Pannoramic 250

HE, TUNEL and immunohistochemical (IHC) staining were carried out as reported by us previously [39 (link)]. HE staining was used to detect the pathological changes. Apoptotic cells in tumor tissues were stained with a TUNEL Apoptosis Detection Kit (Beyotime) according to the manufacturer’s protocol. For histological analysis, tumors were fixed overnight in 10% neutral buffered formalin, embedded in paraffin and sectioned at 5-μm thickness using a Leica RM2265 microtome. IHC staining was carried out with an EnVision Detection System HRP. A rabbit/mouse (DAB+) kit (Agilent) was used following the manufacturer’s instructions. Endogenous peroxidase was blocked by incubation with 0.3% hydrogen peroxide for 15 min. Antigen retrieval was performed by boiling the slides in citrate buffer (10 mM, pH 6.0) in a water bath for 20 min. After being rinsed and blocked with 5% bovine serum albumin (BSA), the slides were incubated overnight at 4 °C with primary antibodies, followed by 1 h with labeled Polymer-HRP at room temperature. Subsequently, the slides were exposed to DAB+ Chromogen. Counterstaining with hematoxylin was carried out. After mounting, the slides were observed under an Olympus CX21 microscope, scanned with a high-resolution digital slide scanner (Pannoramic 250, 3DHistech), and quantified by ImageJ software.

TILs and CD⁸⁺ lymphocyte analysis was performed by one experienced pathologist (S.A). TILs were scored as a percentage of immune cells in tumor-associated stromal areas^{28 (link)}. All mononuclear cells (including lymphocytes and plasma cells) were scored; polymorphonuclear leukocytes were excluded^{28 (link)}. CD⁸⁺ stained slides were scanned using a high-resolution digital slide scanner up to 400 × magnification (3DHISTECH Pannoramic 250; 3DHISTECH Ltd., Budapest, Hungary), and an area of 1 mm² was marked per tumor. For tumors with heterogeneous distribution of CD⁸⁺ lymphocytes, one hotspot area was selected per sample. The absolute number of CD⁸⁺ cells was manually counted.

GLUT-1 IHC images were obtained by slide scanner (Pannoramic 250, 3D histech, Budapest, Hungary) and interpreted on 3D histech software (caseViewer). Red blood cells were used as an internal positive control. Only membrane staining was considered positive. The evaluation was performed using a semi-quantitative score inspired by Sakashita et al. [31 (link)], which grades according to the proportion of GLUT-1 positive cells. We defined a four-grade IHC score: Score 0 negative (no membrane staining by tumor cells); Score 1+, weakly positive (membrane staining in <10% of tumor cells); Score 2+, moderately positive (membrane staining in ≥10 and <50% of tumor cells); Grade 3, strongly positive (membrane staining in ≥50% of tumor cells). Tumors classified as score 2+ or 3+ were considered to overexpress GLUT-1 (Figure 1). All cases were interpreted independently by two pathologists unaware of the patients’ associated medical data. In case of disagreement about GLUT-1 IHC score, cases were discussed until consensus was reached.

H&E and IHC staining were performed as previously reported^{46 (link)} with a small modification. H&E staining was used to detect pathological changes. For histological analysis, formalin-fixed, paraffin-embedded tumors were sectioned, and slides were deparaffinized using xylenes (Thermo Fisher Scientific Inc.). Endogenous peroxidases were quenched with 3% hydrogen peroxide in methanol. Staining was performed using antibodies against BRCA1 (1: 100), BRCA2 (1: 100), RAD51 (1: 50), γ-H2AX (1: 50), P-gp (1: 200), BCRP (1: 200), PAR (1: 250), or Ki67 (1: 500). Counterstaining was performed using Mayer’s hematoxylin (Dako, Glostrup, Denmark). Images were observed under an Olympus Cx21 microscope, scanned with a high-resolution digital slide scanner (Pannoramic 250, 3DHistech), and quantified using ImageJ software.