Patients and tissue specimens

This study included 44 PDAC who underwent endoscopy at the Hepatology and Gastroenterology Department at Theodor Bilharz Research Institute (TBRI), Giza, Egypt, between the years 2021 and 2023.

The inclusion criteria for this study involved cases of primary PDAC with comprehensive clinical, radiological, and operative data properly documented in their files. None of the included cases had received radiation or chemotherapy before specimen collection. Conversely, cases that did not meet the criteria for PDAC, patients with > 180 degrees superior mesenteric artery encasement, cases involving celiac abutment, unreconstructable superior mesenteric vein/portal occlusion, aortic invasion or encasement, tumors extending to the uncinate or exhibiting distant metastasis, patients with significant cardiorespiratory comorbidities, or cases with insufficient data in their files were excluded from the study.

To confirm the diagnosis of PDAC, the biopsies were sent to the Pathology Department, TBRI. After that, patients underwent a Whipple operation at the Department of Surgery, at TBRI (Winter et al. 2006).

Tissue and blood samples from PDAC patients were sent to the immunology Department and Biochemistry and Molecular Biology Department at TBRI, and stored at – 20 °C till processing. The Whipple operation tissues were processed to prepare formalin-fixed paraffin-embedded tissue (FFPE) blocks from pancreatic cancer in the pathology Department, TBRI.

Additionally, for IHC, ELISA, and qPCR on tissue: the control was the normal pancreatic tissue adjacent to the cancer pancreas (n = 44). For ELISA on serum: controls were blood samples from 37 normal volunteers.

Ethical approval

The protocol of this study was approved by the Institutional Review Board (IRB] of Theodor Bilharz Research Institute, for the Protection of Human Subjects and adopted by the 18th World Medical Assembly, Helsinki, Finland (2013). Informed consent was obtained from all participants and/or their legal guardians. All experiments were performed following relevant guidelines and regulations.

Histopathological technique

One FFPE block was selected from each of the 44 PDCA lesions, including tumor areas and an adjacent area of histologically normal pancreatic tissue, cut in a thickness of 4 μm, and spread on positively charged glass slides. Sections were stained using hematoxylin and eosin stain (H&E stain) for routine histopathological examination at the Pathology Department, TBRI. The histological diagnoses and staging of PDAC were based on the TNM 8th edition classification of The American Joint Committee on Cancer (AJCC) criteria (Chun et al. 2018).

Immunohistochemical (IHC) technique for SATB2 and β-catenin

IHC for SATB2 and β-catenin was performed on the FFPE tissues. The tissue sections were put in the oven at 60 °C for 4 h, deparaffinized in xylene, rehydrated in a graded ethanol series, and treated with 3% hydrogen peroxide solution for 10 min. Antigen retrieval was done by microwaving the tissue in 10 mmol/L citric acid buffer for 12 min, then cooling it at room temperature for 2 h. The sections were incubated with SATB2 at a dilution of 1:100 (code YAP2448, Biospes, Chongqing, China) and β-catenin at a dilution of 1:100 (catalog no. sc-376841, Santa Cruz, CA, USA) for overnight at 4 °C. Sections were then washed three times for 5 min in PBS. Slides were incubated with ImmunoDetector DAB HBR detection system (BioSB, Santa Barbra, USA). Finally, staining was developed with 3,3’- diaminobenzidine (DAB) solution, and sections were counterstained with hematoxylin, dehydrated with graded ethanol, and mounted. For each setting, positive and negative controls were routinely used. Normal colorectal mucosa was used as a positive control for SATB2. Negative controls were carried out in which phosphate-buffered saline (PBS) was used instead of the primary antibody (Muniz Partida and Walters 2023).

