Abstract
Background/Aim: Pancreatic ductal adenocarcinoma (PDAC) is one of the most common cancers worldwide, with a poor prognosis. Owing to the difficulty of early diagnosis, the aim of this study was to isolate biomarkers from extracellular vesicles (EVs) that can lead to early diagnosis. Materials and Methods: EVs in the culture supernatant were isolated from a pancreatic cancer cell line (PK-1) and expanded by using two-dimensional gel electrophoresis, and protein identification from each spot was performed by using matrix-assisted laser desorption ionization mass spectrometry. The identified proteins were classified and compared with previously reported results for EVs from murine pancreatic cancer PAN02 cells, and their expression specificity was examined using PDAC cell lines and patient-derived PDAC tissues. In addition, the significance of selected biomarker(s) was examined based on the changes in biomarkers in the blood EVs of PDAC patients after surgery.
Results: We found that the ITGA6A splice variant was predominantly expressed in several pancreatic cancer cell lines and blood EVs from patients with PDAC, whereas the ITGA6B splice variant was predominantly expressed in EVs from the blood of normal volunteers. In the expression pattern of ITGA6 in EVs from blood samples of two PDAC patients before and after resection surgery, the expression of ITGA6A in EVs significantly decreased after surgery and increased several months before clinical recurrence. Furthermore, the increased expression of ITGA6A in EVs occurred much earlier than that of CA19-9. Conclusion: Determination of ITGA6A expression in blood EVs in PDAC patients could be a useful blood marker for the early diagnosis of PDAC recurrence.
Pancreatic ductal adenocarcinoma (PDAC) is still considered a disease with a poor prognosis, ranking fifth among men and fourth among women in terms of annual deaths worldwide, and the number of patients with pancreatic cancer is still increasing (1). PDAC tends to be detected late owing to a lack of clinical symptoms, and many cases are unresectable at the time of diagnosis because of distant metastasis or local progression (2). Therefore, curative resection can be expected in only approximately 35% of cases, with poor five-year survival rates of 2%-9%, ranking firmly last among all cancer sites in terms of prognostic outcomes for patients. Even if resection is possible, the 5-year survival rate for resection alone is as low as 14.5%, and surgical resection is the only treatment method expected to ensure long-term survival (3).
A variety of tumor markers and related genes have been reported for PDAC, mainly based on recent genomic analyses (4). Although several promising gene clusters have been isolated, early diagnostic and/or prognostic markers for PDAC have yet to be established in clinical practice. There are high expectations for the development of a new detection method that is superior to conventional methods of measuring serum CEA and CA19-9 levels in terms of both sensitivity and specificity. Although new promising biomarkers have been identified, CA 19-9 currently remains the only regularly used and validated biomarker for pancreatic cancer in routine clinical practice (5, 6).
In contrast, exosomes are durable, cell-specific lipid microvessels (40-100 nm in diameter) that are present in various body fluids such as urine, blood, and saliva. In recent years, the presence of specific proteins and miRNAs in tumor cell-derived extracellular vesicles (EVs) has led to a worldwide movement to use them as new biomarkers for the diagnosis and prognosis of diseases (7). CD44v6, Tspan 8, EpCAM, CD104, and Glypican-1 have been reported as novel diagnostic markers for pancreatic cancer (8, 9). Moreover, macrophage migration inhibitory factor (MIF) is highly expressed in PDAC-derived exosomes (10). MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis (10). Tumor-specific exosome-like EVs have also been shown to promote cancer recurrence and metastasis, which are key to prognosis and treatment selection (11). Therefore, if EVs can be isolated from blood samples collected from patients with PDAC and novel pancreatic cancer-specific molecules can be identified, we may be able to detect pancreatic cancer or postoperative recurrence at an early stage and develop new treatment methods.
