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A comparative proteomics analysis identified differentially expressed proteins in pancreatic cancer–associated stellate cell small extracellular vesicles

Open AccessPublished:November 01, 2022DOI:https://doi.org/10.1016/j.mcpro.2022.100438

      Highlights

      • PDAC-associated human pancreatic stellate cells (HPSCs) trending to secrete more small extracellular vesicles (sEVs) compared to normal stellate cells (HPaStec)
      • sEVs reduced α-SMA expressions in normal stellate cells
      • Membrane-associated proteins may be essential for stellate cell sEVs uptake by cancer cells
      • LC-MS/MS analysis revealed that 87 protein groups were differentially expressed between HPSC and HPaStec sEVs
      • sEVs could potentially be targeted as a cargo vehicle for the safe delivery of drugs or other biological materials to the cancer cells

      ABSTRACT

      Human pancreatic stellate cells (HPSCs) are an essential stromal component and mediators of pancreatic ductal adenocarcinoma (PDAC) progression. Small extracellular vesicles (sEVs) are membrane-enclosed nanoparticles involved in cell-to-cell communications and are released from stromal cells within PDAC. A detailed comparison of sEVs from normal pancreatic stellate cells (HPaStec) and from PDAC-associated stellate cells (HPSCs) remains a gap in our current knowledge regarding stellate cells and PDAC. We hypothesized there would be differences in sEVs secretion and protein expression that might contribute to PDAC biology. To test this hypothesis, we isolated sEVs using ultracentrifugation followed by characterization by electron microscopy and Nanoparticle Tracking Analysis. We report here our initial observations. First, HPSC cells derived from PDAC tumors secrete a higher volume of sEVs when compared to normal pancreatic stellate cells (HPaStec). Although our data revealed that both normal and tumor derived sEVs demonstrated no significant biological effect on cancer cells, we observed efficient uptake of sEVs by both normal and cancer epithelial cells. Additionally, intact membrane-associated proteins on sEVs were essential for efficient uptake. We then compared sEV proteins isolated from HPSCs and HPaStecs cells using liquid chromatography–tandem mass spectrometry. Most of the 1,481 protein groups identified were shared with the exosome database, ExoCarta. Eighty-seven protein groups were differentially expressed (selected by 2-fold difference and adjusted p value ≤ 0.05) between HPSC and HPaStec sEVs. Of note, HPSC sEVs contained dramatically more CSE1L (chromosome segregation 1–like protein), a described marker of poor prognosis in patients with pancreatic cancer. Based on our results, we have demonstrated unique populations of sEVs originating from stromal cells with PDAC and suggest that these are significant to cancer biology. Further studies should be undertaken to gain a deeper understanding that could drive novel therapy.

      Graphical abstract

      Abbreviations:

      PDAC ((Pancreatic ductal adenocarcinoma)), HPSC sEVs ((PDAC associated human pancreatic stellate cells small extracellular vesicles)), HPaStec sEVs ((Normal human pancreatic stellate cells small extracellular vesicles)), CSE1L/CAS ((chromosome segregation 1-like/cellular apoptosis susceptibility)), CD63 ((cluster of differentiation 63)), CD9 ((cluster of differentiation 9)), CD81 ((cluster of differentiation 81)), TSG101 ((tumor susceptibility gene 101 protein)), Alix ((apoptosis-linked gene 2-interacting protein X)), FACS ((fluorescence activated cell sorting)), BSA ((bovine serum albumin)), MAPK ((mitogen-activated protein kinase)), ERK ((extracellular signal-regulated kinase)), EGFR ((epidermal growth factor receptor)), RAS ((rat sarcoma)), GO:BP ((gene ontology: biological process)), GO:CP ((gene ontology: cellular component)), PC ((principal component))

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      • Zuba-Surma E.K.
      Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.
      ).
      We report here that HPSC cells secreted more sEVs than primary stellate cells (HPaStec). Interestingly, we have shown that intact membrane-associated proteins may be essential for sufficient uptake of sEVs by both normal and cancer cells. Our data revealed that sEVs have no significant biological effects on the cancer cells, but we see this as a benefit since several studies have suggested that sEVs are promising tools for drug delivery across different biological barriers and can be used as next generation therapeutics. Therefore, we performed a detailed comparative proteomics analysis between the 2 different sEVs, and 87 proteins were differentially expressed. Our results suggested that both sEVs are biologically different from each other and may be utilized as a cargo vehicle for the safe delivery of drugs or other biological materials to pancreatic or other cancer cells.

      EXPERIMENTAL PROCEDURES

      Cell lines and reagents

      Immortalized HPSCs harvested from primary PDAC tumors were given by Dr. Rosa F. Hwang (
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      ) and maintained in RPMI (Invitrogen, Grand Island, NY) medium supplemented with 10% fetal bovine serum (FBS, Invitrogen). Primary HPaStecs isolated from normal pancreases were purchased from ScienCell Research Laboratories (Carlsbad, CA) and maintained in culture using recommended media (SteCM) at 37 °C in 5% CO2. Established cancer and normal pancreas cell lines, such as, Panc1, MiaPaCa2 and HPNE were purchased from ATCC (Manassas, VA). All experiments were performed using cell lines at less than ten passages and tested negative for mycoplasma contamination using MycoALert Plus Mycoplasma Detection Kit (Lonza). The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX): anti-CD63 (sc-5275), anti-ALIX (sc-53540), and anti-TSG 101 (sc-7964). APC anti-human CD63 (#353007) was purchased from Biolegends (CA). The following antibodies were used from Cell Signaling Technology (Danvers, MA): anti–β tubulin antibody (#2128), anti-Calnexin (#2433), anti-p44/42 MAPK (Erk1/2) (#9102), anti-phospho-p44/42 MAPK (Erk1/2) (#4376), α4 integrin (#4600), β1 integrin (#9699), c-PARP (#5625), c-caspase3 (#9664) and EGFR (D38B1XP) (#4267). Anti-CSE1L/CAS/Exportin-2 (JU34-33) (#NBP2-75451), anti-smooth muscle actin (#MAB1420) and anti–β-actin (#A2228) were purchased from Novus Biologicals (Littleton, CO), R and D systems (Minneapolis, MN) and Sigma-Aldrich (St. Louis, MO), respectively. Gemcitabine hydrocholoride was purchased from Sellekchem (Houston, TX).

      Growth rate and cellular morphology of HPSC and HPaStec cells

      Equal number of HPSC and HPaStec cells (5X10ˆ6) were seeded to assess their growth rate by Trypan blue (Gibco) exclusion method at 24 and 48hrs. Cellular morphology of both the cells were captured using Incucyte S3 Live Imager with 10x magnification in-phase channel.

