanied by lamin A/C cleavage and suggests that the anticancer agents promote a similar capsase-dependant cell death pathway as observed in the spheroids. In addition to these spheroids being better tumour models on which to study drug response, further proteomic analysis could help to identify protein regulation and signalling pathways that may reveal new targets for effective cancer therapies.

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