Evaluation of glypican-1 in extracellular vesicles from serum and pancreatic tissue as a biomarker for pancreatic cancer

Evaluation of glypican-1 in extracellular vesicles from serum and pancreatic tissue as a biomarker for pancreatic cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Evaluation of glypican-1 in extracellular vesicles from serum and pancreatic tissue as a biomarker for pancreatic cancer Rongrong Ren, Lu Cheng, Huan Zhang, Linggong Zeng, Lvhu Shan, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5675979/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Pancreatic cancer (PC) is a lethal disease, and early detection is crucial for reducing mortality. Blood exosome glypican-1 (GPC1) has been reported as a powerful diagnostic and screening tool for detecting pancreatic ductal adenocarcinomas (PDAC) even at early stages; however, results from subsequent studies on extracellular vesicles (EVs) or exosome GPC1 have been conflicting. We hypothesized that if EVs GPC1 serves as a biomarker for PC, it may be enriched in pancreatic tumor tissues compared to adjacent non-tumor tissues. Methods Dissociated tumor tissues and paratumor tissues were treated with collagenase D and DNase I to release EVs from the extracellular matrix. Both serum-derived EVs and tissue-derived EVs were isolated by ultracentrifugation, and EVs GPC1 levels were analyzed by flow cytometry. The expression of EV GPC1 was compared between patients and controls, pre- and post-surgery, and between tumor tissues and adjacent non-tumor tissues. Results EVs were successfully isolated from pancreatic tissue. Serum EVs GPC1 levels showed no significant difference between PC patients and healthy controls, nor between pre-operative and post-operative samples. EVs GPC1 derived from tumor tissue showed no significant difference compared to matched paratumor tissue. Conclusion Although EVs GPC1 was found not to be a reliable biomarker for pancreatic cancer, we successfully isolated EVs from pancreatic tissue. Further research is needed to explore the potential of tissue-derived EVs as sources of screening biomarkers and to standardize methods for isolating and detecting EVs biomarkers. Pancreatic cancer Extracellular vesicles Glypican-1 Biomarker Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Pancreatic cancer (PC) ranks as the twelfth most prevalent malignancy, accounting for 510,566 new cases globally in 2022, and is the sixth leading cause of cancer-related mortality, with 467,005 deaths worldwide (Bray et al., 2024). More than 90% of PCs are categorized as pancreatic ductal adenocarcinomas (PDAC) (Jiang et al., 2022). The incidence rate of PC has manifested a consistent annual escalation of approximately 1% from 2015 to 2019 in the United States (Siegel et al., 2024). It was projected that this upward trend in incidence will persist over the forthcoming decades (Klein et al., 2021). Although the five-year relative survival rate for PC has improved from 4% during the period of 1995–1997 to 13% between 2013 and 2019, it continues to represent the lowest survival rate among all malignancies (Siegel et al., 2024). The low survival rates associated with PC can be partially attributed to the advanced stage at which it is diagnosed, significantly hindering resectability; only approximately 10% of patients are diagnosed with localized and surgically resectable disease (Khalaf et al., 2021). PDAC is rarely detected at an early stage due to its rapid progression (Yu et al., 2015), and most early-stage PDAC cases are asymptomatic (Kanno et al., 2018). For stage IA PDAC, the five-year overall survival rate improved from 44.7% in 2004 to 83.7% in 2012; these trends may be attributed to advancements in early diagnosis and detection (Blackford et al., 2020). However, current screening strategies for PC remain far from optimal, and a combination of imaging techniques with biomarkers could represent a promising approach (Stoffel et al., 2023). The early detection of PC is particularly challenging due to the lack of specific biomarkers(O'Neill & Stoita, 2021). Clinical routine biomarkers, such as carcinoembryonic antigen and carbohydrate antigen 19 − 9, are primarily utilized for monitoring early recurrences rather than serving as effective screening or diagnostic tools because of their low sensitivity and specificity (Bhat et al., 2012). Extracellular vesicles (EVs) are a heterogeneous group of cell-derived, nano-sized vesicles that play a crucial role in intercellular communication (Colombo et al., 2014; Zaborowski et al., 2015). EVs can be classified into microvesicles, exosomes, oncosomes, and apoptotic bodies based on differences in biogenesis and size. (Colombo et al., 2014; Zaborowski et al., 2015). These vesicles are present in various biological fluids and are enriched with cytosolic proteins, lipids, and nucleic acids, serving as carriers of disease biomarkers (Doyle & Wang, 2019). Melo and colleagues have reported that serum glypican-1 (GPC1) positive circulating exosomes represent an 'ideal' biomarker, demonstrating 100% specificity and sensitivity for PDAC. In their view, this may serve as a potential non-invasive diagnostic and screening tool for the early detection of PDAC (Melo et al., 2015). However, subsequent studies investigating GPC1 in exosomes or EVs related to PC have yielded contradictory results(Buscail et al., 2019; Frampton et al., 2018; Lai et al., 2017; Li et al., 2024; Lucien et al., 2019; Qian et al., 2020; Yan et al.,, 2023; Yang et al., 2017; Yu et al., 2023; Zhao et al., 2022), raising controversy over whether blood-derived GPC1 is a reliable biomarker for PC. A protocol described in detail the methodology for isolating EVs from tissues (Crescitelli et al., 2021). To our knowledge, no studies have yet reported on EVs derived from PC tissue. We hypothesized that if EVs GPC1 serves as a biomarker for PC, then EVs GPC1 levels may be enriched in tumor tissues compared to adjacent non-tumor tissues. We isolated EVs from both tumor and paratumor tissues of patients with PDAC and refined the protocol to detect GPC1 in EVs derived from serum and tissue samples. The expression of EVs GPC1 was compared between patients and controls, pre- and post-surgery, and between tumor tissues and adjacent non-tumor tissues. Materials and methods Serum and tissue samples In our study, we collected blood samples from 49 PC patients and 38 healthy donors, dividing them into two cohorts: one cohort consisting of 35 patients (including 18 PDAC patients undergoing surgery and 17 late-stage PC patients) and 24 healthy donors, quantified using Glypican-1 Polyclonal Antibody (PA5-28055) to assess serum EVs GPC1; the second cohort comprised 14 late-stage PC patients and 14 healthy donors, analyzed with Glypican-1 Polyclonal Antibody (PA5-86043). 10 ml of fasting whole blood were collected via venipuncture and centrifuged at 2,000 g for ten minutes to obtain serum samples, which were stored at -80℃ until analysis. Among 18 PDAC patients undergoing pancreatic surgery, 11 PDAC patients provided an additional peripheral blood sample at a mean of 5.6 ± 1.9 days post-surgery. Tumor tissues along with matched paratumor tissues were obtained intra-operatively from 6 PDAC patients. Informed consent was obtained from all participants, and the study was approved by the Ethics Review Committee of Zhejiang Hospital under approval numbers 2020(84k) and 2023(90k). Main antibodies and reagents Collagenase D (from Clostridium histolyticum, Roche) and DNase I (grade II, from bovine pancreas, Roche) were obtained from Sigma-Aldrich. Aldehyde/sulfate latex beads with a diameter of 4.0 µm (4% w/v, A37304) were purchased from Invitrogen. The bicinchoninic acid (BCA) protein assay kit was purchased from Mei5Bio Biotechnology. The following antibodies were acquired from Abcam: anti-CD63 (ab193349, 0.2 mg/ml, mouse monoclonal, reacts with human), anti-CD63 (ab252919, 0.598 mg/ml, rabbit monoclonal, reacts with human), anti-CD9 (ab236630, rabbit monoclonal, reacts with human), and anti-ALIX antibody (ab275377, rabbit monoclonal). Additionally, the following antibodies were sourced from Invitrogen: PE-conjugated CD9 antibody (eBioSN4; SN4 C3-3A2; mouse monoclonal IgG1; reacts with human; eBioscience™), Glypican-1 antibody (PA5-28055; rabbit polyclonal), Glypican-1 antibody (PA5-86043; rabbit polyclonal), FITC-conjugated goat anti-rabbit IgG secondary antibody, rabbit IgG isotype control (FITC; eBioscience™), and mouse IgG1 kappa isotype control (PE; eBioscience™) Isolation of EVs from tissue In brief, dissociated fresh tissues were treated with collagenase D and DNase I to release EVs from the extracellular matrix (Crescitelli et al. 2021). This was followed by differential ultracentrifugation: first centrifuging at 300 g for 10 minutes, then at 2,000 g for 20 minutes. The supernatant was transferred to ultracentrifuge tubes and subjected to centrifugation at 10,000 g for 30 minutes at 4℃ using a Ti41 rotor in an Optima XPN-100 Ultracentrifuge (Beckman Coulter) to eliminate cells, dead cells, and cellular debris. The final supernatant was filtered through a 0.22 µm filter (Millipore), after which it underwent further ultracentrifugation in the Optima XPN-100 Ultracentrifuge at 120,000 g for 120 minutes at 4℃. Following this step, the supernatant was discarded and the pellet re-suspended in 12 ml of phosphate buffered saline (PBS) before being centrifuged again for 70 minutes at 120,000 × g at 4℃. After removing the supernatant once more, the pellet was finally re-suspended in 100–200µl of PBS; the collected EVs were either used for analysis or stored at -80℃. Isolation of EVs from serum Serum samples collected from patients and healthy donors were stored at -80℃. Prior to the isolation of EVs, serum was thawed on ice and