Cell-Cycle Analysis of Vismodegib-Treated Fibroblasts and Basal Cell Carcinoma Cells

Objectives: Gorlin Syndrome is characterized by the formation of numerous skin basal cell carcinomas (BCC) and multiple keratocystic odontogenic tumors. Vismodegib, a chemotherapy agent small-molecule inhibitor, targets abnormal activity of the Sonic Hedgehog pathway in BCC cells, but its effect on supportive fibroblasts within these tumors is unclear. We explored the effect of Vismodegib on the cell cycle of human dermal fibroblasts and BCC cells in vitro, using different cell-fixation methods and flow cytometry analysis.
Methods: Cultures of primary fibroblasts and BCC cells (ATCC TE 354.T) were treated with DMSO (control), 10nM or 10μM Vismodegib for 4-12 days. Trypsinized cells were fixed by 4% Paraformaldehyde or by 70% Ethanol, followed by nuclear staining with Propidium-Iodide/RNAase solution. Flow cytometry analysis was performed with BD FACSAria II Flow Cytometer, and cell cycle phases were analyzed with BD FACSDiva Software. Statistical analysis was performed with one-way ANOVA using Stata 13.1.
Results: Fixation of control, 10nM or 10μM Vismodegib-treated fibroblasts or BCC cells with Paraformaldehyde resulted in partially overlapping bell-curve graphs with different fluorescence intensity of PI when analyzed with flow cytometry. This approach did not allow gating defined singlet and doublet cell sub-populations that underwent G0/G1, S and M phases of the cell cycle. In contrast, cell culture fixation with Ethanol resulted in characteristic cell-cycle graphs, in which the mean of PI fluorescence intensity was twice as high in cells in the M phase compared to cells in G0/G1 phase. We were able to demonstrate that the distribution of sub-populations of fibroblasts and BCC cells in G0/G1, S and M phases was affected by drug concentration and duration of treatment.
Conclusions: Ethanol fixation, unlike Paraformaldehyde fixation, allows for an accurate representation of the cell cycle via flow cytometry, permitting the analysis of the effects of concentration and duration of Vismodegib treatment on BCC keratinocytes and dermal fibroblasts.