In this study, we collected EVs secreted from pancreatic cancer cell lines and comprehensively analyzed their proteins contained in them. From the EV data obtained from the supernatants of pancreatic cancer cell lines in this study, ITGA6, a member of the integrin family, was a poor prognostic factor because it was associated with metastatic recurrence of pancreatic cancer (12). Furthermore, two isoforms of ITGA6 (ITGA6A/B), and ITGA6A are mainly expressed in pancreatic cancer cells, and ITGA6B is mainly expressed in non-cancerous pancreatic tissues. This study suggests that the difference between the two isoforms in EVs may be a diagnostic system for detecting the recurrence of pancreatic cancer.
Materials and Methods
Patient samples. The study protocol for collecting tissues and blood samples from PDAC patients who underwent pancreatic resection was approved by the Human Ethics Review Board of the University of Miyazaki (the accepted numbers and dates were #O-0050 and 03/08/2016, respectively), and agreement by patients to participate in this study was obtained. This study was carried out in accordance with the principles established by the Declaration of Helsinki. Written informed consent was obtained from all the participants. Normal pancreatic tissues were derived from the non-cancerous part of the excised PDAC tissue from patients with pancreatic cancer or the normal pancreatic tissue of excised tissue from patients with bile duct cancer. Blood and tissue samples were collected and stored between 2002 and 2015. Peripheral blood samples were collected from each patient in the early morning, under stable conditions. The blood samples were centrifuged at 1,800 × g for 10 min, and the supernatant was stored at –80°C until use. Patient characteristics are summarized in Table I.
Cell lines. Seven human PDAC cell lines (PK-1, PK-8, PK-45P, KLM-1, SUIT-2, and PANC-1) were purchased from RIKEN Bioresource Center (Tsukuba, Ibaraki, Japan). PK-45P and KLM-1 cells were cultured in RPMI-1640 medium supplemented with 5% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 mg/l streptomycin. PK-1, PK-8, SUIT-2, and PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% FBS, 100 U/ml penicillin, and 100 mg/l streptomycin. All the cells were incubated at 37°C with 5% CO2 in a humidified atmosphere.
Isolation of EVs. Lyophilized and purified exosomes (HBM-PEP-30/2, HansaBioMed Life Sciences, Tallinn, Estonia) from the plasma of healthy donors were used as control samples for EVs. The lyophilized EV product was extracted from mixed plasma of approximately 100 healthy volunteers. The patient’s plasma was centrifuged at 16,500 × g and 4°C for 30 min, and the supernatant was collected and ultracentrifuged at 100,000 × g and 4°C for 70 min. After removing the supernatant, the EV pellet was resuspended in phosphate buffer saline (PBS) or sodium dodecylsulfate (SDS) sample buffer and stored at –80°C. For the isolation of EVs from cell culture, EVs were depleted by ultracentrifugation at 200,000 × g at 4°C for 16 h, and the supernatants were filtered through a 0.22 μm filter (Merck Millipore, Burlington, MA, USA). The cells were cultured in RPMI 1640 medium or DMEM supplemented with 10% EV-depleted FBS, 100 U/ml penicillin, and 100 mg/l streptomycin at 37°C and 5% CO2 for 48 h before collecting the conditioned medium for EV isolation. The conditioned medium was centrifuged at 300 × g for 10 min at 4°C and the supernatant was collected. Additional centrifugation was performed at 12,000 × g for 35 min at 4°C, and the supernatants were filtered through a 0.22 μm filter (Merck Millipore) to remove cell debris and ultracentrifuged at 100,000 × g at 4°C for 70 min. After removing the supernatant, the EV pellet was resuspended in PBS or SDS sample buffer and stored at –80°C.
