The colony forming ability of HNSCC lines in-vitro

Objective:To assess colony forming abilities of individual cells within three cell lines generated from head and neck cancers and characterize the colonies formed by staining for cell surface markers. Materials and Methods:The Ca1, CaLH2 and CaLH3 cell lines were plated at low densities and grown for 2 to 4 weeks to develop colonies. Using cloning rings, single cells were isolated from holoclones, the type of colonies putatively containing stem cells. Individual cells, collected using direct vision, were plating singly in 24-well plates in a supplemented growth medium (fresh RM⁺:condition medium=1:1). Once colonies had formed they were stained with Rhodamine red or by immunocytochemistry for CD44, E-Cadherin, EGFR, Pan-Cytokeratin, CD133, and Involucrin. Results:Compared with CaLH2 and CaLH3, the Ca1 cell line showed significantly higher colony forming potential with a greater proportion of single cells, either from individual holoclones or pooled populations, forming colonies. The CaLH2 cell line showed the lowest level of colony forming potential. Plating individual cells on a 3T3 feeder layer increased the proportion of colonies formed by Ca1 and CaLH2 holoclone and pooled cells. Colonies formed from single cells showed high expression of cytokeratins and the cell surface markers CD44 and EGFR, typical of malignant stem cells. The colonies generated by CaLH2 formed sphere-like morphologies. Conclusions:The varying colony-forming potentials, assessed as the proportion of cells forming continuously expanding colonies with strong staining for potential stem cell surface markers CD44 and EGFR, suggests a varying proportion of potential cancer stem cells within each cell line. The use of a feeder layer provided additional support for cell survival and colony formation. It is anticipated that these cell culture methods can provide a way of assessing the stem cell fraction of tumours and relating this to tumour behaviour.