Clonogenic cell assay for anchorage-dependent squamous carcinoma cell lines using limiting dilution

Clonogenic assays under either anchorage-dependent or -independent conditions are very useful for testing the sensitivity of tumor cells to cytotoxic drugs and radiation. These assays have not been widely used with squamous-cell carcinomas (SCC) because of poor tumor-cell viability and poor cloning efficiency, especially in semi-solid media. To find a clonogenic assay suitable for use with human squamous cancers we tested SCC lines, derived in our laboratory from patients with head and neck cancer, for the capacity to form colonies in soft agar and in 96-well plates. Of 13 UM-SCC lines tested for colony formation in agarose, only UM-SCC-11A was capable of growth in conventional semi-solid media. One other line, UM-SCC-14C, produced colonies in agarose only in the presence of epidermal growth factor. In contrast, all 17 of the SCC lines tested exhibited colony formation in adherent cell culture using limiting dilution in 96-well plates. The plating efficiencies of the SCC lines in the 96-well plate assay ranged from 0.02 to 0.52 colonies (wells)/cell whereas the PE values in soft agar were lower, ranging from 0.0055 to 0.0086 colonies/cell. The 96-well plate assay is not affected by cell migration, a problem encountered with some cell lines when clonogenic assays are performed in Petri dishes. UM-SCC-11A was tested for radiation sensitivity both in soft agar and in the 96-well plate assay. Comparable results were obtained. In summary, the majority of SCC cell lines did not form viable colonies in soft agar but the 96-well plate assay was applicable to a broad spectrum of anchorage-dependent human SCC cell lines and provides an efficient method for evaluating clonogenic cell survival.