then centrifuged at 12,000 g for 45 minutes at 4℃. One milliliter of serum was added to approximately 11 ml of PBS and mixed thoroughly before being filtered through a 0.22 µm filter. The resulting solution was transferred to ultracentrifuge tubes and subjected to centrifugation at 150,000 g for 120 minutes using a Ti41 rotor. After discarding the supernatant, the pellet was resuspended in approximately 12 ml of PBS and centrifuged again at 150,000 g for 70 minutes; the final pellet was re-suspended in between 50 and 150 µl of PBS. The collected EVs were either used for analysis or stored at -80℃. Transmission Electron Microscope (TEM) EVs stored at -80℃ were thawed on ice, after which 5 µl of resuspended EVs were deposited onto carbon-coated copper grids with a mesh size of 200. One minute later, filter paper was used to absorb excess liquid from the edges of the grid while leaving a thin layer of liquid on its surface. The grids were cleaned twice with a solution of 2% uranyl acetate before an additional drop of this solution was applied to each grid; one minute later, excess liquid was absorbed with filter paper prior to observation under an electron microscope (Thermo Fisher Scientific Talos L120C operating at 120 kV). Nanoparticle Tracking Analysis (NTA) The size of EVs derived from serum and tissues was measured using the ZetaView® PMX110 particle tracking analyzer (Particle Metrix, Diessen, Germany). EVs isolated from serum were diluted 1:300, while those derived from tissue were diluted 1:1500 with PBS. Data analysis was performed using ZetaView® analysis software version 8.05.14SP7, with a minimum size threshold set at 5 nm, a maximum size limit of 1000 nm, and a minimum brightness setting of 20. Preparation of the anti-CD63-coated bead working solution Aldehyde/sulfate latex beads with a diameter of 4.0 µm were 1:10 diluted with PBS. Subsequently, 10 µl of the diluted beads were subjected to incubation in combination with 10 µl of anti-CD63 monoclonal antibody (ab193349, Abcam) within a 1.5 ml microcentrifuge tube at ambient temperature for 30 minutes. Subsequently, the volume was adjusted to 500 µl with PBS and subjected to overnight incubation at 4℃ on a test tube rotator. The reaction was terminated by supplementing the mixture with 55 µl of glycine (1000 mM) and conducting an additional incubation at ambient temperature for 30 minutes. Thereupon, the mixture underwent centrifugation for five minutes at 10,000 × g at 4℃; the supernatant was meticulously discarded, and the pellet was rinsed twice with 1% PBS/BSA before being re-suspended in 500 µl of 1% PBS/BSA solution. This preparation yielded a working solution of anti-CD63-coated latex beads, which was stored at 4℃ with a dilution factor of 1:500. Flow cytometry analysis of EVs EVs derived from 1.0 ml of serum were incubated for 20 minutes at room temperature with either 3.33 µl of a working solution of anti-CD63-coated latex beads or 3.33 µl of uncoated latex bead solution diluted in PBS at a ratio of 1:500. Additionally, 2.5 µg of EVs derived from tissue were incubated under the same conditions with the working solution of anti-CD63-coated latex beads. The total volume was then adjusted to 500 µl with PBS and incubated overnight at 4℃ on a test tube rotator. For the uncoated latex bead solution diluted to 1:500 in PBS, an aliquot of 55 µl glycine (1000 mM) was added and allowed to stand at room temperature for an additional 30 minutes. Subsequently, both coated and uncoated beads were centrifuged for five minutes at 10,000 g at 4℃. The supernatant was carefully discarded, followed by two washes with a solution containing 1% PBS/BSA. It is important to minimize bead loss, approximately 50 µl of liquid remained at the bottom after each discarding step. Subsequently, 1.66 µl of PE-conjugated anti-CD9 monoclonal antibody, 6.66 µl of 1:250 diluted rabbit monoclonal like anti-CD63(ab252919), or anti-ALIX (ab275377) or anti-glypican-1(PA5-28055 or PA5-86043) were added to each sample. After incubation for 45 minutes at room temperature, an additional volume of 500 µl containing 1% PBS/BSA was added and the samples were centrifuged for five minutes at a speed of 10,000 g at 4℃; the supernatant was carefully discarded while approximately 50 µl remained in the bottom and washed twice with a solution containing 1% PBS/BSA. Following this step, 6.66 µl of FITC-conjugated goat anti-rabbit secondary antibody diluted to 1:250 was added and incubated for another 45 mintutes at room temperature; after two washes with 1% PBS/BSA, 200 µl PBS was added for flow cytometry analysis. Single beads and doublets were gated based on Forward Scatter and Side Scatter parameters(Thery, Amigorena, Raposo, & Clayton, 2006), with a total of 2,000 latex beads set for analysis using either a flow cytometryCalibur flow cytometer (BD Biosciences) or FlowJo™10 software (BD Biosciences). Western blot analysis of EVs The EVs obtained were lysed in RIPA Lysis Buffer supplemented with a cocktail of protease inhibitors (Thermo Scientific, A32965). Following centrifugation at 12,000 g for 15 minutes at 4℃, the supernatants were collected for Western blotting according to standard procedures. For each lane, 3 µg of protein was loaded for analysis. Antibodies against CD63 (ab193349, 1:1000), ALIX (ab275377, 1:1000), and CD9 (ab236630, 1:1000) were purchased from Abcam. Anti-GPC1 antibodies PA5-28055 and PA5-86043 were acquired from Invitrogen at a dilution of 1:1000 each. Chemiluminescence detection was performed using a ChemiScope3300 system (Clinx). Statistics Statistical analyses were carried out using GraphPad Prism software version 10 (GraphPad Software), and the data were presented as means ± standard deviation. Unpaired two-tailed Student’s t-tests were employed to compare the percentages of CD9 and CD63 positive beads between anti-CD63-coated latex beads and uncoated latex beads, as well as the percentage of serum EVs GPC1 positive beads, GPC1 mean fluorescence intensity, and the ratio of GPC1 positive beads to CD9 positive beads in late-stage PC patients and healthy donors. One way analysis of variance (ANOVA) was adopted to compare the differences among late-stage PC patients, pre-operative PDAC patients, and healthy donors. Paired two-tailed Student’s t-tests were performed to assess the differences between tumor tissues and adjacent non-tumor tissues, along with pre-operative PDAC patients and post-operative PDAC patients. A p-value < 0.05 was considered statistically significant. Results Morphological characterization of EVs via TEM and size measurement by NTA EVs isolated from serum and tissue, as characterized by electron microscopy, exhibited typical EVs morphology (see Fig.1 a and b). NTA revealed a median diameter of 151.6 nm for serum-derived EVs (see Fig.1 c) and 142.5 nm for tissue-derived EVs(see Fig.1 d). Biochemical characterization of EVs by western blot and flow cytometry The presence of canonical EVs markers CD9, CD63, and Alix (Thery et al. 2006) was confirmed through Western blotting and flow cytometry. In conjunction with results from TEM and NTA, these findings indicate the successful isolation of EVs from serum and tissue in our study. Furthermore, the expression of GPC1 protein in EVs derived from both serum and tissue was validated using Western blotting and flow cytometry with two distinct antibodies (PA5-28055 and PA5-86043). EVs captured by anti-CD63-latex beads were subsequently stained with antibodies against CD9, CD63, Alix, and GPC1 (PA5-28055 or PA5-86043). The data were analyzed using FlowJo™ 10 software. a Scatter plot illustrating the distribution of anti-CD63-latex beads, including both single beads and doublets. b Histogram representing flow cytometry analyses of anti-CD63-latex beads in conjunction with EVs derived from a representative serum sample and paratumor tissue; green histograms indicate FITC fluorescence while red histograms represent PE fluorescence (gray histograms serve as negative controls obtained using an appropriate isotype). FITC fluorescein isothiocyanate , PE phycoerythrin Both serum and tissue-derived EVs were captured more efficiently using anti-CD63-coated latex beads compared to uncoated latex beads We evaluated the efficacy of various beads in capturing EVs derived from serum or tissue by quantifying the proportion of CD63- or CD9-positive beads as an indicator of EV attachment. For serum-derived EVs, the percentage of CD63-positive beads was 8.3% ± 2.2% for uncoated latex beads, compared to 86.9% ± 5.6% for anti-CD63-coated latex beads. Similarly, the percentage of CD9-positive beads was 10.3% ± 2.1% for uncoated latex beads, versus 89.2% ± 2.9% for anti-CD63-coated latex beads. For tissue-derived EVs, the percentages of CD63- and CD9-positive beads were 26.5% ± 2.0% and 36.0% ± 3.5%, respectively, for uncoated latex beads, compared to 89.8% ± 4.5% and 93.5% ± 4.0% for anti-CD63-coated latex beads. These results (see Fig. 4a) demonstrate that a substantial majority of the beads tested positive when utilizing anti-CD63-coated latex beads, whereas most uncoated latex beads yielded negative outcomes. Collectively, these findings indicate that significantly more tissue and serum-derived EVs were captured by anti-CD63-coated latex beads compared to their uncoated counterparts. Glypican-1 in serum EVs showed no significant difference between PC and control We compared the percentage of GPC1 positive EVs (abbreviated as GPC1%), the GPC1 mean fluorescence intensity (abbreviated as GPC1 MFI), and the ratio of GPC1-positive beads to CD9-positive beads (abbreviated as GPC1/CD9) among late-stage PC patients (n = 17), pre-operative PDAC patients (n = 18), and healthy donors (n = 24) in the first cohort using the GPC1 antibody PA5-28055. One-way ANOVA showed no significant