      Ultracentrifugation to isolate sEVs

      A classical differential ultracentrifugation protocol (
      • Thery C.
      • Amigorena S.
      • Raposo G.
      • Clayton A.
      Isolation and characterization of exosomes from cell culture supernatants and biological fluids.
      ) was used with minor modifications to isolate sEVs. 5X10ˆ6 HPSC/HPaStec cells were seeded in a T150 cm2 flasks (corning) at 50% confluence in each respective medium with 10% exosome-free FBS (Thermo Scientific, #A2720803, MA), and the conditioned medium was harvested when cells were 70% to 80% confluent (48hrs.). Cell-conditioned media was cleared of cells, cell debris, and large membrane vesicles by sequential centrifugation at 500 x g for 30 minutes followed by 12,000 x g for an additional 30 minutes and was then filtered through a 0.22 μM filter unit (Millipore, #SCGP00525). This step was done to maximize quality over quantity to exclude the apoptotic bodies and other debris supported by previous publications (
      • Melo S.A.
      • Luecke L.B.
      • Kahlert C.
      • Fernandez A.F.
      • Gammon S.T.
      • Kaye J.
      • LeBleu V.S.
      • Mittendorf E.A.
      • Weitz J.
      • Rahbari N.
      • Reissfelder C.
      • Pilarsky C.
      • Fraga M.F.
      • Piwnica-Worms D.
      • Kalluri R.
      Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.
      ,
      • Han S.
      • Gonzalo D.H.
      • Feely M.
      • Rinaldi C.
      • Belsare S.
      • Zhai H.
      • Kalra K.
      • Gerber M.H.
      • Forsmark C.E.
      • Hughes S.J.
      Stroma-derived extracellular vesicles deliver tumor-suppressive miRNAs to pancreatic cancer cells.
      ,
      • Han S.
      • Gonzalo D.H.
      • Feely M.
      • Delitto D.
      • Behrns K.E.
      • Beveridge M.
      • Zhang D.
      • Thomas R.
      • Trevino J.G.
      • Schmittgen T.D.
      • Hughes S.J.
      The pancreatic tumor microenvironment drives changes in miRNA expression that promote cytokine production and inhibit migration by the tumor associated stroma.
      ).To sort by density, sEVs were collected from the cleared supernatants after centrifugation at 100,000 x g for 2 hrs. in SWT32i (Beckman, IN) swinging buckets using a Beckman Coulter ultracentrifuge (Beckman). The sEVs were washed with phosphate-buffered saline (PBS) and repurified by centrifugation at 100,000 x g for 2 hrs.. The pellets were resuspended in 0.22 μM–filtered PBS. Exosomal protein quantity was estimated by Pierce BCA protein assay reagent (Thermo Scientific, #23227) and Nanodrop assay (Thermo Scientific), according to the manufacturer’s instructions.

      Electron microscopy

      sEV proteins were quantified by BCA protein assay, and an aliquot of 10 μg protein of the sEVs was fixed with 2.5% glutaraldehyde in HEPES buffer for imaging with transmission electron microscopy, using a previously described procedure with minor modifications (
      • Thery C.
      • Amigorena S.
      • Raposo G.
      • Clayton A.
      Isolation and characterization of exosomes from cell culture supernatants and biological fluids.
      ). Briefly, 3 μl of fixed sEV suspension was placed on a formvar-coated copper grid (Electron Microscopy Sciences, Hartfield, PA) and allowed to settle for 2 minutes before adding 3 μl of 2% aqueous uranyl acetate for contrast enhancement. After 30 seconds, excess liquid was wicked off with filter paper and the grid was allowed to dry overnight before TEM micrograph capture with a JEOL1400 transmission electron microscope (JEOL, Japan) equipped with a side mounted Gatan Orius digital camera.

      Western blot

      Cells were lysed in NP40 buffer containing 50 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, 1.0% NP40, and 1× Proteinase Inhibitor Cocktail Set (Thermo Scientific, #1861281). Proteins were estimated using the Pierce BCA protein assay reagent (Thermo Scientific, #23227). Whole-cell protein lysates (40 μg) and sEV proteins (20 μg) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to Immobilon-P PVDF membranes (Millipore, Danvers, VA), and incubated overnight in the corresponding primary antibody at 4 °C. Secondary antibodies conjugated to horseradish peroxidase (GE Healthcare, Chicago, IL) were used, and chemiluminescence (Thermo Fisher Scientific) was used for detection.

      Nanoparticle tracking analysis

      The size distribution and concentration of particles in the EV preparations were analyzed using dynamic light-scattering technology with a NanoSight NS300 instrument (Malvern Panalytical, Malvern, UK) and the nanoparticle tracking analysis (NTA) 3.4 software (Malvern Panalytical, Malvern). The instrument was equipped with a 488 nm blue laser module, flow-cell top plate, integrated temperature control, and a single-syringe pump module. The syringe pump module allowed for flow-controlled detection of moving particles. By tracking the Brownian motion of particles in the suspension, the software calculated the theoretical hydrodynamic diameter of single particles based on the Stokes-Einstein equation. By measuring the light-scattering intensity and the size of individual particles, NTA provided better resolution for heterogeneous mixtures of particles (
      • Gandham S.
      • Su X.
      • Wood J.
      • Nocera A.L.
      • Alli S.C.
      • Milane L.
      • Zimmerman A.
      • Amiji M.
      • Ivanov A.R.
      Technologies and Standardization in Research on Extracellular Vesicles.
      ). Samples were diluted using cell culture grade water (Corning, #25-005-CI) to produce a particle concentration in the range of 107 to 109 particles/ml (as determined during an initial quick static measurement), which represented approximately 20 to 100 particles/frame (particles in the instrument’s field of view). Final measurements consisted of 5 standard measurements of 1 minute of duration each at a controlled temperature of 25 °C and under constant automatic flow (continuous syringe pump speed set to 50 arbitrary units). Camera level for video capture was set to 13 and detection threshold to 5 for all sample measurements.

      Flow cytometry

      Aliquots of 20 μg of sEVs were attached to 10 μl of 4 μm aldehyde/sulfate latex beads (Invitrogen) as described previously (
      • Melo S.A.
      • Luecke L.B.
      • Kahlert C.
      • Fernandez A.F.
      • Gammon S.T.
      • Kaye J.
      • LeBleu V.S.
      • Mittendorf E.A.
      • Weitz J.
      • Rahbari N.
      • Reissfelder C.
      • Pilarsky C.
      • Fraga M.F.
      • Piwnica-Worms D.
      • Kalluri R.
      Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.
      ) with minor modifications. Briefly, to stop the reaction, sEVs-bound beads were treated with 100 mM glycine and 2% BSA in PBS and were blocked with 10% BSA and incubated with anti-CD63-APC antibody (BioLegend, #353007). Beads alone and beads + sEVs without antibody and beads+sEVs+isotype control (APC igG1, ƙ isotype Ctrl (FC) antibody (Biolegends, #400121) were used as controls to gate the beads with CD63-bound sEVs. The median fluorescence intensity of CD63 (∼100,000 events) was recorded in FACS Canto (BD Biosciences) and results were analyzed using the FlowJo software (BD Biosciences). Experiments were done in three replicates of each conditions.

      Imaging of sEVs by image stream

      Samples were run on an Amnis Image Stream X MKII (ISXII) (Luminex Corporation, Austin, TX) with dual charge-coupled device (CCD) camera system; multiple magnifications (20×, 40×, and 60× objectives); and 405 nm, 488 nm, 561 nm, 642 nm, and 785 nm lasers. The red laser (642 nm, 150 mW) was used to excite CD63-APC–positive sEVs (conjugated to aldehyde/sulfate latex beads) at the power settings of 90 mW, and channel 11 (702/85 nm filter) was used to collect the APC signals. Channels 1 and 9 were used as the brightfield (BF) channels, and channel 12 (762/35 nm filter) was used to detect the SSC from the 785 nm laser. All other lasers and channels were disabled during data acquisition. Samples were acquired at low speed and high sensitivity at 60x magnification (7 μm core size, image pixels are 0.33 μm2). For all samples, 10 to 20,000 events of CD63-APC+ beads were collected using a data acquisition template created in the INSPIRETM acquisition software integrated in ISXII. All data were saved as raw image files (RIFs) as well as FCS files and analyzed using a data analysis template created in IDEAS v6.2 software.