Two-dimensional (2D) gel electrophoresis and protein identification. To identify PDAC-specific EV proteins, control non-culture medium (AIM-V serum-free medium, Thermo Fisher Scientific, Waltham, MA, USA) and PK-1/PDAC cell line culture media were stained with different fluorescent dyes and compared with each other using specific software. Labelling was performed at a ratio of 50 μg total protein to 400 pmol dye. Equal amounts of sample aliquots were labelled with either IC3 (red) or IC5 (green) fluorescent dye (Dojindo Laboratories, Kumamoto, Japan) following the manufacturer’s protocols. Samples labelled with IC3 or IC5 were mixed before isoelectric focusing electrophoresis (IEF). After treating 50 μg of each protein sample with TCA-acetone precipitate, it was mixed with solubilization buffer (sample buffer 100 μl, 4 μl 1.875 M DDT, and 1 μl). The immobilized pH gel (IPG) strip gel was swollen over 12 h, the sample was applied, and equilibration was performed. After equilibration and isoelectric focusing (pH 3–10), the IPG strip gel was placed on a 10% acrylamide gel, and SDS-PAGE was performed. After 2D-gel electrophoresis, the IC3 images were scanned on a ProXPRESS 2D (Perkin Elmer, Waltham, MA, USA) using a green laser at 540 nm and a 590 nm emission filter, whereas the IC5 image was scanned using a red laser at 625 nm and a 680 nm emission filter. Identical cropped images for both IC3 and IC5 were analyzed using the default detection parameters in Progenesis SameSpots PG240 (Nonlinear Dynamics, Newcastle upon Tyne, UK).
After identifying PDAC-specific EV protein spots by differential fluorescent labelling analysis, 200 μg of PDAC cell line culture medium was run by 2D electrophoresis under the same conditions as described above. The gel was stained with Coomassie Brilliant Blue and 84 protein spots were identified and resected. After reduction and alkylation, samples were subjected to trypsin digestion. The peptides were then extracted, concentrated, and stored at –80°C until analysis by using Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Shimadzu, Kyoto, Japan) as previously described. Mass spectrometry (MS) was performed, and the MS spectrum data were automatically retrieved from the protein database using MASCOT search. In general, multiple peptides were detected in each protein, adding reliability to protein identification.
Pathway analysis. To identify which biological terms/functions were specifically enriched in proteins from EVs of the PK-1 PDAC cell line, we conducted pathway analysis of the proteomic results using the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms. Modified Fisher’s exact test was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 (13). Pathways with a false discovery rate (FDR) <0.05 were considered significant.
Antibodies. A mouse monoclonal antibody (AC-15) against β-actin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Rabbit monoclonal antibodies against ITGA6 (138G6) and CD9 were obtained from Cell Signaling Technology (Danvers, MA, USA).
Western blotting. Cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% NP-40) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitors (PhosStop, Roche Diagnostics, Basel, Switzerland). Protein samples were electrophoresed on 8% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Merck Millipore). After blocking for 1 h with Tris-buffered saline (TBS)-Tween (0.1%) and 5% bovine serum albumin (BSA), the membranes were incubated with each primary antibody diluted in TBS containing 0.1% Tween 20 supplemented with 5% BSA. Bound antibodies were detected using a Lumi-light Plus kit, according to the manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland).
Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. One microgram of RNA was converted to cDNA using an RNA PCR kit (Takara Bio Inc., Kusatsu, Shiga, Japan). Amplifications were performed in a 20-μl reaction volume containing 1 μl of template cDNA, 2 μl Ex Taq Buffer (Takara Bio Inc., Kusatsu, Japan), 1.6 μl dNTP (each 25 mM), 1 μl each primer (each 10 μM), and 0.1-μl TaKara Ex Taq (5 U/μl, Takara Bio Inc.). Thermal cycling conditions comprised an initial denaturation at 95°C for 30 s, 30 cycles of denaturation at 95°C for 30 s, annealing at 45°C for 30 s, and elongation at 72°C for 45 s, and a final elongation at 72°C for 7 min. The oligonucleotide primer sequences used are listed in Table II.
Primer list for semiquantitative reverse transcription-polymerase chain reaction.
Statistical analysis. All continuous data are expressed as the mean±standard deviation. The data for the different groups were compared using one-way analysis of variance (ANOVA), which was examined using Student’s t-test. The correlation of continuous data was tested using Spearman’s rank correlation test, and the correlation coefficient (r) was determined. All statistical analyses were performed using GraphPad Prism software (GraphPad Software, San Diego, CA, USA). *p<0.05, **p<0.01 were considered statistically significant.