differences among these three groups for the serum EVs GPC1%, GPC1 MFI, and GPC1/CD9 (see Fig.4 b-d). Specifically, for late-stage PC patients, GPC1%, GPC1 MFI, and GPC1/CD9 were 27.2% ± 18.5%, 111.6 ± 79.5, and 0.34 ± 0.22, respectively; for pre-operative PDAC patients, they were 32.1% ± 19.2%, 122.2 ± 66.8, and 0.41 ± 0.23; for healthy donors, they were 29.5% ± 18.7%, 106.7 ± 52.6, and 0.34 ± 0.21. Similarly, no significant differences were observed in the second cohort using GPC1 antibody PA5-86043; when comparing late-stage PC patients (n=14) with healthy donors (n=14), the serum EVs GPC1% was 35.2% ± 22.4% vs. 31.0% ± 21.6%, GPC1 MFI was 135.1 ± 82.1 vs. 134.1 ± 70.2, and GCP1/CD9 was 0.38 ± 0.23 vs. 0.34 ± 0.22(see Fig.4 e-g). EVs glypican-1 showed no significant difference between pancreatic tumor and paratumor tissues There was no significant difference in tissue-derived EVs GPC1% (56.0% ± 28.9% vs. 51.5% ± 25.5%), GPC1 MFI (234.8 ± 116.4 vs. 216.3 ± 108.8), and GCP1/CD9 (0.59 ± 0.30 vs. 0.56 ± 0.24) for GPC1 PA5-28055 between tumor tissues and matched paratumor tissues collected from 6 patients with PDAC. Similarly, no significant difference was found when another GPC1 antibody PA5-86043 was used. For GPC1%, it was 66.8% ± 31.4% vs. 70.9% ± 32.5%; for GPC1 MFI, it was 302.7 ± 164.2 vs. 377.0 ± 213.3; and for GCP1/CD9, it was 0.71 ± 0.33 vs. 0.79 ± 0.36 between tumor tissues and paratumor tissues (see Fig.4 h-i). Glypican-1 levels in serum EVs exhibited no significant difference between pre-operative and post-operative samples No significant differences were detected in the serum EVs GPC1% (37.9% ± 18.8% vs. 33.8% ± 20.3%), GPC1 MFI (140.0 ± 73.9 vs. 120.3 ± 54.9), and GPC1/CD9 (0.46 ± 0.24 vs. 0.37 ± 0.21) between the pre-operative and post-operative blood samples from 11 PDAC patients, which were assessed an average of 5.6 ± 1.9 days (range: 4-9 days) after surgical resection (see Fig.4 k-m). Discussion Glypican-1 is a member of the glypican family, consisting of six members, and is anchored to the cell membrane. It regulates various signaling pathways, including Wnt, Hedgehog (Hh), fibroblast growth factors (FGF), and bone morphogenic protein (BMP) (Pan & Ho, 2021). Glypican-1 is highly expressed at both mRNA and protein levels in human PC (Duan et al., 2013; Kleeff et al., 1998; Lu et al., 2017) and is overexpressed in other malignancies such as breast cancer (Matsuda et al., 2001), hepatocellular carcinoma (Chen et al., 2020), and esophagogastric cancer (Pratap et al., 2022). A study evaluated GPC1 expression using immunohistochemical staining in a cohort of 140 PDAC patients, 55% of tissue microarrays exhibited weak GPC1 expression while one-third were negative for GPC1(Lucien et al., 2019). Immunohistochemical assessments conducted in two additional studies demonstrated that GPC1 levels in PC tissues were significantly higher than those found in normal pancreatic tissues (Duan et al., 2013; Lu et al., 2017). Faint GPC1 immunoreactivity was observed in some PDAC cells, with faint to moderate reactivity present in a few fibroblasts within normal pancreas or chronic pancreatitis samples (Kleeff et al., 1998). However, no significant difference was detected between GPC1 protein levels in PDAC tissues and adjacent normal pancreas using enzyme-linked immunosorbent assay (ELISA) methods (Frampton et al., 2018). These findings suggest that GPC1 protein expression may be enriched within specific structures of cancer cells rather than uniformly distributed across total tissue among certain PDAC patients; however, it remains unclear whether GPC1 levels are more concentrated in EVs derived from PDAC tissues compared to those from paratumor tissues. One study reported that GPC1 levels were elevated in breast cancer and PC cells compared to three non-tumorigenic cell lines, along with over 90% of GPC1-positive exosome beads found in cancer cells versus less than 3% in non-cancerous cells (Melo et al., 2015). GPC1-positive circulating exosomes (crExos) were identified as an excellent biomarker for PDAC, demonstrating an area under the curve (AUC) of 1.00; crExos GPC1 effectively distinguished PDAC from healthy donors, even in cases of histologically validated PC precursor lesions, where crExos GPC1 levels remained consistently higher than those in the healthy donor group. A significant decrease in exosomal GPC1 levels was also observed at seven days post-surgical resection in PDAC patients, suggesting that blood-derived exosomal GPC1 may serve as a powerful diagnostic and screening tool for detecting PDAC even at early stages (Melo et al., 2015). These results would be exceptionally valuable if replicated across other laboratories. However, subsequent studies is still controversial (Buscail et al., 2019; Frampton et al., 2018; Lai et al., 2017; Li et al., 2024; Lucien et al., 2019; Qian et al., 2020; Yan et al., 2023; Yang et al., 2017; Yu et al., 2023; Zhao et al., 2022). Three studies employed differential ultracentrifugation for the isolation of exosomes or EVs (Frampton et al., 2018; Lai et al., 2017; Yang et al., 2017), one utilized density gradient ultracentrifugation (Xiao et al., 2020), one adopted a spin column-based method (Qian et al., 2018), one employed size-exclusion chromatography (Yan et al., 2023), four utilized a total exosome isolation kit (Buscail et al., 2019; Li et al., 2024; Yu et al., 2023; Zhao et al., 2022), and one directly detected EVs GPC1 without prior isolation (Lucien et al., 2019). The detection of EVs or exosomal GPC1 was carried out using flow cytometry (Buscail et al., 2019; Qian et al., 2018; Xiao et al., 2020), multiplexed plasmonic assay (Yang et al., 2017), nanoscale flow cytometry (Lucien et al., 2019), LC/MS (Lai et al., 2017), ELISA (Frampton et al., 2018; Zhao et al., 2022), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Yan et al., 2023), nanoliquid biopsy (Yu et al., 2023), and immune lipoplex nanoparticle biochip assay (Li et al., 2024). Four reports failed to differentiate PDAC from controls based on EVs or exosomal GPC1 levels (Frampton et al., 2018; Lai et al., 2017; Lucien et al., 2019; Yang et al., 2017); however, other studies reported significantly elevated levels of EVs GPC1 in PDAC patients compared to the control group (Buscail et al., 2019; Qian et al., 2018; Xiao et al., 2020; Yan et al., 2023; Yu et al., 2023; Zhao et al., 2022). Interestingly, one study conducted experiments using ultracentrifugation followed by latex beads coupling were unable to distinguish PDAC from controls, which contrasts with the situation when a density-based separation kit coupled to magnetic beads decorated with anti-CD63 was used (Buscail et al., 2019). Most recently, a study found that EVs GPC1 protein expression can effectively discriminate between PDAC and controls through tumor-associated microvesicles rather than through exosomes (Li et al., 2024). We conjecture that these observed disparities might be partly attributed to the variations in EVs isolation techniques and detection methods adopted by different studies or the small sample size in some studies. Hence, We utilized ultracentrifugation for EVs isolation and flow cytometry to detect EVs GPC1 following the methodology established by Melo, and employed two GPC1 antibodies, one of which was the same as reported (Melo et al., 2015). Caby and colleagues initially demonstrated the existence of EVs in human blood by isolating plasma EVs through ultracentrifugation and analyzing their biochemical characteristics via flow cytometry after vesicle adsorption onto anti-CD63-coated latex beads (Caby et al., 2005). Among the studies on EVs GPC1, one study utilized anti-CD63-coupled magnetic beads to capture serum EVs (Buscail et al., 2019), while three other studies incubated exosomes or EVs with uncoated latex beads (Melo et al., 2015; Qian et al., 2018; Xiao et al., 2020). We compared anti-CD63-coated latex beads with uncoated latex beads using the percentages of CD9- or CD63-positive beads as indicators of captured EVs quantities; our results indicated that anti-CD63-coated latex beads pulled down significantly more tissue and serum-derived EVs than uncoated latex beads. This finding aligns with a previous report which showed that EVs bound to the surface of anti-CD63-coated latex beads while no vesicles were observed on uncoated latex beads via electron microscopy (Caby et al., 2005). Consequently, we employed anti-CD63-coated latex beads for capturing both serum- and tissue-derived EVs for flow cytometry analysis. Current studies primarily utilize cell lines or body fluids to isolate EVs for research; however, EVs isolated from tissues may provide more informative insights than those derived from cell lines due to the potential alterations in characteristics following extensive cell culture passages and the loss of influence from co-existing cells (Crescitelli et al., 2021). Additionally, tumor tissue may serve as a superior source of biomarkers compared to body fluids, as tumor-derived EVs can accumulate in interstitial spaces while non-cancerous cell-derived EVs may predominate in body fluids, potentially interfering with biomarker identification (Jang et al., 2019). A study isolated tissue-derived EVs from melanoma metastatic patients and found an enrichment of mitochondrial membrane proteins in melanoma tissue-derived EVs compared to non-melanoma-derived counterparts; these mitochondrial membrane proteins are detectable in plasma and are elevated in conditions such as melanoma, ovarian cancer, and breast cancer (Jang et al., 2019). A protocol was reported involving the treatment of dissociated tissues with collagenase D and DNase I to release EVs from the extracellular matrix, followed by differential ultracentrifugation and density separation for isolation purposes (Crescitelli et al., 2021). We adopted this protocol to extract EVs from both pancreatic tumor tissue and paratumor tissue before isolating them through differential ultracentrifugation. Subsequently, we characterized their morphology using TEM, measured sizes via NTA, and detected specific EVs markers including CD63, CD9, and ALIX through Western blotting and flow cytometry. These results verified the successful harvest of EVs derived from pancreatic tissue, this is the first study on isolating EVs from pancreatic tissue. In our study, there was no significant difference in serum EVs GPC1%, GPC1 MFI, and GPC1/CD9 between PC patients and healthy donors using two distinct GPC1 antibodies. In the first cohort, we divided PC patients into late-stage PC patients and pre-operative PDAC patients as the relative early-stage group, using the same GPC1 antibody (PA5-28055) as Melo et al. did. No significant differences in serum EVs GPC1 expression were found among late-stage PC, early-stage PDAC, and healthy controls. In the second cohort, we employed another GPC1 antibody (PA5-86043), and no differences were shown either. Regarding tissue-derived EVs GPC1 expression, no differences were detected between tumor tissue and paratumor tissue using the two antibodies. Also no differences of serum EVs GPC1 expression were found between the pre-operative and post-operative in PDAC patients at an average of 5.6 ± 1.9 days after surgical resection. This finding is in line with one report (Lai et al., 2017), but contrasts with two studies which showed a significant decrease after pancreatic resection (Frampton et al., 2018; Melo et al., 2015) and another report that demonstrated significantly higher serum exosomal GPC1 expression in the post-operative period compared with the pre-operative serum exosomal (Zhao et al., 2022). Collectively, these findings suggest that EVs GPC1 is not a reliable biomarker for the differential diagnosis or monitoring of PC, despite certain limitations in our research. Given the laborious and time-consuming nature of the entire experimental procedure, the clinical feasibility of EVs GPC1 for PC is relatively low. However, it should be noted that the limited size of the tissue samples and post-operative serum samples may constrain the statistical significance of our findings, and the relatively short duration of sampling after the operation could restrict the significance of our discoveries. We have successfully demonstrated the extraction of EVs from pancreatic tissue, and EVs isolated from the tissue may potentially serve as sources to uncover novel biomarkers for PC in future studies. Declarations Acknowledgements We thank Xiaoxia Wan for her technical assistance on Transmission Electron Microscopy in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University. Funding This research was supported by Zhejiang Provincial Natural Science Foudation of China under Grant No.LGC20H200002. Conflict of interest The authors have no relevant financial or non-financial interests to disclose. Author contributions All authors contributed to the study conception and design. Material preparation was performed by LC, HZ, LZ, LS and RR. Data collection and analyses were performed by RR and QW. The first draft of the manuscript was written by QW and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics approval This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the the Ethics Review Committee of Zhejiang Hospital under approval numbers 2020(84k) and 2023(90k). Consent to participate Informed consent was obtained from all individual participants included in the study. 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J Gastrointest Oncol , 13 (5), 2082–2104. https://doi.org/10.21037/jgo-22-240. Qian, J., Tan, Y., Zhang, Y., Yang, Y., & Li, X. (2018). Prognostic value of glypican-1 for patients with advanced pancreatic cancer following regional intra-arterial chemotherapy. Oncol Lett , 16 (1), 1253–1258. https://doi.org/10.3892/ol.2018.8701. Siegel, R. L., Giaquinto, A. N., & Jemal, A. (2024). Cancer statistics, 2024. Ca Cancer J Clin , 74 (1), 12–49. https://doi.org/10.3322/caac.21820. Stoffel, E. M., Brand, R. E., & Goggins, M. (2023). Pancreatic Cancer: Changing Epidemiology and New Approaches to Risk Assessment, Early Detection, and Prevention. Gastroenterology , 164 (5), 752–765. https://doi.org/10.1053/j.gastro.2023.02.012. Thery, C., Amigorena, S., Raposo, G., & Clayton, A. (2006). Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol , Chap. 3, 3–22. https://doi.org/10.1002/0471143030.cb0322s30. Xiao, D., Dong, Z., Zhen, L., Xia, G., Huang, X., Wang, T., Guo, H., Yang, B., Xu, C., Wu, W., Zhao, X., & Xu, H. (2020). Combined Exosomal GPC1, CD82, and Serum CA19-9 as Multiplex Targets: A Specific, Sensitive, and Reproducible Detection Panel for the Diagnosis of Pancreatic Cancer. Mol Cancer Res , 18 (2), 300–310. https://doi.org/10.1158/1541-7786.MCR-19-0588. Yan, S., Zheng, H., Zhao, J., Gao, M., & Zhang, X. (2023). Quantification of GPC1(+) Exosomes Based on MALDI-TOF MS In Situ Signal Amplification for Pancreatic Cancer Discrimination and Evaluation. Anal Chem , 95 (27), 10196–10203. https://doi.org/10.1021/acs.analchem.3c00193. Yang, K. S., Im, H., Hong, S., Pergolini, I., Del Castillo, A. F., Wang, R., Clardy, S., Huang, C., Pille, C., Ferrone, S., Yang, R., Castro, C. M., Lee, H., Del Castillo, C. F., & Weissleder, R. (2017). Multiparametric plasma EV profiling facilitates diagnosis of pancreatic malignancy. Sci Transl Med , 9 (391). https://doi.org/10.1126/scitranslmed.aal3226. Yu, J., Blackford, A. L., Dal Molin, M., Wolfgang, C. L., & Goggins, M. (2015). Time to progression of pancreatic ductal adenocarcinoma from low-to-high tumour stages. Gut , 64 (11), 1783–1789. https://doi.org/10.1136/gutjnl-2014-308653. Yu, Z., Yang, Y., Fang, W., Hu, P., Liu, Y., & Shi, J. (2023). Dual Tumor Exosome Biomarker Co-recognitions Based Nanoliquid Biopsy for the Accurate Early Diagnosis of Pancreatic Cancer. Acs Nano , 17 (12), 11384–11395. https://doi.org/10.1021/acsnano.3c00674. Zaborowski, M. P., Balaj, L., Breakefield, X. O., & Lai, C. P. (2015). Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study. Bioscience , 65 (8), 783–797. https://doi.org/10.1093/biosci/biv084. Zhao, J., Guo, M., Song, Y., Liu, S., Liao, R., Zhang, Y., Zhang, Y., Yang, Q., Gu, Y., & Huang, X. (2022). Serum exosomal and serum glypican-1 are associated with early recurrence of pancreatic ductal adenocarcinoma. Front Oncol , 12 , 992929. https://doi.org/10.3389/fonc.2022.992929. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5675979","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":392371253,"identity":"4634cb8a-8190-4eb3-b2d2-0a7054fa10c0","order_by":0,"name":"Rongrong Ren","email":"","orcid":"","institution":"Zhejiang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Rongrong","middleName":"","lastName":"Ren","suffix":""},{"id":392371254,"identity":"e84d8b0b-440b-4413-b5b6-cd6dd503baa8","order_by":1,"name":"Lu Cheng","email":"","orcid":"","institution":"Zhejiang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Cheng","suffix":""},{"id":392371255,"identity":"51b91c68-414f-40f1-9d4f-83e384783b9b","order_by":2,"name":"Huan Zhang","email":"","orcid":"","institution":"Zhejiang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Huan","middleName":"","lastName":"Zhang","suffix":""},{"id":392371256,"identity":"b2ede290-7618-4e30-b40f-f1cf45b8ed1f","order_by":3,"name":"Linggong Zeng","email":"","orcid":"","institution":"Zhejiang Hospital","correspondingAuthor":false,"prefix":"","firstName":"Linggong","middleName":"","lastName":"Zeng","suffix":""},{"id":392371257,"identity":"95da3f8a-40e2-4e0f-9ba2-b36a2f563d09","order_by":4,"name":"Lvhu Shan","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lvhu","middleName":"","lastName":"Shan","suffix":""},{"id":392371258,"identity":"400751fa-02fc-481e-b30c-e8342b27cf30","order_by":5,"name":"Qiang Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYDCCA2AkAWQxHzjwoYI0LWyJB2ecIVILFPAYH+ZtIUIH3/Hegwd/lFnIm/Ov+XCAt4FBnl/sAH4tkmfOJRzmOSdhuHPG2w0HJHcwGM6cnYBfi8GNHIPDjG0SjBtunN1wwPAMQ4LBbUJa7r8xOPizTcJ+w40zDw4kthGj5QaPwQHeNonEDed7GA4cJEaL5Bmgw4B+Sd5wg83gYMMZCcJ+4Tt+xvjjj7I62w3nDz/+/KfCRp5fmoAWCGADYgmwSglilMO08B8gVvUoGAWjYBSMNAAAGNBSCGwgWCwAAAAASUVORK5CYII=","orcid":"","institution":"Zhejiang Hospital","correspondingAuthor":true,"prefix":"","firstName":"Qiang","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2024-12-19 10:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5675979/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5675979/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":72363206,"identity":"e48317a0-ef0b-4310-9b7d-91055383f44c","added_by":"auto","created_at":"2024-12-26 06:13:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":523784,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological characterization of EVs by TEM(A and B) and NTA of EVs(C and D)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ea.EVs isolated from serum b. EVs isolated from tissue c.NTA of serum derived EVs d.NTA of tissue derived EVs TEM: Transmission Electron Microscope NTA:Nanoparticle Tracking Analysis\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5675979/v1/192fb9fca2d319fc5d517e1e.png"},{"id":72363795,"identity":"a2e3e5f6-d546-48ed-9855-b6e474071f9d","added_by":"auto","created_at":"2024-12-26 06:21:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":155999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical characterization of EVs by western blot. \u003c/strong\u003e\u0026nbsp;Lane A. serum EVs derived from a PDAC patient Lane B.serum EVs derived from a healthy donor Lane C.EVs derived from pancreatic tumor tissue Lane D. EVs derived from pancreatic paratumor