      sEV uptake

      sEVs were labeled with fluorescent lipid dye PKH26 (Sigma) according to the manufacturer’s recommendations with modifications. sEVs were incubated with 2 μM PKH-26, the reaction was stopped using 1% BSA, and extensive washing was done to remove residual lipid dye followed by ultracentrifugation to precipitate labeled sEVs (red). For uptake studies, we have chosen the incubation time and the concentrations of sEVs based on previous publications (
      • Christianson H.C.
      • Svensson K.J.
      • van Kuppevelt T.H.
      • Li J.P.
      • Belting M.
      Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.
      ,
      • Nakase I.
      • Kobayashi N.B.
      • Takatani-Nakase T.
      • Yoshida T.
      Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.
      ). Subconfluent HPNE, Panc1, and Miapaca2 cells were incubated with PKH-labeled sEVs (20 μg/mL) for 24 hrs. After incubation, cells were washed twice with 1 M NaCl and twice with PBS to remove cell surface-associated sEVs (
      • Christianson H.C.
      • Svensson K.J.
      • van Kuppevelt T.H.
      • Li J.P.
      • Belting M.
      Cancer cell exosomes depend on cell-surface heparan sulfate proteoglycans for their internalization and functional activity.
      ). Cells were fixed with 100% pre-chilled methanol, permeabilized with 0.2% Triton X-100 (Thermo Scientific), blocked with 10% goat serum (Invitrogen) in PBS, and incubated overnight at 4 °C with the anti-β tubulin antibody (Cell Signaling Technologies). On the next day, secondary antibody (Alexa Fluor 647 anti-rabbit IgG, Invitrogen) was added to the cells prior to counterstaining with 4’,6-diamidino-2-phenylindole (DAPI; Invitrogen) and mounting with antifade. Micrograph images were taken with a Leica SP8 AOBS laser scanning confocal microscope through a 63x/1.4NA Plan Apochromat Oil Immersion Objective Lens (Leica Microsystems CMS GmbH, Germany). To excite the samples, 405, 488, and 552 nm laser lines were applied, and tunable emissions were used to minimize crosstalk between fluorochromes. Images were captured with photomultiplier detectors and LAS AF software version 2.6 (Leica Microsystems, Germany).
      For live cell imaging, HPSC sEVs were digested with proteinase K (Sigma) at the concentration of 40 mAU/mg protein in 37°C water bath for 1 hour followed by heat inactivation of proteinase K in a 60°C water bath for 20 minutes. Next, sEVs were labeled with PKH-26 lipid dye as described above. Normal HPNE, Panc1, and Miapaca2 cells were treated with PKH-26 dye only, 20 μg/ml PKH26-labeled HPSC sEVs, or 100 ng of proteinase K–digested PKH26-labeled HPSC sEVs for 6 hrs. Live imaging was performed on EVOS auto microscope (Leica) at magnification 10x.

      Protein lysis, digestion, and label‐free proteomics—sample preparation

      Using equal amounts of protein (10 μg), sEVs were lysed in denaturing buffer containing 8 M urea, 20 mM HEPES (pH 8), 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, and 1 mM β-glycerophosphate. Protease inhibitors were not added because of the use of 8 M urea and to avoid trypsin inhibition during proteolysis. Phosphatase inhibitors were used for future phosphor proteomics experiments. A Bradford assay was carried out to determine the protein concentration. The proteins were reduced with 4.5 mM dithiothreitol (DTT) and alkylated with 10 mM iodoacetamide. Trypsin digestion was carried out at room temperature overnight, and tryptic peptides were then acidified with 1% trifluoroacetic acid (TFA) and desalted with C18 MicroSpin Columns (The Nest Group, Inc. Southborough, MA) according to the manufacturer’s procedure.

      sEV LC-MS/MS

      A nanoflow ultra-high-performance liquid chromatograph (RSLC, Dionex, Sunnyvale, CA) interfaced with an electrospray benchtop quadrupole-orbitrap mass spectrometer (Q Exactive HF-X, Thermo Fisher Scientific, San Jose, CA) was used for tandem mass spectrometry peptide sequencing experiments. The sample was first loaded onto a precolumn (100 μm ID × 2 cm in length packed with C18 reversed-phase resin, 5 μm particle size, 100 Å pore size) and washed for 8 minutes with aqueous 2% acetonitrile and 0.1% formic acid. The trapped peptides were eluted onto the analytical column (C18, 75 μm ID × 50 cm in length, 2 μm particle size, 100 Å pore size, Dionex, Sunnyvale, CA). The 120-minute gradient was programmed as: 95% solvent A (aqueous 2% acetonitrile + 0.1% formic acid) for 8 minutes, solvent B (aqueous 90% acetonitrile + 0.1% formic acid) from 5% to 38.5% in 90 minutes, then solvent B from 50% to 90% B in 7 minutes and held at 90% for 5 minutes, followed by solvent B from 90% to 5% in 1 minute and re-equilibration for 10 minutes. The flow rate on the analytical column was 300 nl/minute. MS resolution was set at 60 000, and MS/MS resolution was set at 15,000 with maximum ion accumulation of 50 ms.

      Data analysis with statistical rationale

      MaxQuant (
      • Cox J.
      • Mann M.
      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
      ) (version 1.6.2.10) was used for protein identification and quantification with the Uniprot database (downloaded March 2020; 20304 total protein entries). Description of the parameters are provided in Supplementary Table S1 and S3. The protease was trypsin, precursor tolerance was 4.5 ppm, and fragment tolerance was 20 ppm. A minimum of one peptide was required for protein identification. At least 7 amino acids per peptide were required, and as many as 2 missed cleavages were allowed. A false discovery rate of 0.01 was used for both peptides and proteins. The match between runs option was selected using a time window of 4 minutes. N-terminal acetylation and methionine oxidation were both modifications allowed in protein quantification. Membrane annotation was added using Uniprot’s ID mapping tool (https://www.uniprot.org/id-mapping; accessed 2022-06-28) by inputting the first entries of the aforementioned protein groups and then toggling the “Intramembrane” and “Transmembrane” columns in the results.
      We used the R programming language (

      R Core Team (2020) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria

      ) (version 4.0.2) with RStudio (

      RStudio Team (2020) RStudio: Integrated Development for R. RStudio, PBC, Boston, MA, USA