Results
Identification of ITGA6 as one of the enriched proteins in EVs from PDAC cells. To identify biomarkers of pancreatic ductal adenocarcinoma (PDAC), EV fractions were ultracentrifuged from the culture medium of the PDAC cell line PK-1 (PK-1 EVs) or from fresh medium as a control. The two samples were stained with different fluorescent labels (IC5, green or IC3, red), mixed together, and subjected to 2D electrophoresis, as described in the Materials and Methods. Several specific spots were identified in the PDAC cell culture medium by differential fluorescent staining (Figure 1A). A total of 84 spots were identified as spots that were not included in the control culture medium but were included in EVs in the culture medium derived from PK-1 cells. Each isolated spot was subjected to MALDI mass spectrometry analysis to identify proteins (Figure 1B).
Differential expression of extracellular vesicles (EVs) from a pancreatic ductal adenocarcinoma (PDAC) cell line by two-dimensional electrophoresis. (A) Ultracentrifuged precipitates from fresh medium (control EVs fraction) were stained with IC5, and those from the culture medium of the PDAC cell line PK-1 (PK-1 EVs fraction) were stained with IC3. Mixed samples were developed by two-dimensional electrophoresis (pH 3 to 10, and 10% SDS-PAGE with 20 to 200 kDa), and the protein spots were identified according to each specific wavelength and compared with each other. (B) The same sample from the PK-1 EVs fraction was subjected to two-dimensional electrophoresis, followed by Coomassie brilliant blue (CBB) staining. Based on the staining in (A), spots found only in the supernatant of the PK-1 culture (numbers in the figure) were picked, and each spot was analyzed by mass spectrometry. The mass spectrometry analysis data are shown in Table III.
List of proteins in extracellular vesicles from SK-1/pancreatic ductal carcinoma cell line.
As a result, 163 proteins were identified using MS analysis (Table III). GO analysis was performed to characterize the isolated protein groups (Table IV). We identified 18 enriched biological pathways for EVs from PDAC cells, which were clustered into two groups by the DAVID functional annotation module: cytoskeleton organization with microtubule-based process and cell-cell adhesion with metabolism and assembly of U. Cluster A has a process that is carried out at the cellular level, which results in the assembly, arrangement of constituent parts, or disassembly of cytoskeletal structures comprising microtubules and their associated proteins in the EVs. Cluster B shows that the EVs may potentially attach to a cell, either to another cell or to an underlying substrate such as the extracellular matrix, via cell adhesion molecules. Clusters A and B might be involved in the formation of a pre-metastatic “niche” (10).
Top pathways from enriched proteins in EVs from PK-1 PDAC cell line from DAVID functional annotation module analysis.
Thirty-four of the 163 proteins were plasma membrane proteins, including α-enolase, integrin α3 (ITGA3), integrin α6 (ITGA6), and others (bold proteins in Table III). In addition, 15 of the 34 proteins identified in this study were found to match the cell surface antigens in the murine PAN02 pancreatic cancer cell line, as described in a previous study (10, 14) (bold proteins in italics in Table III). ITGA6 was found in EVs from both PANC02 and PK1 cell lines, and high expression of ITGA6 has been reported to be associated with poor prognosis, invasion, and metastasis in patients with PDAC (12). Furthermore, the isoform of ITGA6 found in the EVs from two pancreatic cancer cells was ITGA6 type A. Therefore, ITGA6 was selected from the 15 cell surface markers for further analysis.
