Ninety samples of colorectal tumors and adjacent normal tissue (at least 10 cm from each tumor) were obtained from patients undergoing surgical resection between 2013 and 2015 at King Khalid University Hospital, King Saud University Riyadh (KSA). The patient clinicopathological characteristics are shown in Table I. Tissues were collected after obtaining the written informed consent of the patients, according to the protocol approved by the Ethics Committee at King Saud University. Paired tissue samples were frozen in liquid nitrogen within 15 min from resection, and stored at −80°C until used. Demographic characteristics, including age and gender, tumor site and histological type of CRC were recorded. The histological stage of the tumor was determined in accordance with the Union International Cancer Control (UICC)-TNM staging system. Tumor localization, UICC stage and tumor differentiation (grading) were in accordance with the WHO classification (25), and documented in a database prospectus. Furthermore, and in order to minimize possible influences of radiotherapy and chemotherapy on CTSB status and prognosis, all patients who had undergone neoadjuvant or adjuvant therapy were excluded from this cohort.

A 5-ml blood sample from each participant patient and healthy control subject was allowed to clot for 1 h at room temperature. Each clotted sample was centrifuged at 5,000 rpm for 10 min. All sera were aliquoted and frozen at −80°C until used. CTSB concentrations in the sera were determined by a human cathepsin B kit using human CTSB ELISA kit (LS-F1026; Life Span Biosciences, Inc., Seattle, WA, USA) according to the manufacturer's instructions. The sera were analyzed in duplicate at a dilution of 1:50.

Total cellular RNA and protein were extracted according to the manufacturer's instructions using the PARIS™ kit (Ambion, Foster City, CA, USA) from frozen paired tumor and adjacent normal tissue as recommended by the manufacturer.

Pure, concentrated RNA was eluted in buffer provided as a kit ingredient, and stored at −80°C for further analysis. The purity of the total RNA was assessed by measuring the A260/280 ratio (1.8–2.0), using an ultraviolet spectrophotometer. To remove the contamination of genomic DNA, RNA samples were treated with RNAse-free DNAase (Ambion). For cDNA synthesis, the High Capacity cDNA kit was used to perform reverse transcriptase reaction (cat. no. 4368814; Applied Biosystems, Foster City, CA, USA).

PCR samples were prepared using Fast SYBR^®-Green Master Mix (cat. no. 4385612; Applied Biosystems), and the following CTSB primers (Integrated DNA Technologies, Leuven, Belgium) were used to detect the expression of CTSB by RT-PCR: forward, 5′-CCAGGGAGCAAGACAGAGAC-3′ and reverse, 5′-GAGACTGGCGTTCTCCAAAG-3′; and GAPDH forward, 5′-TGCACCACCAACTGCTTAGC-3′ and reverse, 5′-GGCATGGACTGTGGTCATGA-3′. The experiments were run on a ViiA™ 7 Real-Time PCR system. Results of CTSB mRNA expression were measured relative to GAPDH gene expression using the 2^−ΔΔCt method.

TMAs were constructed from paraffin-embedded tumor blocks using a manual tissue arrayer (Array Mold kit D; IHC World, Woodstock, MD, USA) as previously described (26). The detection of CTSB expression was performed using streptavidin-biotinylated horseradish peroxidase (S-ABC) kit (NovoLink Max Polymer Detection System; Novocastra, Milton Keynes, UK) according to the manufacturer's instructions. As a negative control, the same procedure was conducted with the omission of the primary antibody. The expression of CTSB in tumor and normal samples was analyzed using the eSlide capture device (ScanScope CS; Aperio Technologies Inc., Vista, CA, USA).