      ) (version 1.3.1093) to analyze the MaxQuant proteinGroups.txt output containing 1,597 rows of protein groups (see supplementary Table S1, maxquant_params). Filtering out the protein groups that consisted of entirely of reverse entries, entirely non-human contaminant entries, or that were identified but not quantified resulted in 1,481 protein groups for further analysis. Sample intensities were normalized using iterative rank order normalization (IRON) (
      • Welsh E.A.
      • Eschrich S.A.
      • Berglund A.E.
      • Fenstermacher D.A.
      Iterative rank-order normalization of gene expression microarray data.
      ). Intensities were log2-transformed and missing values were left as NA. We performed Welch’s t tests to test for differences in the relative expression of protein groups between HPSC and HPaStec samples. T test values were adjusted using the Benjamini-Hochberg procedure (
      • Benjamini Y.
      • Hochberg Y.
      Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing.
      ). We defined differentially expressed protein groups as those ≥ 2-fold differences (or ±1 log2 ratio of HPSC to HPaStec) with an adjusted P value ≤ 0.05. Ratios were calculated for HPSC over HPaStec, so positive values indicate higher expression in HPSC whereas negative values indicated lower expression. Pathway enrichment was performed using the Enrichr (
      • Kuleshov M.V.
      • Jones M.R.
      • Rouillard A.D.
      • Fernandez N.F.
      • Duan Q.
      • Wang Z.
      • Koplev S.
      • Jenkins S.L.
      • Jagodnik K.M.
      • Lachmann A.
      • McDermott M.G.
      • Monteiro C.D.
      • Gundersen G.W.
      • Ma'ayan A.
      Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.
      ) R package. For protein groups comprised of more than one protein, the first or parent protein of the group was used for enrichment analysis and other comparisons unless noted otherwise. ExoCarta (
      • Keerthikumar S.
      • Chisanga D.
      • Ariyaratne D.
      • Al Saffar H.
      • Anand S.
      • Zhao K.
      • Samuel M.
      • Pathan M.
      • Jois M.
      • Chilamkurti N.
      • Gangoda L.
      • Mathivanan S.
      ExoCarta: A Web-Based Compendium of Exosomal Cargo.
      ) Protein/mRNA data (ExoCarta Download - 5 [release date: July 29, 2015]) were downloaded (http://www.exocarta.org; accessed 2020/09/02). The web-based tool (
      • Anaya J.
      OncoLnc: linking TCGA survival data to mRNAs, miRNAs, and lncRNAs.
      ) Oncolnc (http://www.oncolnc.org/; accessed 2020/10/28), which provides an interface for multivariate Cox regression, was used to perform a survival analysis on 174 pancreatic adenocarcinomas from The Cancer Genome Data Atlas. Patients were stratified into low and high groups based on median CSE1L expression (
      • Anaya J.
      OncoLnc: linking TCGA survival data to mRNAs, miRNAs, and lncRNAs.
      ). Significantly different proteins were queried with the STRING protein-protein interaction database (
      • Szklarczyk D.
      • Gable A.L.
      • Nastou K.C.
      • Lyon D.
      • Kirsch R.
      • Pyysalo S.
      • Doncheva N.T.
      • Legeay M.
      • Fang T.
      • Bork P.
      • Jensen L.J.
      • von Mering C.
      The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets.
      ). Homo sapiens was selected as the organism, and the minimum required interaction score was set to 0.400. The network was exported as a tab separated variable file and imported into Cytoscape version 3.8.2 (
      • Shannon P.
      • Markiel A.
      • Ozier O.
      • Baliga N.S.
      • Wang J.T.
      • Ramage D.
      • Amin N.
      • Schwikowski B.
      • Ideker T.
      Cytoscape: a software environment for integrated models of biomolecular interaction networks.
      ). Circular layout and degree sorted circular layout options were used to organize the nodes and edges.

      FBS LC-MS/MS

      Using a Bradford assay (Pierce™ Coomassie Plus Bradford Assay 23236), approximately 10 μg of protein from FBS was aliquoted and diluted with 25 mM of ammonium bicarbonate. The sample was subjected to reduction and alkylation of cysteine residues using DTT and iodoacetamide, followed by trypsin at a 1:20 enzyme:protein ratio. After overnight digestion, the sample was acidified with 20% Trifluoroacetic acid (TFA) and peptide clean-up was performed using a ZipTip (ZTC18S096 Millipore). Eluted peptides were dried and re-suspended in 2% acetonitrile/0.1% formic acid and analyzed by LC-MS/MS in Q-Exactive plus (Thermo Scientific). Data analysis was performed using MaxQuant version 1.6.14 with the Uniprot human database (downloaded March, 2021).

      Fluorescence microscopy and Western blot to detect α-smooth muscle actin

      Fluorescence microscopy

      HPaStec cells (1×104) were seeded in 8-well chamber slides (Lab Tek II system, Thermo Fisher Scientific), and the next day were left untreated or were treated with HPSC or HPaStec sEVs (20 μg/ml) for 72 hrs. Cells were fixed with 100% pre-chilled methanol, permeabilized with 0.2% Triton X-100, blocked with 10% goat serum (Invitrogen) in PBS, and incubated overnight at 4 °C with the anti-SMA antibody (Thermo Scientific,). Cells were incubated with secondary antibody (Alexa Fluor 647 anti-rabbit IgG, Invitrogen) and were counterstained and mounted with antifade containing DAPI (Invitrogen). Micrograph images were acquired as described above in the sEVs uptake section. Three independent experiments have been done and the immunofluorescence signal of αSMA in control and treated wells have been quantified using image J software. Western blot: For an additional validation of αSMA expressions, HPaStec cells were left untreated or were treated with HPSC or HPaStec sEVs (20 μg/ml) for 72 hrs. After 72hrs. cells were harvested, lysed and Western blot was carried out to determine αSMA expression as described above in the Western blot section.

      Cell viability assays using sEVs

      The dose dependent response of sEVs was evaluated using the CellTiter-Glo assay (Promega). Briefly, either HPNE, Panc1 or Miapaca2 cells (4,000 cells/well) were seeded into 96-well plates overnight. The next day, vehicle control (PBS) and increasing doses of sEVs were added to the cells and incubated for 24 and 72 hrs. For coculture experiments, cancer cells (1,000 cells) and stellate cells (1,000 cells) were seeded at 1:1 ratio, and the next day sEVs were added to the cells for 24 hrs. Cells were incubated at 37°C with 5% CO2 prior to analysis with CellTiter-Glo chemiluminescent reagent (Promega). Data was recorded using Flexstation 3 plate reader (Molecular Devices), and cell viability was normalized to vehicle-treated wells and fit to a sigmoidal dose-response curve using GraphPad Prism 6. All the experiments were performed in triplicate. The data was statistically analyzed using one way ANOVA.

      Cell viability assays using conditioned media (CM) and gemcitabine

      Five million cells (HPSC and HPaStec) were seeded in T150 cm2 (corning) flasks in a respective media without serum and after 48hrs. conditioned media were collected, centrifuged to remove the cellular debris and concentrated using Amicon ultra centrifugal filtration unit of 100KD cut off (Millipore) (
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      ). Protein contents of the concentrated HPSC and HPaStec conditioned media (CM) were determined by Bradford protein assay (Nanodrop) and aliquots were stored at -80°C until use. Panc1 and Miapaca2 cell lines were seeded in 96-well plates at a density of ∼4000 cells/well were treated for 48hr to determine the proliferation of Panc1 and Miapaca2 cells in presence of HPaStec and HPSC CM. Next, for cytotoxicity assay, the cells were treated with gemcitabine (concentration ranging from 0.01–100μM) alone or in combination with 1μg/ml HPaStec or HPSC CM (
      • Amrutkar M.
      • Vethe N.T.
      • Verbeke C.S.
      • Aasrum M.
      • Finstadsveen A.V.
      • Santha P.
      • Gladhaug I.P.
      Differential Gemcitabine Sensitivity in Primary Human Pancreatic Cancer Cells and Paired Stellate Cells Is Driven by Heterogenous Drug Uptake and Processing.
      ). To determine gemcitabine-induced cytotoxicity, cell viability in response to varying concentrations of gemcitabine and the corresponding EC50 values were determined using the CellTiter-Glo chemiluminescent assay (Promega). Data analysis was done as described above.

      Live cell imaging

      The Incucyte (Sartorius Corporation, NY) is a system of exploring in vitro biological changes in real-time, automated live-cell imaging and analysis inside the incubator. 1,000 Panc1 or Miapaca2 cells were seeded in 96-well plates and the next day, the cells were treated with either vehicle control (PBS) or indicated amount of either HPaStec or HPSC-sEVs. Automated live-cell imaging was done for 72 hrs. to explore in vitro biological changes in real-time, using Incucyte S3 Live Imager with 10x magnification in-phase channel. Triplicate images were acquired and analyzed from each well.