Expression of ITGA6 isoforms in PDAC tumors and EVs derived from patients with PDAC. To determine the expression of ITGA6 in PDAC cell lines using RT-PCR, we initially found that normal pancreatic cells expressed two forms of ITGA6 mRNA. Sequence analysis confirmed that the two forms of ITGA6 mRNA were derived from the two isoforms of ITGA6 (6A and 6B) (15) (Figure 2A). Therefore, to examine the expression of ITGA6 mRNA in PDAC cells, we performed semi-quantitative RT-PCR analysis using primers that identified the two isoforms of ITGA6 (6A, 382 nt and 6B, 252 nt). ITGA6A mRNA contained exon 25 (130 nt) and was longer than the ITGA6B mRNA (Figure 2A). Intriguingly, normal pancreatic tissues expressed both ITGA6A and 6B, and ITGA6B mRNA was more highly expressed than ITGA6A mRNA. However, in PDAC tissues, the expression of ITGA6A mRNA was significantly higher than that of ITGA6B mRNA (Figure 2B, C, and D). Additionally, we determined the expression of ITGA6 in six PDAC cell lines (PK-1, PK-8, PK-45P, KLM-1, SUIT-2, and Panc-1). ITGA6A mRNA was predominantly expressed in all PDAC cell lines (Figure 2E). Next, we determined the expression of ITGA6 protein in whole cell lysates or EVs from the six PDAC cell lines using western blot analysis. The deletion of 130 nucleotides of the ITGA6 coding sequences resulted in a frameshift that eliminated the original stop codon, generating an alternative splicing variant (6B) that is 18 amino acids longer than 6A, which results in a high number of charged amino acids (24 out of 54) in the 6B isoform (16).
High expression of ITGA6A in pancreatic ductal adenocarcinoma (PDAC) cells and in extracellular vesicles (EVs) released from PDAC cells. (A) Structure of the ITGA6 gene and two transcripts and proteins by alternative transcription. As alternative transcripts for exon 25 of the ITGA6 gene, there are two types of ITGA6: ITGA6A, which has exon 25, and ITGA6B, which skips exon 25. Since ITGA6A has 130 nt of exon 25, the PCR product of ITGA6A is 382 bp and that of ITGA6B is 252 bp, which are derived from ITGA6-F (exon 24) and ITGA6-R (exon 26). On the other hand, the termination codon of ITGA6A is located in exon 25, and the termination codon of ITGA6B is in the next exon 26, which is 54 nt longer than the open reading frame (ORF) of ITGA6A. Therefore, the protein is approximately 150 kDa for ITGA6B and 130 kDa for ITGA6A in western blot analysis. (B) Expression study of ITGA6A/6B mRNA by reverse transcription-polymerase chain reaction (RT-PCR) in pancreatic tissues from 6 healthy volunteers and 6 tumor samples from patients with PDAC. The expression of β-actin was used as a control. (C) Comparison of the expression levels of ITGA6A/6B in normal pancreatic tissue and PDAC tissue based on the data in B. *p<0.05; n.s.: not significant. (D) Based on the data in C, the expression ratios of ITGA6A/6B in normal pancreatic tissue and in PDAC tissue were compared. *p<0.05. (E) The expression levels of ITGA6A/6B were examined by RT-PCR using six PDAC cell lines. The bar graph below shows the ratio of the expression levels of ITGA6A/6B in each sample. (F) The expression of ITGA6A/6B was examined by western blotting using cell extracts from six PDAC cell lines. (G) The expression of ITGA6A/6B in EVs contained in the culture supernatant of six PDAC cell lines was examined by western blotting. The expression of CD9 was used as an internal control for EVs.
The protein abundance of the ITGA6A isoform (130 kDa) was much higher than that of the ITGA6B isoform (150 kDa) (Figure 2A) in cell lysates from all six PDAC cell lines tested (Figure 2F). In addition, ITGA6A protein was enriched in purified EVs from the culture media of all PDAC cell lines (Figure 2G). These results suggest that the ITGA6A form in EVs may be used as a potential biomarker in the blood of PDAC patients.
The expression level of integrin α6A (ITGA6A) in EVs secreted by PDAC cells reflects the pathology of PDAC. To examine the expression of ITGA6A protein in plasma EVs of PDAC patients, EVs were isolated by ultracentrifugation from plasma samples of three PDAC patients (#1 to #3, stage IVa or IVb) (Table I). A mixture of EVs isolated from dozens of healthy volunteers was also purchased and used as a control for western blot analysis. ITGA6B was mainly detected in mixed EVs from healthy volunteers; however, ITGA6A was predominantly detected in the plasma of PDAC patients (Figure 3A). Next, we investigated how the isoforms of ITGA6 change during the clinical course of patients with PDCA before and after surgery, compared with typical cancer markers. Western blot analysis was performed on EVs extracted from plasma samples collected preoperatively and several months postoperatively from PDAC patients. Two cases of PDAC were examined, and in these cases, CEA (as a reference with an upper limit of 5.0 ng/ml) and CA19-9 (as a reference with an upper limit of 37.0 U/ml) in serum were regularly measured as tumor markers during postoperative follow-up (1, 3, 6, 9 months, and a year after surgery). In addition, abdominal CT imaging was performed 1, 3, and 6 months and 1 year after surgery.