tissue\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5675979/v1/87e06ef691aea5e926d295d4.png"},{"id":72363208,"identity":"dd238d84-ad08-4f47-9afb-0ddbf4339b89","added_by":"auto","created_at":"2024-12-26 06:13:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":167823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBiochemical characterization by flow cytometry after anti-CD63-latex bead immunoisolation of EVs\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5675979/v1/e60ad4eff2f34928ac54184a.png"},{"id":72363210,"identity":"4181e9aa-2dee-4905-bffe-3328f1574fdf","added_by":"auto","created_at":"2024-12-26 06:13:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":523492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of flow cytomety results (a-m). a\u003c/strong\u003e Assessment of the binding capacity of anti-CD63-coated latex beads versus uncoated latex beads for both tissue and serum-derived EVs (n=3 replicates). \u003cstrong\u003eb-d \u003c/strong\u003eComparison percentage of serum derived EVs GPC1 positive beads(GPC1%) , GPC1 mean fluorescence intensity(GPC1 MFI) and GPC1 positive beads/CD9 positive beads（GPC1/CD9）among late-stage PC patients(PC), pre-operative PDAC patients(pre-op), and healthy donors using GPC1 antibody PA5-28055. \u003cstrong\u003ee-g \u003c/strong\u003eComparison of serum derived EVs GPC1% , GPC1 MFI and GPC1/CD9 between late-stage PC and healthy donors using GPC1 antibody PA5-86043. \u003cstrong\u003eh-j \u003c/strong\u003eComparison of tissue derived EVs GPC1% , GPC1 MFI and GPC1/CD9 between tumor tissue and paratumor tissue using two GPC1 antibodies(PA5-28055 and PA5-86043). \u003cstrong\u003ek-m\u003c/strong\u003e Comparison of serum derived EVs GPC1%, GPC1 MFI and GPC1/CD9 between pre-op samples and matched post-operative (post-op) samples using GPC1 antibody PA5-28055.\u003cstrong\u003e \u003c/strong\u003e\u0026nbsp;ns indicates no significance. Scatterplots represent results from each sample, with horizontal lines indicating mean values and standard deviations.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5675979/v1/765407eae4e7de21e4e7980c.png"},{"id":72429439,"identity":"aad5329e-c10f-4fb9-9964-54a7cc9a91a2","added_by":"auto","created_at":"2024-12-27 03:31:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2152254,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5675979/v1/8b7a25c8-f383-4e63-bf17-9df5888179e0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of glypican-1 in extracellular vesicles from serum and pancreatic tissue as a biomarker for pancreatic cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePancreatic cancer (PC) ranks as the twelfth most prevalent malignancy, accounting for 510,566 new cases globally in 2022, and is the sixth leading cause of cancer-related mortality, with 467,005 deaths worldwide (Bray et al., 2024). More than 90% of PCs are categorized as pancreatic ductal adenocarcinomas (PDAC) (Jiang et al., 2022). The incidence rate of PC has manifested a consistent annual escalation of approximately 1% from 2015 to 2019 in the United States (Siegel et al., 2024). It was projected that this upward trend in incidence will persist over the forthcoming decades (Klein et al., 2021). Although the five-year relative survival rate for PC has improved from 4% during the period of 1995\u0026ndash;1997 to 13% between 2013 and 2019, it continues to represent the lowest survival rate among all malignancies (Siegel et al., 2024).\u003c/p\u003e \u003cp\u003eThe low survival rates associated with PC can be partially attributed to the advanced stage at which it is diagnosed, significantly hindering resectability; only approximately 10% of patients are diagnosed with localized and surgically resectable disease (Khalaf et al., 2021). PDAC is rarely detected at an early stage due to its rapid progression (Yu et al., 2015), and most early-stage PDAC cases are asymptomatic (Kanno et al., 2018).\u003c/p\u003e \u003cp\u003eFor stage IA PDAC, the five-year overall survival rate improved from 44.7% in 2004 to 83.7% in 2012; these trends may be attributed to advancements in early diagnosis and detection (Blackford et al., 2020). However, current screening strategies for PC remain far from optimal, and a combination of imaging techniques with biomarkers could represent a promising approach (Stoffel et al., 2023). The early detection of PC is particularly challenging due to the lack of specific biomarkers(O'Neill \u0026amp; Stoita, 2021). Clinical routine biomarkers, such as carcinoembryonic antigen and carbohydrate antigen 19\u0026thinsp;\u0026minus;\u0026thinsp;9, are primarily utilized for monitoring early recurrences rather than serving as effective screening or diagnostic tools because of their low sensitivity and specificity (Bhat et al., 2012).\u003c/p\u003e \u003cp\u003eExtracellular vesicles (EVs) are a heterogeneous group of cell-derived, nano-sized vesicles that play a crucial role in intercellular communication (Colombo et al., 2014; Zaborowski et al., 2015). EVs can be classified into microvesicles, exosomes, oncosomes, and apoptotic bodies based on differences in biogenesis and size. (Colombo et al., 2014; Zaborowski et al., 2015). These vesicles are present in various biological fluids and are enriched with cytosolic proteins, lipids, and nucleic acids, serving as carriers of disease biomarkers (Doyle \u0026amp; Wang, 2019).\u003c/p\u003e \u003cp\u003eMelo and colleagues have reported that serum glypican-1 (GPC1) positive circulating exosomes represent an 'ideal' biomarker, demonstrating 100% specificity and sensitivity for PDAC. In their view, this may serve as a potential non-invasive diagnostic and screening tool for the early detection of PDAC (Melo et al., 2015). However, subsequent studies investigating GPC1 in exosomes or EVs related to PC have yielded contradictory results(Buscail et al., 2019; Frampton et al., 2018; Lai et al., 2017; Li et al., 2024; Lucien et al., 2019; Qian et al., 2020; Yan et al.,, 2023; Yang et al., 2017; Yu et al., 2023; Zhao et al., 2022), raising controversy over whether blood-derived GPC1 is a reliable biomarker for PC.\u003c/p\u003e \u003cp\u003eA protocol described in detail the methodology for isolating EVs from tissues (Crescitelli et al., 2021). To our knowledge, no studies have yet reported on EVs derived from PC tissue. We hypothesized that if EVs GPC1 serves as a biomarker for PC, then EVs GPC1 levels may be enriched in tumor tissues compared to adjacent non-tumor tissues. We isolated EVs from both tumor and paratumor tissues of patients with PDAC and refined the protocol to detect GPC1 in EVs derived from serum and tissue samples. The expression of EVs GPC1 was compared between patients and controls, pre- and post-surgery, and between tumor tissues and adjacent non-tumor tissues.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSerum and tissue samples\u003c/h2\u003e \u003cp\u003eIn our study, we collected blood samples from 49 PC patients and 38 healthy donors, dividing them into two cohorts: one cohort consisting of 35 patients (including 18 PDAC patients undergoing surgery and 17 late-stage PC patients) and 24 healthy donors, quantified using Glypican-1 Polyclonal Antibody (PA5-28055) to assess serum EVs GPC1; the second cohort comprised 14 late-stage PC patients and 14 healthy donors, analyzed with Glypican-1 Polyclonal Antibody (PA5-86043). 10 ml of fasting whole blood were collected via venipuncture and centrifuged at 2,000 g for ten minutes to obtain serum samples, which were stored at -80℃ until analysis. Among 18 PDAC patients undergoing pancreatic surgery, 11 PDAC patients provided an additional peripheral blood sample at a mean of 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 days post-surgery. Tumor tissues along with matched paratumor tissues were obtained intra-operatively from 6 PDAC patients. Informed consent was obtained from all participants, and the study was approved by the Ethics Review Committee of Zhejiang Hospital under approval numbers 2020(84k) and 2023(90k).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMain antibodies and reagents\u003c/h3\u003e\n\u003cp\u003eCollagenase D (from Clostridium histolyticum, Roche) and DNase I (grade II, from bovine pancreas, Roche) were obtained from Sigma-Aldrich. Aldehyde/sulfate latex beads with a diameter of 4.0 \u0026micro;m (4% w/v, A37304) were purchased from Invitrogen. The bicinchoninic acid (BCA) protein assay kit was purchased from Mei5Bio Biotechnology. The following antibodies were acquired from Abcam: anti-CD63 (ab193349, 0.2 mg/ml, mouse monoclonal, reacts with human), anti-CD63 (ab252919, 0.598 mg/ml, rabbit monoclonal, reacts with human), anti-CD9 (ab236630, rabbit monoclonal, reacts with human), and anti-ALIX antibody (ab275377, rabbit monoclonal). Additionally, the following antibodies were sourced from Invitrogen: PE-conjugated CD9 antibody (eBioSN4; SN4 C3-3A2; mouse monoclonal IgG1; reacts with human; eBioscience\u0026trade;), Glypican-1 antibody (PA5-28055; rabbit polyclonal), Glypican-1 antibody (PA5-86043; rabbit polyclonal), FITC-conjugated goat anti-rabbit IgG secondary antibody, rabbit IgG isotype control (FITC; eBioscience\u0026trade;), and mouse IgG1 kappa isotype control (PE; eBioscience\u0026trade;)\u003c/p\u003e\n\u003ch3\u003eIsolation of EVs from tissue\u003c/h3\u003e\n\u003cp\u003eIn brief, dissociated fresh tissues were treated with collagenase D and DNase I to release EVs from the extracellular matrix (Crescitelli et al. 2021). This was followed by differential ultracentrifugation: first centrifuging at 300 g for 10 minutes, then at 2,000 g for 20 minutes. The supernatant was transferred to ultracentrifuge tubes and subjected to centrifugation at 10,000 g for 30 minutes at 4℃ using a Ti41 rotor in an Optima XPN-100 Ultracentrifuge (Beckman Coulter) to eliminate cells, dead cells, and cellular debris. The final supernatant was filtered through a 0.22 \u0026micro;m filter (Millipore), after which it underwent further ultracentrifugation in the Optima XPN-100 Ultracentrifuge at 120,000 g for 120 minutes at 4℃. Following this step, the supernatant was discarded and the pellet re-suspended in 12 ml of phosphate buffered saline (PBS) before being centrifuged again for 70 minutes at 120,000 \u0026times; g at 4℃. After removing the supernatant once more, the pellet was finally re-suspended in 100\u0026ndash;200\u0026micro;l of PBS; the collected EVs were either used for analysis or stored at -80℃.