      Apoptosis and caspase 3 and 7 assays

      Cells were incubated with HPSC sEVs at 40 μg/ml and 80 μg/ml at various time points following cell lysis and Western blot as described above to probe for apoptosis markers, cleaved PARP and cleaved caspase 3 (antibodies’ information described in the reagent section). Next, for caspase 3 and 7 assays, HPSC sEVs were incubated with cancer cells at 40 μg/ml and 80 μg/ml for 24 hrs. followed by immunofluorescence to detect the caspase 3 and 7 in the cancer cells using Image-iTTM Live Red caspase 3 and 7 detection kit (Invitrogen) following the manufacturer’s instructions. Images were captured at 63x magnification using a Leica SP8 AOBS laser scanning confocal microscope through a 63x/1.4NA Plan Apochromat Oil Immersion Objective Lens (Leica Microsystems CMS GmbH, Germany).

      Scratch assay

      The cancer cells were seeded with HPaStec cells in 1:1 ratio into 96-well tissue culture dishes overnight at 37C, at a concentration of 2.5×104 cells/ml each and cultured in respective medium containing 10% FBS to confluent cell monolayers. The next day, a linear wound was generated in the monolayer with a commercially available tool called wound maker (Essen Bioscience, MI). Cellular debris was removed by washing the cells two times with phosphate buffer saline (PBS). HPSC sEVs were added to the cells at a concentration of 40 μg/ml and time lapse imaging was performed by scratch wound assay analysis module in an Incucyte S3 Live imager (Sartorius Corporation, NY) with 4X magnification in-phase channel. Random images were chosen for measurement at 0 hrs. up to 24 hrs. Experiments were done in quadruplicates and wound confluency and width were plotted in GraphPad Prism.

      RESULTS

      Isolation and physical characterization of sEVs secreted from stellate cells

      HPSCs are like hepatic stellate cells, which are important effector cells in hepatic fibrosis, and stain positive for vimentin, desmin, and α-SMA. Our previous publications described the detailed characterization of immortalized HPSCs isolated from tissues adjacent to PDAC tumors (
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      ,
      • Li X.
      • Lee Y.
      • Kang Y.a.
      • Dai B.
      • Perez M.R.
      • Pratt M.
      • Koay E.J.
      • Kim M.
      • Brekken R.A.
      • Fleming J.B.
      Hypoxia-induced autophagy of stellate cells inhibits expression and secretion of lumican into microenvironment of pancreatic ductal adenocarcinoma.
      ,
      • Kang Y.
      • Roife D.
      • Lee Y.
      • Lv H.
      • Suzuki R.
      • Ling J.
      • Rios Perez M.V.
      • Li X.
      • Dai B.
      • Pratt M.
      • Truty M.J.
      • Chatterjee D.
      • Wang H.
      • Thomas R.M.
      • Wang Y.
      • Koay E.J.
      • Chiao P.J.
      • Katz M.H.
      • Fleming J.B.
      Transforming Growth Factor-beta Limits Secretion of Lumican by Activated Stellate Cells within Primary Pancreatic Adenocarcinoma Tumors.
      ,
      • Li X.
      • Kang Y.
      • Roife D.
      • Lee Y.
      • Pratt M.
      • Perez M.R.
      • Dai B.
      • Koay E.J.
      • Fleming J.B.
      Prolonged exposure to extracellular lumican restrains pancreatic adenocarcinoma growth.
      ,
      • Li X.
      • Roife D.
      • Kang Y.
      • Dai B.
      • Pratt M.
      • Fleming J.B.
      Extracellular lumican augments cytotoxicity of chemotherapy in pancreatic ductal adenocarcinoma cells via autophagy inhibition.
      ). Primary HPaStecs were characterized for the presence of fibroblast marker, α-SMA, as described by the vendor (ScienCell Research Laboratories, CA). In our current study we have evaluated the morphology and growth rate of both cell lines at 24 and 48 hrs. by seeding equal number of cells. (supplementary Fig. S1 a,b,c). There is no significant difference (HPSC vs. HPaStec, unpaired t test, p=0.80 and 0.77 at 24 and 48hrs.) in growth rate between HPSC (1.33X10ˆ7 at 48hrs.) and HPaStec (1.13X10ˆ7 at 48hrs.) cells as determined by the cell number (supplementary Fig. S1a). For sEV isolation, an equal number of cells were cultured in the respective media using commercially available exosome-free FBS (Invitrogen) until 80% confluency. sEVs were isolated from HPSC and HPaStec-conditioned media using differential ultracentrifugation steps as described previously (
      • Thery C.
      • Amigorena S.
      • Raposo G.
      • Clayton A.
      Isolation and characterization of exosomes from cell culture supernatants and biological fluids.
      ) with minor modifications (Figure 1a). Protein content of the sEVs were then quantified. To analyze the shape and size distributions, 10 μg of sEVs were fixed with glutaraldehyde and stained with uranyl acetate and imaged using transmission electron microscopy (TEM). Representative electron micrographs of the clear, round, or ellipsoidal-shaped particles of HPSC and HPaStec sEVs, mostly with a diameter of 50 nm to 130 nm, were visible and were captured using TEM as shown in Figure 1b (magnifications ranging from 200,000×-400,000×, with a scale bar of 100 or 50 nm, respectively). Although most of the sEVs in both categories fell in the size range of 50 nm to 200 nm, mostly smaller sized HPSC sEVs were captured in the electron micrographs compared to the HPaStec sEVs (Figure 1b).
      Figure thumbnail gr1
      Fig. 1Isolation and physical characterization of sEVs. (a) Flow chart of sEVs Isolation and characterization procedure from stellate cells. sEVs were isolated from the conditioned media of HPSC and HPaStec cells by differential ultracentrifugation and further characterization was done. (b) Electron microscopic characterization of exosomes secreted from stellate cells. 10 μg of the sEVs (HPSC and HPastec) were fixed with 4% paraformaldehyde and 1% glutaraldehyde in PBS buffer overnight for imaging in the transmission electron microscope (TEM) using a previously described procedure with minor modification as described in the methods section. Representative electrographs are captured using TEM of HPSC and HPastec sEVs as shown. Red arrows are pointing towards the different sizes of sEVs. Image Magnifications and scale bars are displayed in the representative images. (c) NanoSight particle analysis of sEVs show the particle sizes of sEVs in culture supernatants isolated from HPSC and HPaStec cells. The horizontal axis represents the particle size (nm), whereas the vertical axis represents the particle concentration (×108 particles/mL). The red bar represents the standard error of the mean.
      Next, to confirm the size distribution and determine the concentration of sEVs released from both cell types, we conducted nanoparticle tracking analysis (NTA) on properly diluted sEV samples (as described in Experimental Procedures) using the NanoSight NS300 (Malvern Panalytical). We have analyzed multiple independent preparations of sEVs using Nanosight NS300 as shown in Table 1. Mean particle diameters of HPSC (six independent preparations) and HPaStec sEVs (five independent preparations) were ranging between 182.2-220.9 nm and 154.4-191 nm (Table 1, Figure 1c) respectively (HPaStec vs HPSC, p=0.02). See Supplementary Video S1 for additional information. Interestingly, NanoSight quantitation revealed that HPSC cells secrete more sEVs (mean conc.4.75×1010 particles/ml) compared to HPaStec cells (mean conc.1.35×1010 particles/ml) from the equal number of cells seeded in an equal volume of conditioned media which is marginally significant (p=0.0569) (Table 1).
      Table 1Sizes and concentrations of sEVs determined by NanoSight
      SampleGroupTotal ConcentrationMean ConcentrationMeanModeSDD10D50D90
      HPaSteC-Prep 1HPaSteC6.65E+091.35E+10184.8125.782.5111.6154.4294.9
      HPaSteC-Prep 2HPaSteC2.02E+10154.4111.975103.4130.8224.3
      HPaSteC-Prep 3HPaSteC1.15E+10191126.9111.7116.1151.1319.3
      HPaSteC-Prep 4HPaSteC2.19E+10189.1122.699.3111.9148.1335.2
      HPaSteC-Prep 5HPaSteC7.53E+09161.8113.272105.8141.6242.8
      HPSC-Prep 1HPSC7.77E+094.75E+10182.2123.299.5104.9145.6319.3
      HPSC-Prep 2HPSC4.63E+10212.2117.6131.5111.9162.1403.1
      HPSC-Prep 3HPSC1.06E+10198.7134.7126.7112.8153.4366.7
      HPSC-Prep 4HPSC8.7E+10210.2138.6111.3127172.9357
      HPSC-Prep 5HPSC5.09E+10191.9114.193.7107.8154.7342.9
      HPSC-Prep 6HPSC8.25E+10220.9119.8141.1111155.9437.7
      HPaSteC vs HPSC T-test0.05690.020
      indicates differentially expressed between HPSC and HPaStec.
      0.4050.025
      indicates differentially expressed between HPSC and HPaStec.
      0.5030.0570.011
      indicates differentially expressed between HPSC and HPaStec.
      SD: standard deviation of mean particle size. D10: 10 percentile diameter. 10% of the particles in the sample are smaller than the D10 diameter. D50: 50 percentile diameter. 50% of the particles in the sample are smaller than the D50 diameter. D90: 90 percentile diameter. 90% of the particles in the sample are smaller than the D90 diameter.
      indicates differentially expressed between HPSC and HPaStec.
      To further characterize differences in these sEVs, Western blot analyses were performed to detect expression of established exosomal surface marker proteins (Alix, CD63, TSG101, EGFR and CSE1L) and any cytoskeletal (Actin) or endoplasmic reticulum (Calnexin) contaminations in the sEVs. These surface markers were present in sEVs, and the extracts were free of cytoskeletal/endoplasmic reticulum contamination (Figure 2a). We have quantified the Western blot analyses using ImageJ bundled with Java 1.8.0_172 software (Figure 2b). HPSC sEVs were significantly more enriched with most of the exosomal markers compared to HPaStec sEVs (Figure 2b). Next, flow cytometry analyses were performed in HPaStec (Figure 2c) and HPSC(Figure 2d)- sEV–bound beads to analyze the expression of CD63, one of the tetraspanin exosomal markers (
      • Datta A.
      • Kim H.
      • McGee L.
      • Johnson A.E.
      • Talwar S.
      • Marugan J.
      • Southall N.
      • Hu X.
      • Lal M.
      • Mondal D.
      • Ferrer M.
      • Abdel-Mageed A.B.
      High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer.
      ,
      • Kaur S.
      • Elkahloun A.G.
      • Arakelyan A.
      • Young L.
      • Myers T.G.
      • Otaizo-Carrasquero F.
      • Wu W.
      • Margolis L.
      • Roberts D.D.
      CD63, MHC class 1, and CD47 identify subsets of extracellular vesicles containing distinct populations of noncoding RNAs.
      ). The representative histogram with proper isotype control in Figure 2c,d and mean bar graph in Figure 2e (three independent experiments) showed that the median fluorescence intensity of CD63 was significantly 2-fold higher (P<0.008) in HPSC than HPaStec sEVs. ImageStream analysis revealed that staining of APC-CD63 bound HPSC sEVs was brighter compared to the HPaStec sEVs (Supplementary Fig. S2a-d).
      Figure thumbnail gr2
      Fig. 2Characterization and quantification of sEV and exosomal markers in sEVs secreted from stellate cells. (a) Western blot analysis was performed on HPSC and HPaStec sEVs with the parental cells to examine exosomal markers (Alix, TSG101, CD63, EGFR and CSE1L) expression and any endoplasmic reticulum (Calnexin) contamination in sEVs using 20 μg of total protein loaded in each lane. (b) Protein expression intensities (a.u.) were compared between HPaStec sEVs and HPSC sEVs using image J software and the fold increase were indicated on the graph (HPSC sEVs vs. HPaStec sEVs, CSE1: 2.78; TSG101:0.67; Alix: 4.37; EGFR: 0.65; CSE1L: 3.50). (c,d) Histogram of median fluorescence intensity of CD63-APC (Y-axis) (blue) of HPaStec (c) and HPSC (d) sEVs on beads of a representative experiment. Aliquots (20 μg) of each type of sEVs were incubated with aldehyde latex beads and incubated with Beads alone (green) and beads + sEVs (red) without antibody and beads+sEVs+isotype control (orange) (APC igG1, ƙ isotype Ctrl (FC) antibody were used as controls to gate the beads with CD63-bound sEVs and with CD63-APC antibody as described in methods section and analyzed in BD FACS Canto (BD Biosciences). (e) GraphPad analysis showed the difference in median fluorescence intensity of CD63 between HPSC SEVs and HPaStec sEVs of three independent experiments by one-way Anova (*p<0.008).