Immunohistochemistry staining was carried out on 5-µm sections of TMA blocks. The detection of CTSB expression was performed using streptavidin-biotinylated horseradish peroxidase (S-ABC) kit (Novo Link Max Polymer Detection System; Novocastra). Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in distilled H₂O for 5 min, and then, the slides were washed in Tris-buffered saline (TBS) for 10 min. Non-specific binding of antibodies was blocked by incubation with protein block (Novocastra) for 5 min. Subsequently, the slides were incubated with human CTSB monoclonal mouse antibody (Abazyme, Cambridge, MA, USA) primary antibody (1:100) for 1 h at room temperature. Slides were washed in 3X TBS for 3 min, and then incubated with biotinylated anti-mouse IgG (Novocastra) for 30 min. Peroxidase was detected using diaminobenzedine (DAB) substrate (Novocastra). Finally, slides were counterstained with Mayer's hematoxylin (Novocastra). As a negative control, the same procedure was conducted with the omission of the primary antibody. The expression of CTSB in tumor and normal samples was analyzed using the eSlide capture device (ScanScope CS).

High-resolution, whole-slide digital scans of all TMA glass slides were created using a ScanScope slide scanner (Aperio Technologies, Inc.) as previously described (26).

Four pairs of CRC tumors and adjacent normal tissue protein samples were isolated from early and late stage CRC using PARIS™ kit. Total protein concentration was determined using Bradford protein reagent (Bio-Rad, Hercules CA, USA). Soluble proteins (20 µg) were loaded on 10% precast TGX gels and were analyzed by immunoblotting with anti-CTSB (dilution 1:100; EMD Millipore, Billerica MA, USA). Reactivity was detected with horseradish peroxidase-conjugated secondary antibodies and chemiluminescence (Bio-Rad). Membranes were developed using C-Digit Blot Scanner (LI-COR, Hamburg, Germany).

Statistical analysis was performed using Graph Pad Prism software (version 5.0). The means between the two groups were compared using Mann-Whitney test and paired t-test in order to compare serum CTSB and mRNA levels in early and late tumor stage. A P-value of ≤0.05 was considered significant.

The present study was approved by the Ethics Committee of King Khalid University Hospital, King Saud University. Patient consent was obtained for the present study.

To evaluate the CTSB level in sera, two groups of patients were analyzed: early and late stage CRC patients. The level of CTSB was significantly increased in the sera of patients with late stage CRC when compared to the healthy controls. The medians were 2.9 and 0.33 ng/ml, respectively (P<0.0001) (Fig. 1). There was no significant difference between early stage cancer patients and healthy control subjects.

CTSB was analyzed at the mRNA level in a series of human paired specimens at early (stage I and II) and late stage (III and VI) CRC. The relative levels of CTSB mRNA in tumor CRC samples were significantly higher than levels in the adjacent normal colorectal tissue (P<0.05). CTSB gene expression was also elevated in late stage tumor samples, including patients with lymph node metastases, when compared to early stage (P=0.0006). The mRNA level in early stage CRC was significantly lower (0.04±0.01) than the mRNA level in late stage tissues (0.07±0.02) (Fig. 2). These findings suggest that CTSB mRNA was significantly higher in late stage CRC similar to the differences noted in the serum CTSB levels.

CTSB protein levels were analyzed in early vs. late stage CRC tumor samples, and vs. adjacent normal tissue. Intracellular CTSB protein expression was increased in late stage when compared to adjacent normal mucosa. No differences were noted in early cancer stages vs. adjacent normal tissue. The intracellular/active form of CTSB was highly expressed in late stage CRC samples (Fig. 3). These findings suggest that CTSB is significantly overexpressed in the late stage of CRC. Therefore active CTSB expression during late disease stage (stage III and IV) may have value as a biomarker for late stage CRC including patients with lymph node metastases.

We performed immunohistochemistry of CTSB on tissue microarray preparations of our paired tumor and adjacent normal specimens. Positive cells displayed red-brown staining in the cytoplasm. Late stage tumor tissues contained many CTSB-positive tumor cells (Fig. 4C). In contrast only a few were noted in early CRC stage samples (Fig. 4B). Normal colorectal tissues contained very few positive epithelial cells or macrophages (Fig. 4C). We performed a semi-quantitative analysis on patient samples, and 80% of late stage CRC samples showed strong positive staining while only 5% samples exhibited strong staining in early stages (P<0.02). The few early stage samples with significant staining may be in transition from early to late stage. These results demonstrate that CTSB expression is significantly increased at both the transcription and translation levels likely giving rise to increased serum CTSB levels observed in late stage CRC.