The use of the tumor 3-D spheroid model for screening for anticancer activity: a strategy for identification anticancer agents with activity on solid tumors


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Walid Fayad1, Rolf Larsson2, Stig Linder1, Mårten Fryknäs2
1Karolinska Institute, Stockholm, Sweden,2Uppsala University, Uppsala, Sweden

Background

There is a definite need for the discovery of more efficient anticancer agents against solid tumors as the cure rates of current clinically used agents are not satisfactory. Cytotoxic agents are generally developed using screening of tumor cell lines grown as monolayers. These monolayers do not reflect the characteristics of solid tumors in vivo. That might explain the failure of monolayers hits when tested in vivo. However, when tumor cell lines are grown as multicellular spheroids they acquire some characteristics of solid tumors. For example, the 3-D conformation, growth kinetics, gradient in diffusion of oxygen, nutrients and metabolites and the presence of hypoxic, quiescent subpopulation. Consequently, spheroids model has better predictability power for penetrating anticancer hits that can work in vivo compared to conventional monolayers.

Method

We established a method for production of spheroids in 96 well plates which make it a possible model for highthroughput screening. We used both colon carcinoma and normal epithelial cell lines in order to identify cytotoxic agents with therapeutic window. General viability was assessed by the acid phosphatase assay, while apoptosis induction was assessed by M30-CytoDeath ELISA. Primary assessment of mechanism of action of identified hits was done through connectivity map analysis. Specific in vitro assay was followed to confirm the predicted mechanism of action.

Results

Spheroid screening was found to enrich for compounds with high hydrophobicity. Mechanistic analysis of hits having therapeutic window revealed that the majority of them are microtubulin inhibitors. Compound 524881 from DIVERSet library from Chembridge was found to be a microtubulin inhibitor with therapeutic window active on colon carcinoma cell line spheroids.

Conclusion

Spheroid screening strategy resulted in the identification of hydrophobic compounds, a finding that has implications in the process of anticancer drug discovery and the selection of libraries for screening of anticancer agents.