      Coculturing of sEVs with normal HPaStec cells modulates α-SMA expression

      To explore the activation of normal stellate cells in presence of HPSC and HPaStec sEVs, both sEVs were co-cultured with normal HPaStecs at a concentration of 20 μg/ml for 72 hrs. followed by α-SMA staining (activation marker for stellate cells). Interestingly, based on our data, by sEVs treatment, both types of EVs reduced the α-SMA expression significantly (p<0.03) relative to untreated control as detected by immunofluorescence and Western blot (Figure 3a,b,c).
      Figure thumbnail gr3
      Fig. 3Effect of sEVs on normal stellate cells and uptake of PKH-26 labeled sEVs by normal epithelial and cancer cells: (a) HPaStec cells were left untreated or treated with either HPSC or HPaStec sEVs (20μg/ml) for 72 hrs. Cells were fixed and stained with anti-α-SMA antibody (red) and DAPI (blue, DNA dye) as described in the Experimental Procedures. Representative 63X images (Scale bar = 50 μm) using confocal microscopy showed significantly decreased staining of α-SMA in HPaStec cells treated with both HPastec sEVs and HPSC sEVs. (b) Representative immunofluorescence intensities (α-SMA) of control and treated wells were quantified using image J software and statistical analysis was done using one way ANOVA (p<0.03). (c) HPaStec cells were treated with 20 μg/ml HPSC-sEVs or HPaStec sEVs for 72 hrs. and Western blot analysis was performed using anti-α-SMA antibody and actin is used as a loading control. Percentage signal intensities of αSMA were normalized against actin as indicated using image J software. (d,h) Immunofluorescence of fixed cells to demonstrate the uptake of PKH-26 labelled HPSC/HPaStec sEVs. Normal HPNE, Panc1 and Miapaca2 cells were treated either with PKH-26 dye (red) only or with 20 μg/ml PKH26-labelled HPSC sEVs for 24 hrs. Following incubation with anti-tubulin antibody to stain the cytoplasmic membrane (green), imaging was done using confocal microscopy (63X Magnification, scale bar=25 μm for HPSC sEVs and scale bar=10 μm for HPastec sEVs). (e) The images at 1890X magnification visualize the cellular localization of PKH-26 labelled HPSC sEVs in HPNE, Panc1 and Miapaca2 cells. White arrows show the localization of sEVs to the cell membranes. (f.g.i) Uptake of both PKH-26 labelled sEVs by cancer cells were quantified using image J software and statistical analysis was done using unpaired t test (HPaStec sEVs uptake in Panc1 cells, p<0.02). (j) Live cell imaging to demonstrate preferential sEV uptake. Normal HPNE, Panc1 and Miapaca2 cells were treated either with PKH-26 dye (red) only or with 20 μg/ml PKH26-labelled HPSC sEVs or with proteinase K digested 100 ng (A260/A280 = 1.84) PKH26-labelled HPSC sEVs for 6 hrs. sEV delivery to normal and cancer cells was higher when undigested sEVs were added to the cells. Live imaging was performed on EVOS auto microscope (Leica) at 10X magnification (scale bar=400μm).