ITGA6A in blood extracellular vesicles (EVs) correlates with tumor burden in pancreatic ductal adenocarcinoma (PDAC) patients. (A) EV fractions from each of the three PDAC patients and, as controls, mixed EVs fractions from a number of healthy individuals were used to measure the expression of ITGA6A/6B and CD9 as a control by western blotting analysis. (left). The right figure shows the expression level of the ratio of ITGA6A/6B (bar graph). (B) In the PDAC patient (Pt 4), the expression of ITGA6 in serum EVs was examined by western blotting before surgery and at 2 weeks, 1 month, and 6 months after surgery. In the lower table, the expression of CEA and CA19-9, which are cancer markers in the blood, are measured and compared with the expression of ITGA6. At the same time, clinical metastasis was examined. #1; CT scan revealing multiple metastases in the liver 12 months after surgery. The figure on the right shows the expression level of ITGA6A at each time point in a bar graph. The line graph also shows the expression level ratio of ITGA6A to ITGA6B. (C) As in B, the expression of ITGA6 in blood EVs (upper left) and the levels of cancer markers in blood (lower left) are shown before and after surgery in PDAC patients (Pt 5). In addition, the right figure shows the expression level of ITGA6A (bar graph) and the ratio of ITGA6A/6B. #2; CT scan revealing metastasis in the intra-abdominal lymph nodes three months after surgery.
Patient #4 was a male with pancreatic head cancer in his 50s. The final diagnosis was stage IVa PDCA and radical resection was performed. The preoperative tumor marker levels were higher than the reference values, with CEA and CA19-9 levels of 12.3 ng/ml and 39.8 U/ml, respectively (Figure 3B). Postoperatively, both tumor markers were below the reference values at one month, CEA was at the upper limit of the reference value at three months postoperatively and continued to rise above the reference value after nine months. Abdominal computed tomography (CT) revealed multiple liver metastases one year after surgery. In patient #4, the relative ratio of ITGA6A/ITGA6B in the EVs from plasma collected preoperatively was 1:1 (0.5), but at 2 weeks postoperatively, only ITGA6B was expressed. At 1 month postoperatively, the relative expression of ITGA6B appeared to be 0.12, and at 6 months postoperatively, it increased sharply to 0.94. Similarly, the relative ratio of ITGA6A/6B was almost zero at 2 weeks postoperatively but was greater than 1 at one month postoperatively, indicating a cancerous pattern of ITGA6A expression exceeding that of ITGA6B. Thereafter, the expression of ITGA6A was further increased and was 2-fold higher than that of ITGA6B. Therefore, ITGA6A expression in EVs may indicate that cancer recurrence had already occurred after one month (Figure 3B).
Patient #5 was a woman with pancreatic head cancer in her 50s. The final diagnosis was stage IVb, and radical resection was performed. The preoperative tumor marker values were 1.8 ng/ml for CEA and 2.0 U/ml for CA19-9. Both CEA and CA19-9 levels were below the reference values in the preoperative stage (Figure 3C). During the postoperative 12 months, both CEA and CA19-9 levels were below reference values. Abdominal CT three months after surgery showed multiple intra-abdominal lymph node metastases. In patient #5, the relative amount of ITGA6A decreased to 0.27 at two weeks postoperatively compared to that preoperatively, and the ratio of the A form/B form was 0.54. The relative amount of ITGA6A had increased to 0.41 at one month postoperatively and again to 0.62 at six months postoperatively. However, the relative ratio of the A form/B form was 1.2 preoperatively, decreased to 0.54 at 2 weeks postoperatively, and increased rapidly to 3.5 at 1 month postoperatively and to 7.44 at 6 months postoperatively (Figure 3C). Therefore, the expression pattern had already changed from a normal pattern to a cancerous pattern one month after surgery, and recurrence or metastasis of PDAC was considered.
