*2.2. Immunohistochemical Staining*

GLUT1 expression was assessed by immunohistochemical staining using a rabbit anti-GLUT1 polyclonal antibody (Abcam, Cambridge, UK; 1:200 dilution). The reaction was visualized using the Histofine Simple Stain MAX-PO (Multi) Kit (Nichirei, Tokyo, Japan), according to the manufacturer's instructions. The detailed protocol for immunostaining has been published elsewhere [4]. Negative controls were incubated without primary antibody, and no staining was observed. GLUT1 expression was considered positive only if distinct cytoplasmic and plasma membrane staining was present. GLUT1 expression was scored as follows: 1, ≤10% of tumor area stained; 2, 11%–25% stained; 3, 26%–50% stained; 4, 51%–75% stained; and 5, ≥76% stained. Tumors in which the stained tumor cells were scored ≥4 were considered as "high-expression" tumors.

Immunohistochemical staining for Ki-67 was performed as described previously [4] using a murine monoclonal antibody against Ki-67 (Dako, Glostrup, Denmark; 1:40 dilution). Highly cellular areas of the immunostained sections were assessed for Ki-67. All epithelial cells with nuclear staining of any intensity were defined as high-expression epithelial cells. Approximately 1000 nuclei were counted on each slide. Proliferative activity was assessed as the percentage of Ki-67-stained nuclei (Ki-67 labeling index) in the sample. The median Ki-67 labeling index value was evaluated, and tumor cells with greater than median Ki-67 labeling index value were defined as high-expression tumor cells. All sections were assessed by light microscopy in a blinded manner by at least two investigators. In case of discrepancies, both investigators evaluated the slides simultaneously until reaching a final consensus. Neither of the investigators had knowledge of the patient outcomes.