      Uptake of sEVs by normal epithelial cells and PDAC cell lines

      Next, to study the uptake of both HPSC and HPaStec sEVs by normal and cancerous cell lines, we labeled the sEVs with a red fluorescent dye (PKH-26) that has long aliphatic tails that are incorporated into the lipid membrane. Next, 20 μg/ml of PKH-26 labeled sEVs were incubated with normal epithelial cells, HPNE, and PDAC cell lines, Miapaca2 and Panc1 for 24 hrs. The cells were fixed with methanol and to demonstrate the localization of sEVs, cells were stained with β-tubulin, which represents the cytoskeletal (green) structure of the individual cells and DAPI (blue) counter stained the nucleus. Immunofluorescence imaging using a confocal microscope determined the overall uptake of PKH-26–labeled HPSC sEVs (red) in normal and pancreatic cancer cells (Figure 3d,3e) and uptake of HPaStec sEVs in panc1 and Miapaca2 cells (Figure 3h). Higher magnifications of the images revealed that most of the sEVs were accumulated on the cellular membranes of cells (Figure 3e,3h). There are no significant differences in HPSC sEVs uptake between the cell lines (Figure 3f,3g). HPaStec sEVs uptake by Panc1 cells are significantly higher than Miapaca2 cells. (Figure 3i).

      Membrane proteins on the sEVs may be essential for better uptake of sEVs in normal and cancer cell lines

      To determine whether HPSC-derived sEVs enriched with membrane proteins are essential for better uptake of sEVs by normal or cancerous cells, undigested PKH-27–labeled HPSC sEVs (20 μg/ml) or 100 ng of proteinase K–digested PKH-26–labeled HPSC sEVs were treated to HPNE, Panc1, and Miapaca2 cells for 6 hrs. Uptake of sEVs by the live cells was visualized via live imaging with the EVOS FL Auto Microscope (Thermo Scientific) (Figure 3j). Digestion of exosomal surface proteins with proteinase K significantly reduced the entry of sEVs into HPNE, PANC1, and Miapaca2 cells (Figure 3j) compared to the higher levels of uptake of undigested sEVs by normal (HPNE) and cancer cells (Panc1 and Miapaca2).

      sEVs have no significant effect on cellular proliferation of cancer cells

      Next, to evaluate the effects of HPSC sEVs and HPaStec sEVs on the proliferation of normal epithelial cells and pancreatic cancer cells, HPNE and Panc1 or Miapaca2 cells were co-incubated with indicated amount of either HPSC sEVs or HPaStec sEVs for 24 and 72 hrs. We could not find any significant effects of either sEVs on proliferation of normal epithelial or cancer cells, as shown in Supplementary Fig S3, a,b,c,d and e. Next, to get better insight on the effect of stellate cells and sEVs on the proliferation of cancer cells in the presence of normal stellate cells, primary HPaStec and PDAC-associated HPSC cells were cocultured with either Panc1 or Miapaca2 cells for 24 hrs. either in the presence or absence of HPSC/HPaStec sEVs. Interestingly, there were no significant changes in cellular proliferation observed upon addition of HPSC or HPaStec sEVs to the coculture system (Supplementary Fig S3, f,g). We also demonstrated that there were no changes in cellular morphology of both primary stellate cells (HPaStec) and Panc1 cells in presence or absence of HPSC sEVs (Supplementary Fig. S3, h).
      We further examined the cellular apoptosis and migration of Panc1 and Miapaca2 cells in the presence of HPSC sEVs. Interestingly, HPSC sEVs did not significantly induce any c-Caspase 3/7 or c-PARP to the cancer cells as shown by Western blots (Supplementary Fig S4, a,b) and immunofluorescence (Supplementary Fig S4, c,d). Cellular migration of Panc1 and HPaStec cells in presence of HPSC sEVs also remained unchanged (Supplementary Fig S4, e-g).
      Next, to examine whether soluble part of the conditioned media but not EV most likely has an effect on cancer cells, HPSC/HPaStec concentrated conditioned media were cocultured with Panc1 and Miapaca2 cells with indicated time as described previously (
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      ) (Supplementary Fig S5, a,b). Coculture of Panc1 and Miapaca2 cells with HPSC stellate cells CM resulted in significant increase (P<0.0001) of the growth of Panc1 cells only (Supplementary Fig S5,a). Effects of either CMs in Miapaca2 cells remained unchanged (Supplementary Fig S5,b). According to the previous report pancreatic stellate cells are capable of provoking chemoresistance of tumor cells (
      • McCarroll J.A.
      • Naim S.
      • Sharbeen G.
      • Russia N.
      • Lee J.
      • Kavallaris M.
      • Goldstein D.
      • Phillips P.A.
      Role of pancreatic stellate cells in chemoresistance in pancreatic cancer.
      ). Therefore, we treated the cancer cells with chemotherapeutic drug gemcitabine, the standard of care for the pancreatic cancer (
      • Park W.
      • Chawla A.
      • O'Reilly E.M.
      Pancreatic Cancer: A Review.
      ), alone or in combination with HPSC/HPaStec CMs. However, according to our findings HPSC /HPaStec CMs are unable to induce significant chemoresistance (no significant changes in the EC50 values. (Supplementary Fig S5 c,d).

      Proteomics identified differences between normal and cancer-associated human pancreatic stellate cell sEVs