\u003c/p\u003e\n\u003ch3\u003eIsolation of EVs from serum\u003c/h3\u003e\n\u003cp\u003eSerum samples collected from patients and healthy donors were stored at -80℃. Prior to the isolation of EVs, serum was thawed on ice and then centrifuged at 12,000 g for 45 minutes at 4℃. One milliliter of serum was added to approximately 11 ml of PBS and mixed thoroughly before being filtered through a 0.22 \u0026micro;m filter. The resulting solution was transferred to ultracentrifuge tubes and subjected to centrifugation at 150,000 g for 120 minutes using a Ti41 rotor. After discarding the supernatant, the pellet was resuspended in approximately 12 ml of PBS and centrifuged again at 150,000 g for 70 minutes; the final pellet was re-suspended in between 50 and 150 \u0026micro;l of PBS. The collected EVs were either used for analysis or stored at -80℃.\u003c/p\u003e\n\u003ch3\u003eTransmission Electron Microscope (TEM)\u003c/h3\u003e\n\u003cp\u003eEVs stored at -80℃ were thawed on ice, after which 5 \u0026micro;l of resuspended EVs were deposited onto carbon-coated copper grids with a mesh size of 200. One minute later, filter paper was used to absorb excess liquid from the edges of the grid while leaving a thin layer of liquid on its surface. The grids were cleaned twice with a solution of 2% uranyl acetate before an additional drop of this solution was applied to each grid; one minute later, excess liquid was absorbed with filter paper prior to observation under an electron microscope (Thermo Fisher Scientific Talos L120C operating at 120 kV).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNanoparticle Tracking Analysis (NTA)\u003c/h2\u003e \u003cp\u003eThe size of EVs derived from serum and tissues was measured using the ZetaView\u0026reg; PMX110 particle tracking analyzer (Particle Metrix, Diessen, Germany). EVs isolated from serum were diluted 1:300, while those derived from tissue were diluted 1:1500 with PBS. Data analysis was performed using ZetaView\u0026reg; analysis software version 8.05.14SP7, with a minimum size threshold set at 5 nm, a maximum size limit of 1000 nm, and a minimum brightness setting of 20.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePreparation of the anti-CD63-coated bead working solution\u003c/h3\u003e\n\u003cp\u003eAldehyde/sulfate latex beads with a diameter of 4.0 \u0026micro;m were 1:10 diluted with PBS. Subsequently, 10 \u0026micro;l of the diluted beads were subjected to incubation in combination with 10 \u0026micro;l of anti-CD63 monoclonal antibody (ab193349, Abcam) within a 1.5 ml microcentrifuge tube at ambient temperature for 30 minutes. Subsequently, the volume was adjusted to 500 \u0026micro;l with PBS and subjected to overnight incubation at 4℃ on a test tube rotator. The reaction was terminated by supplementing the mixture with 55 \u0026micro;l of glycine (1000 mM) and conducting an additional incubation at ambient temperature for 30 minutes. Thereupon, the mixture underwent centrifugation for five minutes at 10,000 \u0026times; g at 4℃; the supernatant was meticulously discarded, and the pellet was rinsed twice with 1% PBS/BSA before being re-suspended in 500 \u0026micro;l of 1% PBS/BSA solution. This preparation yielded a working solution of anti-CD63-coated latex beads, which was stored at 4℃ with a dilution factor of 1:500.\u003c/p\u003e\n\u003ch3\u003eFlow cytometry analysis of EVs\u003c/h3\u003e\n\u003cp\u003eEVs derived from 1.0 ml of serum were incubated for 20 minutes at room temperature with either 3.33 \u0026micro;l of a working solution of anti-CD63-coated latex beads or 3.33 \u0026micro;l of uncoated latex bead solution diluted in PBS at a ratio of 1:500. Additionally, 2.5 \u0026micro;g of EVs derived from tissue were incubated under the same conditions with the working solution of anti-CD63-coated latex beads. The total volume was then adjusted to 500 \u0026micro;l with PBS and incubated overnight at 4℃ on a test tube rotator. For the uncoated latex bead solution diluted to 1:500 in PBS, an aliquot of 55 \u0026micro;l glycine (1000 mM) was added and allowed to stand at room temperature for an additional 30 minutes. Subsequently, both coated and uncoated beads were centrifuged for five minutes at 10,000 g at 4℃. The supernatant was carefully discarded, followed by two washes with a solution containing 1% PBS/BSA. It is important to minimize bead loss, approximately 50 \u0026micro;l of liquid remained at the bottom after each discarding step. Subsequently, 1.66 \u0026micro;l of PE-conjugated anti-CD9 monoclonal antibody, 6.66 \u0026micro;l of 1:250 diluted rabbit monoclonal like anti-CD63(ab252919), or anti-ALIX (ab275377) or anti-glypican-1(PA5-28055 or PA5-86043) were added to each sample. After incubation for 45 minutes at room temperature, an additional volume of 500 \u0026micro;l containing 1% PBS/BSA was added and the samples were centrifuged for five minutes at a speed of 10,000 g at 4℃; the supernatant was carefully discarded while approximately 50 \u0026micro;l remained in the bottom and washed twice with a solution containing 1% PBS/BSA. Following this step, 6.66 \u0026micro;l of FITC-conjugated goat anti-rabbit secondary antibody diluted to 1:250 was added and incubated for another 45 mintutes at room temperature; after two washes with 1% PBS/BSA, 200 \u0026micro;l PBS was added for flow cytometry analysis. Single beads and doublets were gated based on Forward Scatter and Side Scatter parameters(Thery, Amigorena, Raposo, \u0026amp; Clayton, 2006), with a total of 2,000 latex beads set for analysis using either a flow cytometryCalibur flow cytometer (BD Biosciences) or FlowJo\u0026trade;10 software (BD Biosciences).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis of EVs\u003c/h2\u003e \u003cp\u003eThe EVs obtained were lysed in RIPA Lysis Buffer supplemented with a cocktail of protease inhibitors (Thermo Scientific, A32965). Following centrifugation at 12,000 g for 15 minutes at 4℃, the supernatants were collected for Western blotting according to standard procedures. For each lane, 3 \u0026micro;g of protein was loaded for analysis. Antibodies against CD63 (ab193349, 1:1000), ALIX (ab275377, 1:1000), and CD9 (ab236630, 1:1000) were purchased from Abcam. Anti-GPC1 antibodies PA5-28055 and PA5-86043 were acquired from Invitrogen at a dilution of 1:1000 each. Chemiluminescence detection was performed using a ChemiScope3300 system (Clinx).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analyses were carried out using GraphPad Prism software version 10 (GraphPad Software), and the data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Unpaired two-tailed Student\u0026rsquo;s t-tests were employed to compare the percentages of CD9 and CD63 positive beads between anti-CD63-coated latex beads and uncoated latex beads, as well as the percentage of serum EVs GPC1 positive beads, GPC1 mean fluorescence intensity, and the ratio of GPC1 positive beads to CD9 positive beads in late-stage PC patients and healthy donors. One way analysis of variance (ANOVA) was adopted to compare the differences among late-stage PC patients, pre-operative PDAC patients, and healthy donors. Paired two-tailed Student\u0026rsquo;s t-tests were performed to assess the differences between tumor tissues and adjacent non-tumor tissues, along with pre-operative PDAC patients and post-operative PDAC patients. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorphological characterization of EVs via TEM and size measurement by NTA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEVs isolated from serum and tissue, as characterized by electron microscopy, exhibited typical EVs morphology (see Fig.1 a and b). NTA revealed a median diameter of 151.6 nm for serum-derived EVs (see Fig.1 c) and 142.5 nm for tissue-derived EVs(see Fig.1 d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiochemical characterization of EVs by western blot and flow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe presence of canonical EVs markers CD9, CD63, and Alix (Thery et al. 2006) was confirmed through Western blotting and flow cytometry. In conjunction with results from TEM and NTA, these findings indicate the successful isolation of EVs from serum and tissue in our study. Furthermore, the expression of GPC1 protein in EVs derived from both serum and tissue was validated using Western blotting and flow cytometry with two distinct antibodies (PA5-28055 and PA5-86043).