A total of 25 (male; mean age, 52.6; age range 35–70 years) human PaC tissue and paired adjacent noncancerous pancreatic tissue samples were acquired after written informed consent was obtained from the participating patients. The patients did not receive partial or systemic treatment prior to tissue sampling, and agreed to receive these treatments at the First Affiliated Hospital of Soochow University (Suzhou, China) between June 2006 and January 2017. Paired adjacent noncancerous pancreatic tissues (≤5 cm from the tumor site) were obtained from patients during surgery. All tissue specimens were immediately snap-frozen in liquid nitrogen following surgery. In addition, peripheral blood specimens were obtained from patients with PaC using vacutainer tubes containing the anticoagulant EDTA. All peripheral blood samples were stored at 4°C and were processed within 3 days. The present study was approved by the Ethics Review Committee of the First Affiliated Hospital of Soochow University.

Human PaC cell lines PANC-1 and SW-1990, and the normal human pancreatic cell line HPDE6-C7 were used in the present study from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were seeded in Dulbecco's modified Eagle's medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), and were cultured at 37°C in a humidified incubator containing 5% CO₂.

Peripheral blood cells (PBCs) and CTCs were isolated and cultured with Isolation of Peripheral Blood Mononuclear Cells kit (Qiagen, Inc., Valencia, CA, USA) and the CellSearch Circulating Tumor Cell kit (Menarini Silicon Biosystems, Inc., Bologna, Italy) according to the manufacturer's protocol, respectively. The peripheral blood samples from patients with PaC were pooled into 10 ml vacutainer tubes containing the anticoagulant EDTA on ice. The 2-ml peripheral blood cells were lysed in a conical tube containing 13 ml 1X red blood cell (RBC) lysis buffer (Beijing Leagene Biotech Co., Ltd., Beijing, China) for 10 min at room temperature, and were then centrifuged at 800 × g for 8 min at room temperature. Subsequently, the cells were washed twice by resuspending the pellets in 6 ml 1X PBS and were centrifuged at 800 × g for 3 min at room temperature. Finally, the remaining cells were cultured in RPMI 1640 medium (HyClone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% FBS and incubated in a cell incubator containing 5% CO₂.

Total RNA was extracted from PaC tissues or cells and peripheral blood cells using a HP Total RNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA) and Blood RNA kit (Omega Bio-Tek, Inc.), respectively, according to the manufacturer's protocols. The RNA levels were analyzed using a NanoDrop spectrophotometer (NanoDrop; Thermo Fisher Scientific, Inc., Wilmington, DE, USA). RNA was reverse transcribed into cDNA with an M-MLV First Strand kit (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. The mRNA expression levels of ROR1 were quantified using a Platinum SYBR Green qPCR SuperMix-UDG kit (Invitrogen; Thermo Fisher Scientific, Inc.) on an ABI Prism 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol. Sequences of the primers were as follows: ROR1 (forward), 5′-AGATCACAGCTGCCTTCACTAT-3′; ROR1 (reverse), 5′-GACATTCTCCAGGATTTCACAT-3′; β-actin (forward), 5′-GGCGGCACCACCATGTACCCT-3′; β-actin (reverse), 5′-AGGGGCCGGACTCGTCATACT-3′. The thermocycling conditions were as follows: 95°C for 10 min, followed by 40 cycles of 50°C for 20 sec and 60°C for 1 min. Quantification cycle (Cq) values of ROR1 mRNA were equilibrated to the internal control β-actin mRNA. Relative expression was calculated using the ΔΔCq method (28). All experiments were performed in triplicate.

Protein lysates from cells and tissues were obtained using radioimmunoprecipitation lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA), which contained protease and phosphatase inhibitors (Sangon Biotech Co., Ltd., Shanghai, China). Protein concentrations were measured using NanoDrop technology (NanoDrop; Thermo Fisher Scientific, Inc.). Protein products (25 µg) were separated by 10% SDS-PAGE and were electrophoretically transferred onto a nitrocellulose membrane (EMD Millipore, Billerica, MA, USA). The membrane was then incubated with primary antibodies at 4°C overnight after blocking with 1.5% bovine serum albumin (Beyotime Institute of Biotechnology, Shanghai, China), followed by incubation with secondary antibodies at room temperature for 2 h. An Pierce™ ECL Western Blotting substrate (Pierce; Thermo Fisher Scientific, Inc.) was used to detect protein bands, which were analyzed using Quantity One 4.6 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA). β-actin was used to normalize target proteins. Antibodies employed in western blotting were: Rabbit anti-ROR1 (1:200, cat. no. ab15148; Abcam, Cambridge, UK), rabbit anti-E-cadherin (1:800, cat. no. ab15148; Abcam), rabbit anti-N-cadherin (1:1,000, cat. no. ab18203; Abcam), rabbit anti-β-actin (1:2,000, cat. no. ab8227; Abcam) and mouse anti-rabbit secondary antibodies (1:2,000, cat. no. sc-2357; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).

