Abstract 5357: Evaluation of colony formation and growth of malignant glioma cells in soft agar using advanced colony counter system

Abstract

Abstract [Introduction] Malignant gliomas are the most common primary tumors of the central nervous system. The prognosis of patients with malignant gliomas is poor in spite of current intensive therapy because of inevitable regrowth or recurrence. Since glioma stem-like cells (GSCs) are thought to play critical role in initiation, regrowth and recurrence, effective therapy against GSCs is sought for. Efficient anticancer therapies should suppress not only the growth of tumor mass but also colony formation initiated by single cancer stem cell. Therefore, it is essential to analyze the effect of anticancer therapies on both tumor growth and colony formation. Regarding tumor growth, multicellular tumor spheroid (MCTS) model is thought to be more plausible method than 2-D culture because MCTSs resemble intervascular regions of solid tumors, that is, MCTSs reproduce 3-D structure-specific features such as intense paracrine, cell-cell adhesion related signaling, direct intercellular transport of materials, gradient of oxygen and nutrients, and lower drug penetration into the spheroids. Since GSCs form MCTSs in the standard stem cell culture media, viability and growth of GSCs have been analyzed mainly in this culture condition. However, regarding analysis of clonogenicity, colony formation assay using GSCs has been a challenge because GSCs aggregate in the stem cell culture media and evaluation of the accurate colony number requires single cell culture system that might need large number of wells/plates, especially for combination treatment, and might not be always appropriate for high-throughput screening. Therefore, a better approach is necessary. GelCountTM colony counter was developed to analyze both number and volume of colonies. In this study, we used soft agar and GelCountTM to analyze colony formation and growth of malignant glioma cells including GSCs. [Materials and Methods] Human glioma stem-like cells GSC11 and GSC20, or human malignant glioma cell line U87-MG were cultured in agarose gel and treated with erlotinib, sorafenib, imatinib or a polynuclear platinum compound BBR3610. The colony number and the calculated total colony volume (total biomass) were analyzed using GelCountTM colony counter system (Oxford Optronix Inc., UK). [Results] Glioma cells formed 3-D colonies in agarose, and the colony number and the total biomass increased according to the Gompertz function that is known to correlate with tumor growth in vivo. Each drug suppressed increase of colony number and total biomass in comparable concentration to that in 2-D culture. [Conclusions] GelCountTM allowed accurate, rapid monitoring of colony number and size at many time points without harm to the cultures. This system can be high throughput screening method using GSCs and also bridge the gap between 2-D culture assays and in vivo tumor formation assays. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5357. doi:1538-7445.AM2012-5357