TdT-mediated dUTP-biotin nick end-labeling assay

For histological examination, tumor tissues were fixed in 10% buffered formalin and embedded in paraffin, and tissue sections (4 μm) were prepared. The TdT-mediated dUTP-biotin nick end-labeling (TUNEL) assay for apoptosis was conducted using an apoptosis detection kit (Roche Diagnostics, Branchburg, NJ, United States) according to the manufacturer’s instructions. Positive cells were counted as the number of TUNEL-labeled cells per 100 epithelial cancer cells in 10 fields of the most affected tumor areas with 400 × magnification and analyzed using light microscopy (Carl Zeiss, Thornwood, NY, United States).