\u003c/p\u003e\n\u003cp\u003eEVs captured by anti-CD63-latex beads were subsequently stained with antibodies against CD9, CD63, Alix, and GPC1 (PA5-28055 or PA5-86043). The data were analyzed using FlowJo\u0026trade; 10 software. \u003cstrong\u003ea\u003c/strong\u003eScatter plot illustrating the distribution of anti-CD63-latex beads, including both single beads and doublets. \u003cstrong\u003eb\u003c/strong\u003eHistogram representing flow cytometry analyses of anti-CD63-latex beads in conjunction with EVs derived from a representative serum sample and paratumor tissue; green histograms indicate FITC fluorescence while red histograms represent PE fluorescence (gray histograms serve as negative controls obtained using an appropriate isotype). FITC\u0026nbsp;fluorescein\u0026nbsp;isothiocyanate\u0026nbsp;,\u0026nbsp;PE phycoerythrin\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBoth serum and tissue-derived EVs were captured more efficiently using anti-CD63-coated latex beads compared to uncoated latex beads\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated the efficacy of various beads in capturing EVs derived from serum or tissue by quantifying the proportion of CD63- or CD9-positive beads as an indicator of EV attachment. For serum-derived EVs, the percentage of CD63-positive beads was 8.3% \u0026plusmn; 2.2% for uncoated latex beads, compared to 86.9% \u0026plusmn; 5.6% for anti-CD63-coated latex beads. Similarly, the percentage of CD9-positive beads was 10.3% \u0026plusmn; 2.1% for uncoated latex beads, versus 89.2% \u0026plusmn; 2.9% for anti-CD63-coated latex beads. For tissue-derived EVs, the percentages of CD63- and CD9-positive beads were 26.5% \u0026plusmn; 2.0% and 36.0% \u0026plusmn; 3.5%, respectively, for uncoated latex beads, compared to 89.8% \u0026plusmn; 4.5% and 93.5% \u0026plusmn; 4.0% for anti-CD63-coated latex beads. These results (see Fig. 4a) demonstrate that a substantial majority of the beads tested positive when utilizing anti-CD63-coated latex beads, whereas most uncoated latex beads yielded negative outcomes. Collectively, these findings indicate that significantly more tissue and serum-derived EVs were captured by anti-CD63-coated latex beads compared to their uncoated counterparts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlypican-1 in serum EVs showed no significant difference between PC and control\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe compared the percentage of GPC1 positive EVs (abbreviated as GPC1%), the GPC1 mean fluorescence intensity (abbreviated as GPC1 MFI), and the ratio of GPC1-positive beads to CD9-positive beads (abbreviated as GPC1/CD9) among late-stage PC patients (n = 17), pre-operative PDAC patients (n = 18), and healthy donors (n = 24) in the first cohort using the GPC1 antibody PA5-28055. One-way ANOVA showed no significant differences among these three groups for the serum EVs GPC1%, GPC1 MFI, and GPC1/CD9 (see Fig.4 b-d). Specifically, for late-stage PC patients, GPC1%, GPC1 MFI, and GPC1/CD9 were 27.2% \u0026plusmn; 18.5%, 111.6 \u0026plusmn; 79.5, and 0.34 \u0026plusmn; 0.22, respectively; for pre-operative PDAC patients, they were 32.1% \u0026plusmn; 19.2%, 122.2 \u0026plusmn; 66.8, and 0.41 \u0026plusmn; 0.23; for healthy donors, they were 29.5% \u0026plusmn; 18.7%, 106.7 \u0026plusmn; 52.6, and 0.34 \u0026plusmn; 0.21. Similarly, no significant differences were observed in the second cohort using GPC1 antibody PA5-86043; when comparing late-stage PC patients (n=14) with healthy donors (n=14), the serum EVs GPC1% was 35.2% \u0026plusmn; 22.4% vs. 31.0% \u0026plusmn; 21.6%, GPC1 MFI was 135.1 \u0026plusmn; 82.1 vs. 134.1 \u0026plusmn; 70.2, and GCP1/CD9 was 0.38 \u0026plusmn; 0.23 vs. 0.34 \u0026plusmn; 0.22(see Fig.4 e-g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEVs glypican-1 showed no significant difference between pancreatic tumor and paratumor tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere was no significant difference in tissue-derived EVs GPC1% (56.0% \u0026plusmn; 28.9% vs. 51.5% \u0026plusmn; 25.5%), GPC1 MFI (234.8 \u0026plusmn; 116.4 vs. 216.3 \u0026plusmn; 108.8), and GCP1/CD9 (0.59 \u0026plusmn; 0.30 vs. 0.56 \u0026plusmn; 0.24) for GPC1 PA5-28055 between tumor tissues and matched paratumor tissues collected from 6 patients with PDAC. Similarly, no significant difference was found when another GPC1 antibody PA5-86043 was used. For GPC1%, it was 66.8% \u0026plusmn; 31.4% vs. 70.9% \u0026plusmn; 32.5%; for GPC1 MFI, it was 302.7 \u0026plusmn; 164.2 vs. 377.0 \u0026plusmn; 213.3; and for GCP1/CD9, it was 0.71 \u0026plusmn; 0.33 vs. 0.79 \u0026plusmn; 0.36 between tumor tissues and paratumor tissues (see Fig.4 h-i).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGlypican-1 levels in serum EVs exhibited no significant difference between pre-operative and post-operative samples\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo significant differences were detected in the serum EVs GPC1% (37.9% \u0026plusmn; 18.8% vs. 33.8% \u0026plusmn; 20.3%), GPC1 MFI (140.0 \u0026plusmn; 73.9 vs. 120.3 \u0026plusmn; 54.9), and GPC1/CD9 (0.46 \u0026plusmn; 0.24 vs. 0.37 \u0026plusmn; 0.21) between the pre-operative and post-operative blood samples from 11 PDAC patients, which were assessed an average of 5.6 \u0026plusmn; 1.9 days (range: 4-9 days) after surgical resection (see Fig.4 k-m).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGlypican-1 is a member of the glypican family, consisting of six members, and is anchored to the cell membrane. It regulates various signaling pathways, including Wnt, Hedgehog (Hh), fibroblast growth factors (FGF), and bone morphogenic protein (BMP) (Pan \u0026amp; Ho, 2021). Glypican-1 is highly expressed at both mRNA and protein levels in human PC (Duan et al., 2013; Kleeff et al., 1998; Lu et al., 2017) and is overexpressed in other malignancies such as breast cancer (Matsuda et al., 2001), hepatocellular carcinoma (Chen et al., 2020), and esophagogastric cancer (Pratap et al., 2022). A study evaluated GPC1 expression using immunohistochemical staining in a cohort of 140 PDAC patients, 55% of tissue microarrays exhibited weak GPC1 expression while one-third were negative for GPC1(Lucien et al., 2019). Immunohistochemical assessments conducted in two additional studies demonstrated that GPC1 levels in PC tissues were significantly higher than those found in normal pancreatic tissues (Duan et al., 2013; Lu et al., 2017). Faint GPC1 immunoreactivity was observed in some PDAC cells, with faint to moderate reactivity present in a few fibroblasts within normal pancreas or chronic pancreatitis samples (Kleeff et al., 1998). However, no significant difference was detected between GPC1 protein levels in PDAC tissues and adjacent normal pancreas using enzyme-linked immunosorbent assay (ELISA) methods (Frampton et al., 2018). These findings suggest that GPC1 protein expression may be enriched within specific structures of cancer cells rather than uniformly distributed across total tissue among certain PDAC patients; however, it remains unclear whether GPC1 levels are more concentrated in EVs derived from PDAC tissues compared to those from paratumor tissues.\u003c/p\u003e \u003cp\u003eOne study reported that GPC1 levels were elevated in breast cancer and PC cells compared to three non-tumorigenic cell lines, along with over 90% of GPC1-positive exosome beads found in cancer cells versus less than 3% in non-cancerous cells (Melo et al., 2015). GPC1-positive circulating exosomes (crExos) were identified as an excellent biomarker for PDAC, demonstrating an area under the curve (AUC) of 1.00; crExos GPC1 effectively distinguished PDAC from healthy donors, even in cases of histologically validated PC precursor lesions, where crExos GPC1 levels remained consistently higher than those in the healthy donor group. A significant decrease in exosomal GPC1 levels was also observed at seven days post-surgical resection in PDAC patients, suggesting that blood-derived exosomal GPC1 may serve as a powerful diagnostic and screening tool for detecting PDAC even at early stages (Melo et al., 2015).\u003c/p\u003e \u003cp\u003eThese results would be exceptionally valuable if replicated across other laboratories. However, subsequent studies is still controversial (Buscail et al., 2019; Frampton et al., 2018; Lai et al., 2017; Li et al., 2024; Lucien et al., 2019; Qian et al., 2020; Yan et al., 2023; Yang et al., 2017; Yu et al., 2023; Zhao et al., 2022). Three studies employed differential ultracentrifugation for the isolation of exosomes or EVs (Frampton et al., 2018; Lai et al., 2017; Yang et al., 2017), one utilized density gradient ultracentrifugation (Xiao et al., 2020), one adopted a spin column-based method (Qian et al., 2018), one employed size-exclusion chromatography (Yan et al., 2023), four utilized a total exosome isolation kit (Buscail et al., 2019; Li et al., 2024; Yu et al., 2023; Zhao et al., 2022), and one directly detected EVs GPC1 without prior isolation (Lucien et al., 2019). The detection of EVs or exosomal GPC1 was carried out using flow cytometry (Buscail et al., 2019; Qian et al., 2018; Xiao et al., 2020), multiplexed plasmonic assay (Yang et al., 2017), nanoscale flow cytometry (Lucien et al., 2019), LC/MS (Lai et al., 2017), ELISA (Frampton et al., 2018; Zhao et al., 2022), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Yan et al., 2023), nanoliquid biopsy (Yu et al., 2023), and immune lipoplex nanoparticle biochip assay (Li et al., 2024). Four reports failed to differentiate PDAC from controls based on EVs or exosomal GPC1 levels (Frampton et al., 2018; Lai et al., 2017; Lucien et al., 2019; Yang et al., 2017); however, other studies reported significantly elevated levels of EVs GPC1 in PDAC patients compared to the control group (Buscail et al., 2019; Qian et al., 2018; Xiao et al., 2020; Yan et al., 2023; Yu et al., 2023; Zhao et al., 2022). Interestingly, one study conducted experiments using ultracentrifugation followed by latex beads coupling were unable to distinguish PDAC from controls, which contrasts with the situation when a density-based separation kit coupled to magnetic beads decorated with anti-CD63 was used (Buscail et al., 2019). Most recently, a study found that EVs GPC1 protein expression can effectively discriminate between PDAC and controls through tumor-associated microvesicles rather than through exosomes (Li et al., 2024).