Immunohistochemical interpretation and scoring for SATB2 and β-catenin

Scoring of SATB2 and β-catenin immunostaining was performed blindly to the patients’ clinicopathological data using × 40 objective. For SATB2, nuclear staining of tumor cells was assessed. For β-catenin; membranous and cytoplasmic expression was assessed. The immunoexpression of the studied protein was evaluated according to the immunoreactive score (IRS) by multiplying the percentage of positively stained cells and staining intensity. The percentage of positive cells was scored as 0 = negative, 1 = 1%-10%; score 2 +  = 11–50%, 3 +  = 51–75%, and 4 +  = 76–100%. The intensity of the stain was scored as 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The final IRS ranged from 0 to 12 and was divided into high expression scores for values > 2 and low expression scores for values ≤ 2 (Elebro et al. 2014).

Detection of serum and tissue-derived SATB2 by sandwich enzyme-linked immunosorbent assay (ELISA)

Blood samples were collected from the studied 44 PDAC cases through a venous puncture in vacutainer tubes for serum collection. The samples were coagulated at RT for 10–20 min, then, centrifuged at the speed of 2000–3000 rpm for 20 min to collect the supernatant (serum), then stored at − 20 °C. For tissue samples, after melting, tissues were homogenized thoroughly, and centrifuged at 2000–3000 rpm for 20 min to collect supernatant then stored at − 20 °C.

Serum samples and tissue homogenate were tested for the presence of SATB2 protein by sandwich-based ELISA using SATB2 ELISA kit, Biospes, China, according to the manufacturer’s instructions. The SATB2 protein that was present in the samples or standards/controls was captured by anti-SATB2 antibody pre-coated onto the test plate. Then, horseradish peroxidase (HRP) conjugated anti-SATB2 antibody was used as a detection antibody. Following incubation and washing procedures, 3,3′,5,5′-Tetramethylbenzidine (TMP) substrates were used to visualize the HRP-enzymatic reaction. O.D absorbance of the produced color was measured at 450 nm using an ELISA reader (MultiscanTMFC, Thermofisher, USA) and then the concentration of SATB2 was calculated from the standard curve (Hayrapetyan et al. 2023).

Quantitative real-time polymerase chain reaction (qPCR) of pancreatic β-catenin level

Total RNA was extracted from pancreatic tissue using an RNeasy Mini kit (Qiagen) in an RNase-free environment according to the manufacturer’s instructions. The RNA quantity and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). RNA (1 μg) was reverse transcribed using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The PCR reactions were done with StepOne™ Real-Time PCR System using 10 μl 2 × PoweUp™ SYBR™ Green/ROX PCR Master Mix (Applied Biosystems, ThermoFisher Scientific, USA). 5 pmol of forward and reverse primers, and 2 μl of cDNA. The thermal cycling conditions were as follows: 3 min at 95 °C, 50 cycles at 95 °C for 15 s, 55 °C for 20 s and 72 °C for 20 s. The sequence of the primers used is as follows: for β-catenin (accession number: XM_047447483.1), forward 5ʹ-CATGAGAAGTATGACAACAGCCT-3ʹ, and reverse 5ʹ-AGTCCTTCCACGATACCAAAGT-3ʹ, for Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (accession number: NM_001357943.2) Forward 5ʹ-CATGAGAAGTATGACAACAGCCT-3ʹ and reverse 5ʹ-AGTCCTTCCACGATACCAAAGT-3ʹ. Relative expression was calculated using the comparative cycle threshold (Ct) (2−ΔCT) method. All values were normalized to GAPDH expression as an endogenous control (Yilmaz et al. 2014; Saber et al. 2021; Salem et al. 2023).

Statistical analysis

Analyses were performed using SPSS version22 (IBM Corp., Armonk, New York, USA). One-way ANOVA was applied to study the effect of T-stage, lymph node involvement, and tumor stage on the concentrations of SATB2 and β-catenin in tissue and serum followed by Duncan's test to illustrate homogeneity among the different levels of each factor. Independent t-test was applied to illustrate the statistical effects of gender, age category, size, nodal stage, perineural invasion, lymphovascular invasion, and fat invasion. The Spearman correlation coefficient with bootstrapped (1000 resamples, bias-corrected) confidence intervals was calculated to find out correlations between β-catenin expression and the studied parameters. A p value < 0.05 represents a significant effect.