These results show that in the two cases of postoperative recurrence, the expression of ITGA6 isoforms in blood EVs showed a cancerous pattern from the very early stage, one month after surgery. This finding suggests that recurrence or metastasis may be detected much earlier than allowed by existing tumor markers in the blood or via imaging diagnosis by determining the ITGA6 isoforms in blood EVs from patients with PDAC.
Discussion
In this study, we identified a number of proteins in EVs from the culture medium of the human pancreatic cancer cell line PK-1, including CD9 and CD63, which are considered to be exosome-like vesicles. A group of proteins similar to those previously reported in mouse pancreatic cancer cells (10, 14) were found in human pancreatic cancer cells. We found that high expression of ITGA6A, an alternative splice form, is specific for human pancreatic cancer cells. Furthermore, the ITGA6A form was highly expressed in EVs from patients with pancreatic cancer. ITGA6 gene expression has been reported to be involved in cancer-specific alternative splicing; the ITGA6A form is expressed and the ITGA6B form is lost in pancreatic cancer. Furthermore, since the ITGA6A form is specifically included in EVs from pancreatic cancer, it is suggested that the expression level of the ITGA6A form in blood EVs and the expression ratio of the ITGA6A form to that of the ITGA6B form can be useful as diagnostic methods to determine the early recurrence and/or metastasis of pancreatic cancer.
The ITGA6/Integrin β-4 (ITGB4) complex is involved in the progression of colorectal cancer (CRC) (17). ITGB4, identified in EVs from PDAC cells, causes mislocalization of plectin to the cell surface and contributes to tumorigenesis by EV secretion (18). In addition, ITGA6A and ITGB4 complexes (ITGA6A/B4) promote cell proliferation, whereas the ITGA6B/B4 complex inhibits cell proliferation (15). Therefore, ITGA6A expression is an important prognostic factor of cancers.
It has been reported that the expression of the ITGA6A form is dependent on the expression of epithelial splicing regulatory protein 1 (ESRP1) and ESRP2 in CRC and that high expression of ESRP is dependent on the expression of C-MYC, which transcriptionally regulates the ESRP promoter (19, 20). We analyzed two sets of data from a comprehensive gene expression analysis using PDAC specimens registered in the Gene Expression Omnibus (GSE16515 and GSE32676). The results showed that ITGA6 and ESRP1 were significantly upregulated in PC cells (data not shown). It is possible that high expression of ESRP1 leads to high expression of ITGA6A, which may represent a mechanism similar to the high expression of ITGA6A in CRC and breast cancer (21). Therefore, further analysis is required to determine the cause of ESRP1 over-expression in PDAC.
In conclusion, identification of the ITGA6A form in blood EVs from patients with recurrent pancreatic cancer is an important discovery that may allow the early diagnosis of PDCA recurrence. In addition, since an increase in ITGA6A can be observed before that of CA19-9 and other markers in pancreatic cancer, it is highly possible that ITGA6A in EVs can be used as a diagnostic marker for early detection of recurrent pancreatic cancer and early diagnosis of metastasis.
Acknowledgements
This work was funded in part by Grants-in-Aid for Scientific Research (B) (25293081 and 17H03581) (KM) from the Japan Society for the Promotion of Science (JSPS) and Takeda Science Foundation (KM).
Footnotes
Authors’ Contributions
Conception and design: K.M. and T.A.; patient sample and data collection: T. A., N.S., Y.F., N.I., and M.H.; acquisition of data: T.A., S.N., Y.R.F., and T.I.; analysis and interpretation of data: T.A., S.N., Y.R.F., T.I., K.I., and K.M.; drafting the manuscript or revising it critically for important intellectual content: K.M., T.A., and S.N.; study supervision: K.M. and A.N.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
- Received February 7, 2022.
- Revision received February 28, 2022.
- Accepted March 2, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
