Histopathological Analysis of Tumor Tissues

For histopathological evaluation, hematoxylin and eosin (H&E) staining was performed on paraffin-embedded tumor tissue sections. For immunohistochemistry, sections were stained with primary antibodies overnight at 4°C. On the next day; sections were incubated for 30 minutes in ImmPRESS AP Reagent (Vector Laboratories, Burlingame, CA), followed by incubating for 2–15 minutes in ImmPACT NOVA-RED (Vector Laboratories). The following primary antibodies were purchased from Agilent (Santa Clara, CA): cancer antigen 19-9 (CA19-9), carcinoembryonic antigen (CEA), cytokeratin 20 (CK20), and Ki-67 [9 (link)]. For terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, In Situ Cell Death Detection Kit (Roche, Penzberg, Germany) was used, followed by the manufacturer’s instruction. Ki-67 positive area or TUNEL positivity in immunohistochemistry (IHC) image was measured by ImageJ software (National Institutes of Health, Bethesda, MD).