The small interfering RNA (siRNA) sequence that directly targets human ROR1 and the scrambled sequence used as a corresponding negative control (NC) were synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China). The target siRNA sequence for ROR1 was (5′-3′): Sense, UGAACCAAUGAAUAACAUCdTdT and antisense, GAUGUUAUUCAUUGGUUCAdTdT. The NC sequence was (5′-3′): Sense, UUCUCCGAACGUGUCACGUTT and antisense, ACGUGACACGUUCGGAGAATT. CTC cells were seeded into 24-well plates at a density of 5×10⁴ cells/well. On the following day, CTCs were transfected with a mixture containing 100 pmol siRNA and Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, incubated in a humidified atmosphere containing 95% air and 5% CO₂ at 37°C. The cells were harvested after 72 h and Transwell assays were performed.

The proliferation of PANC-1 and SW-1990 cells, as well as CTCs obtained from patient blood samples, was assessed using MTT assay (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), according to the manufacturer's protocol. A total of 3,000 cells/well were seeded in a 96-well plate and were incubated for 4 days in a humidified atmosphere containing 95% air and 5% CO₂ at 37°C. Subsequently, 10 µl MTT reagent was added to each well and cultured at 37°C for 2 h in darkness. Dimethyl sulfoxide was added to dissolve the crystals, and the absorbance was measured at 570 nm every 24 h. Three independent experiments were performed.

Cell invasion assays were performed using Transwell plates (BD Biosciences, Franklin Lakes, NJ, USA) Cell invasion assays were performed in 24-well Transwell chambers containing polycarbonate filters with 8 mm pores coated with Matrigel. According to the manufacturer's protocol, 5×10⁴ cells were suspended in DMEM containing 1% FBS and were placed into the top chamber, which contained a Matrigel-coated filter. DMEM supplemented with 10% FBS was added to the bottom chamber to be used as a chemoattractant. After 48 h at 37°C, the DMEM was discarded and cells adhering to the upper side of the membrane were removed with a cotton swab. Cells that had invaded onto the lower side of the membrane were stained with 1% crystal violet for 30 min at room temperature and observed by a light microscope (magnification, ×100; Olympus Corporation, Tokyo, Japan). Invasive cells were stained and counted in at least three microscopic fields (magnification, ×100). The experiments were independently repeated three times.

Differences between two groups were analyzed using Student's t-test (two-tailed). Statistical analyses were conducted using GraphPad Prism 5.02 (GraphPad Software, Inc., La Jolla, CA, USA) and SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). Representative data from three independent experiments were presented as the means ± standard deviation or means ± standard error. P<0.05 was considered to indicate a statistically significant difference.

Numerous studies have indicated that ROR1 is increased in several types of blood and solid malignancies (18,27). To investigate whether the expression levels of ROR1 were elevated in PaC, ROR1 expression was detected in 25 paired PaC tissues and adjacent noncancerous tissues using RT-qPCR and western blotting. The mRNA expression levels of ROR1 in PaC tissues were significantly increased compared with in paired noncancerous pancreatic tissues (Fig. 1A). Furthermore, the protein expression levels of ROR1 were detected, and the results demonstrated that PaC tissues exhibited significantly increased ROR1 protein expression compared with in the paired noncancerous pancreatic tissues (Fig. 1B). CTCs, also known as metastasis-derived cells, move into the circulatory system from the primary tumor site. Therefore, the difference in ROR1 mRNA expression between CTCs and PaC blood cells was investigated. The results indicated that the abundance of ROR1 mRNA in CTCs was ~10-fold higher compared with in peripheral blood cells obtained from patients with PaC (Fig. 1C). These findings indicated that ROR1 expression was increased in PaC, particularly in CTCs.

Increased ROR1 expression serves key roles in cell proliferation and invasion (18,27). To investigate the role of ROR1 in PaC cells, and in CTC growth and invasion, MTT and Transwell assays were performed. Initially, the expression levels of ROR1 were detected in CTCs and PANC-1 and SW-1990 cells by western blotting. As illustrated in Fig. 2A, ROR1 expression was markedly increased in CTCs compared with in PANC-1 and SW-1990 cells. Furthermore, compared with in the PANC-1 and SW-1990 cells, CTCs with high ROR1 expression exhibited significantly higher cell viability (Fig. 2B). Consistent with the MTT assay results, CTCs with high ROR1 expression displayed enhanced cell invasive ability compared with PANC-1 and SW-1990 cells (Fig. 2C). Taken together, these results suggested that ROR1 may serve key roles in the ability of CTCs to mediate PaC growth and invasion.

In order to further demonstrate the role of ROR1 in PaC metastasis, ROR1 was knocked down in CTCs by siRNA (Fig. 3). Western blot analysis demonstrated that ROR1 expression in ROR1 siRNA-transfected CTCs was markedly reduced compared with in NC siRNA-transfected CTCs (Fig. 3A). Subsequently, a Transwell assay was performed to determine the invasive ability of ROR1 siRNA-transfected CTCs and NC siRNA-transfected CTCs cells. Knockdown of ROR1 expression markedly prevented CTC invasion compared with in the control cells (Fig. 3C).

The present study explored the mechanism by which ROR1 regulates cell invasion. EMT serves a key role in tumor cell invasion and metastasis (29). To investigate whether the typical molecular markers of EMT were altered, the protein expression levels of E-cadherin and N-cadherin were examined by western blotting. ROR1 knockdown by siRNA increased E-cadherin protein expression, but decreased N-cadherin protein expression in CTCs (Fig. 3B). Taken together, these results indicated that knockdown of ROR1 in CTCs markedly decreased the invasive ability by regulating the EMT process.