\u003c/p\u003e \u003cp\u003eWe conjecture that these observed disparities might be partly attributed to the variations in EVs isolation techniques and detection methods adopted by different studies or the small sample size in some studies. Hence, We utilized ultracentrifugation for EVs isolation and flow cytometry to detect EVs GPC1 following the methodology established by Melo, and employed two GPC1 antibodies, one of which was the same as reported (Melo et al., 2015).\u003c/p\u003e \u003cp\u003eCaby and colleagues initially demonstrated the existence of EVs in human blood by isolating plasma EVs through ultracentrifugation and analyzing their biochemical characteristics via flow cytometry after vesicle adsorption onto anti-CD63-coated latex beads (Caby et al., 2005). Among the studies on EVs GPC1, one study utilized anti-CD63-coupled magnetic beads to capture serum EVs (Buscail et al., 2019), while three other studies incubated exosomes or EVs with uncoated latex beads (Melo et al., 2015; Qian et al., 2018; Xiao et al., 2020). We compared anti-CD63-coated latex beads with uncoated latex beads using the percentages of CD9- or CD63-positive beads as indicators of captured EVs quantities; our results indicated that anti-CD63-coated latex beads pulled down significantly more tissue and serum-derived EVs than uncoated latex beads. This finding aligns with a previous report which showed that EVs bound to the surface of anti-CD63-coated latex beads while no vesicles were observed on uncoated latex beads via electron microscopy (Caby et al., 2005). Consequently, we employed anti-CD63-coated latex beads for capturing both serum- and tissue-derived EVs for flow cytometry analysis.\u003c/p\u003e \u003cp\u003eCurrent studies primarily utilize cell lines or body fluids to isolate EVs for research; however, EVs isolated from tissues may provide more informative insights than those derived from cell lines due to the potential alterations in characteristics following extensive cell culture passages and the loss of influence from co-existing cells (Crescitelli et al., 2021). Additionally, tumor tissue may serve as a superior source of biomarkers compared to body fluids, as tumor-derived EVs can accumulate in interstitial spaces while non-cancerous cell-derived EVs may predominate in body fluids, potentially interfering with biomarker identification (Jang et al., 2019). A study isolated tissue-derived EVs from melanoma metastatic patients and found an enrichment of mitochondrial membrane proteins in melanoma tissue-derived EVs compared to non-melanoma-derived counterparts; these mitochondrial membrane proteins are detectable in plasma and are elevated in conditions such as melanoma, ovarian cancer, and breast cancer (Jang et al., 2019).\u003c/p\u003e \u003cp\u003eA protocol was reported involving the treatment of dissociated tissues with collagenase D and DNase I to release EVs from the extracellular matrix, followed by differential ultracentrifugation and density separation for isolation purposes (Crescitelli et al., 2021). We adopted this protocol to extract EVs from both pancreatic tumor tissue and paratumor tissue before isolating them through differential ultracentrifugation. Subsequently, we characterized their morphology using TEM, measured sizes via NTA, and detected specific EVs markers including CD63, CD9, and ALIX through Western blotting and flow cytometry. These results verified the successful harvest of EVs derived from pancreatic tissue, this is the first study on isolating EVs from pancreatic tissue.\u003c/p\u003e \u003cp\u003eIn our study, there was no significant difference in serum EVs GPC1%, GPC1 MFI, and GPC1/CD9 between PC patients and healthy donors using two distinct GPC1 antibodies. In the first cohort, we divided PC patients into late-stage PC patients and pre-operative PDAC patients as the relative early-stage group, using the same GPC1 antibody (PA5-28055) as Melo et al. did. No significant differences in serum EVs GPC1 expression were found among late-stage PC, early-stage PDAC, and healthy controls. In the second cohort, we employed another GPC1 antibody (PA5-86043), and no differences were shown either. Regarding tissue-derived EVs GPC1 expression, no differences were detected between tumor tissue and paratumor tissue using the two antibodies.\u003c/p\u003e \u003cp\u003eAlso no differences of serum EVs GPC1 expression were found between the pre-operative and post-operative in PDAC patients at an average of 5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 days after surgical resection. This finding is in line with one report (Lai et al., 2017), but contrasts with two studies which showed a significant decrease after pancreatic resection (Frampton et al., 2018; Melo et al., 2015) and another report that demonstrated significantly higher serum exosomal GPC1 expression in the post-operative period compared with the pre-operative serum exosomal (Zhao et al., 2022).\u003c/p\u003e \u003cp\u003eCollectively, these findings suggest that EVs GPC1 is not a reliable biomarker for the differential diagnosis or monitoring of PC, despite certain limitations in our research. Given the laborious and time-consuming nature of the entire experimental procedure, the clinical feasibility of EVs GPC1 for PC is relatively low. However, it should be noted that the limited size of the tissue samples and post-operative serum samples may constrain the statistical significance of our findings, and the relatively short duration of sampling after the operation could restrict the significance of our discoveries. We have successfully demonstrated the extraction of EVs from pancreatic tissue, and EVs isolated from the tissue may potentially serve as sources to uncover novel biomarkers for PC in future studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e We thank Xiaoxia Wan for her technical assistance on Transmission Electron Microscopy in the Center of Cryo-Electron Microscopy (CCEM), Zhejiang University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was supported by Zhejiang Provincial Natural Science Foudation of China under Grant No.LGC20H200002.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation was performed by LC, HZ, LZ, LS and RR. Data collection and analyses were performed by RR and QW. The first draft of the manuscript was written by QW and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e This study was performed in line with the principles of the Declaration of Helsinki. 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Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study. \u003cem\u003eBioscience\u003c/em\u003e, \u003cem\u003e65\u003c/em\u003e(8), 783\u0026ndash;797. https://doi.org/10.1093/biosci/biv084.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, J., Guo, M., Song, Y., Liu, S., Liao, R., Zhang, Y., Zhang, Y., Yang, Q., Gu, Y., \u0026amp; Huang, X. (2022). Serum exosomal and serum glypican-1 are associated with early recurrence of pancreatic ductal adenocarcinoma. \u003cem\u003eFront Oncol\u003c/em\u003e, \u003cem\u003e12\u003c/em\u003e, 992929. https://doi.org/10.3389/fonc.2022.992929.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pancreatic cancer, Extracellular vesicles, Glypican-1, Biomarker","lastPublishedDoi":"10.21203/rs.3.rs-5675979/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5675979/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003ePurpose\u003c/strong\u003e Pancreatic cancer (PC) is a lethal disease, and early detection is crucial for reducing mortality. Blood exosome glypican-1 (GPC1) has been reported as a powerful diagnostic and screening tool for detecting pancreatic ductal adenocarcinomas (PDAC) even at early stages; however, results from subsequent studies on extracellular vesicles (EVs) or exosome GPC1 have been conflicting. We hypothesized that if EVs GPC1 serves as a biomarker for PC, it may be enriched in pancreatic tumor tissues compared to adjacent non-tumor tissues.\u003cbr\u003e\n\u003cstrong\u003eMethods \u003c/strong\u003eDissociated tumor tissues and paratumor tissues were treated with collagenase D and DNase I to release EVs from the extracellular matrix. Both serum-derived EVs and tissue-derived EVs were isolated by ultracentrifugation, and EVs GPC1 levels were analyzed by flow cytometry. The expression of EV GPC1 was compared between patients and controls, pre- and post-surgery, and between tumor tissues and adjacent non-tumor tissues.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eEVs were successfully isolated from pancreatic tissue. Serum EVs GPC1 levels showed no significant difference between PC patients and healthy controls, nor between pre-operative and post-operative samples. EVs GPC1 derived from tumor tissue showed no significant difference compared to matched paratumor tissue.\u003cbr\u003e\n\u003cstrong\u003eConclusion \u003c/strong\u003eAlthough EVs GPC1 was found not to be a reliable biomarker for pancreatic cancer, we successfully isolated EVs from pancreatic tissue. Further research is needed to explore the potential of tissue-derived EVs as sources of screening biomarkers and to standardize methods for isolating and detecting EVs biomarkers.\u003c/p\u003e","manuscriptTitle":"Evaluation of glypican-1 in extracellular vesicles from serum and pancreatic tissue as a biomarker for pancreatic cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-26 06:13:19","doi":"10.21203/rs.3.rs-5675979/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"244ae2f9-04b4-46b7-a823-da0728951471","owner":[],"postedDate":"December 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-27T03:23:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-26 06:13:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5675979","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5675979","identity":"rs-5675979","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}