A New Preclinical 3-Dimensional Agarose Colony Formation Assay

Abstract

The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount™ technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (μm2 × OD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount™ scanning at 3–4 days, while it took 6–7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5–6 days; glioma cells grew more slowly, with 100 colonies counted in 9–10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount™ method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.