      PDAC-associated HPSC cells secrete more exosomal sEVs than HPaStec and intact membrane protein containing sEVs are better up taken by cancer cells. Therefore, to test whether the membrane protein expression levels were different in sEVs of HPSC and HPaStec cells, both the cells were grown in biological triplicates. sEVs were harvested and analyzed using LC-MS/MS proteomics. After processing with MaxQuant for quantification (
      • Cox J.
      • Mann M.
      MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.
      ) and quality control, we identified 1,481 protein groups for further analysis. Of these 1,481 protein groups, 355 proteins had transmembrane annotation and 9 had intramembrane annotation in Uniprot (Supplementary Table S2). We compared our findings to the supplemental data available that have also compared vesicles from tumor-associated and normal stellate cells and in total, we have 76% protein identification overlap with findings from Servage et al (Supplementary Table S2 and S3, Supplemental Fig. S6) (
      • Servage K.A.
      • Stefanius K.
      • Gray H.F.
      • Orth K.
      Proteomic Profiling of Small Extracellular Vesicles Secreted by Human Pancreatic Cancer Cells Implicated in Cellular Transformation.
      ). Data were log2 transformed and normalized using IRON (
      • Welsh E.A.
      • Eschrich S.A.
      • Berglund A.E.
      • Fenstermacher D.A.
      Iterative rank-order normalization of gene expression microarray data.
      ) (Supplementary Fig. S7, a,b). Principal component analysis showed that HPSC and HPaStec sEVs separated in principal component 1 (Supplementary Fig. S7, c). Consistent with exosome enrichment, 1,239 protein groups were listed in the ExoCarta database (
      • Keerthikumar S.
      • Chisanga D.
      • Ariyaratne D.
      • Al Saffar H.
      • Anand S.
      • Zhao K.
      • Samuel M.
      • Pathan M.
      • Jois M.
      • Chilamkurti N.
      • Gangoda L.
      • Mathivanan S.
      ExoCarta: A Web-Based Compendium of Exosomal Cargo.
      ) and 1,088 ExoCarta proteins were identified in exosomes from both cell types (Figure 4a and Supplementary Table S3). Sixty or 91 ExoCarta proteins were exclusively identified in HPSC or HPaStec sEVs, respectively (Figure 4a).
      Figure thumbnail gr4
      Fig. 4Venn diagrams and Volcano plot of differential protein expression levels of HPSC and HPastec sEVs: (a) Venn diagram of exosomal proteins found in Exocarta. Overlap of identified protein groups by sample type and ExoCarta proteins. A large percentage of identified protein groups was also found in ExoCarta, which was consistent with sEV enrichment. (b) Significant proteins and overlap of the differentially expressed protein groups and ExoCarta. (c) Volcano plot of the 1,481 proteins from HPSC and HPastec sEVs. The 50 protein groups with at least two-fold higher expression (or 1 log2 ratio) with an adjusted p-value ≤ 0.05 are colored red and 37 protein groups with two-fold lower expression (or -1 log2 ratio) with adjusted p-value ≤ 0.05 are colored green. Ratios are given relative to HPSC. CSE1L was noticeably higher (log2 ratio 11.25) in HPSC sEVs.
      During our sEVs isolation we did not use serum-free media, so we washed the pellets with PBS after sEVs precipitation. Since there is a minor risk of FBS contamination, we independently ran an FBS aliquot through LC-MS/MS, searched with the human Uniprot database, and intersected with our results. Overlap with our findings and FBS was small (2% of identifications or 30 proteins), as seen in Supplementalary Table S3 and Supplementary Fig. S8.
      We performed Welch’s t tests and adjusted the P values using Benjamini-Hochberg (
      • Benjamini Y.
      • Hochberg Y.
      Controlling the False Discovery Rate - a Practical and Powerful Approach to Multiple Testing.
      ) methodology (Supplementary Fig. S7) to test for differences in protein content between HPSC and HPaStec sEVs. We identified 87 protein groups that were differentially expressed (±1 log2 ratio of HPSC to HPaStec and adjusted P value ≤ 0.05; Figure 4b and volcano plot in Figure 4c, Supplementary Table S3). Among the top 10 proteins elevated in HPSC sEVs (Table 2, Supplementary Table S3), the chromosome segregation 1–like protein (CSE1L), also known as cellular apoptosis susceptibility protein (CAS) and Exportin-2 (XPO2), was dramatically higher (log2 ratio = 11.25, Padj = 0.009). Other notable results included that HPSC sEVs were enriched with several DNA binding histones (Table 2, Supplementary Table S3), such as histones H2A type 1, core histone macro-H2A.1, H2A.V, H2B type 1, H1.5, and histone H4. Other than histones, chromatin regulatory barrier to auto integrin factors (BANF1) and BRO1 domain-containing protein (BROX) were highly enriched in HPSC sEVs (Table 2, Supplementary Table S3). There were significantly lower amounts of an aminopeptidase, ANPEP, and a matrix metalloprotease, MMP14, (Supplementary Table S3). Notably, except for annexins 2 and 3, HPSC sEVs were enriched with annexins 1, 4, 5, 6, 7, and 11 compared to HPaStec sEVs (Table 3). Several integrins were elevated and differentially expressed in HPaStec sEVs: integrin alpha-2 (ITGA2), integrin alpha-4 (ITGA4), integrin 1 (ITGB1), integrin beta-6 (ITGB6), and integrin alpha-3 (ITGA3) (Table 4). To further confirm the higher integrin expression in HPaStec sEVs, Western blot analyses were carried out using representative integrin α and β antibodies. These results again confirmed that both integrins were highly expressed in HPaStec sEVs compared to the HPSC sEVs (Supplementary Figure S9). Next, we identified well-known exosomal markers enriched in HPSC sEVs, including programmed cell death 6-interacting protein (PDCD6IP) or Alix, tumor susceptibility gene, syntenin 1 (SDCBP), Annexin 7 (ANAX7), flotillin 1 and 2 (FLOT1 and FLOT2), annexin 11 (ANAX11), and heat shock 70 kDa protein (HSPA1A/HSPA1B). On the other hand, Ezrin (EZR) and moesin (MSN) were contained at higher levels in HPaStec sEVs (Supplementary Table S3).
      Table 2Top 10 proteins differentially expressed in HPSC sEVs compared to HPaStec sEVs
      Gene Symbol(s)HPSC-HPaStec Log2 RatioStandard DeviationAdjusted P-value
      CSE1L11.256.168.55E-03
      H2AC14;H2AC12;H2AJ9.024.971.20E-02
      MACROH2A18.24.584.96E-02
      H2AZ2;H2AZ18.134.482.94E-02
      H2AC6;H2AW;H2AC47.784.34.41E-02
      H17.64.313.85E-02
      H2BC14;H2BC15;H2BC97.163.951.15E-02
      H4C16.683.732.94E-02
      H16.443.572.94E-02
      BANF15.933.313.23E-02
      Table 3Annexins found in sEVs
      Gene Symbol(s)HPSC-HPaSteC Log2 RatioStandard DeviationAdjusted P-value
      ANXA112.451.354.96E-02
      indicates differentially expressed between HPSC and HPaStec.
      ANXA72.081.172.83E-02
      indicates differentially expressed between HPSC and HPaStec.
      ANXA3-0.650.835.55E-01
      ANXA2;ANXA2P2-0.460.271.11E-01
      ANXA100.460.394.53E-01
      ANXA50.690.417.43E-02
      ANXA10.970.601.24E-01
      ANXA61.040.627.71E-02
      ANXA41.260.736.80E-02
      indicates differentially expressed between HPSC and HPaStec.
      Table 4Integrins found in sEVs
      Gene Symbol(s)HPSC-HPaSteC Log2 RatioStandard DeviationAdjusted P-value
      ITGA2-3.221.778.55E-03
      indicates differentially expressed between HPSC and HPaStec.
      ITGA4-3.521.941.30E-02
      indicates differentially expressed between HPSC and HPaStec.
      ITGA5-3.051.728.20E-02
      ITGB5-2.631.571.29E-01
      ITGB3-2.581.521.24E-01
      ITGB1-2.571.422.47E-02
      indicates differentially expressed between HPSC and HPaStec.
      ITGAV-2.351.338.59E-02
      ITGB6-2.221.231.96E-02
      indicates differentially expressed between HPSC and HPaStec.
      ITGA3-2.211.232.94E-02
      indicates differentially expressed between HPSC and HPaStec.
      ITGA6-1.250.841.70E-01
      ITGA1-0.280.375.55E-01
      indicates differentially expressed between HPSC and HPaStec.
      We next performed pathway enrichment analyses of the 87 differentially expressed protein groups using the Enrichr R package (
      • Kuleshov M.V.
      • Jones M.R.
      • Rouillard A.D.
      • Fernandez N.F.
      • Duan Q.
      • Wang Z.
      • Koplev S.
      • Jenkins S.L.
      • Jagodnik K.M.
      • Lachmann A.
      • McDermott M.G.
      • Monteiro C.D.
      • Gundersen G.W.
      • Ma'ayan A.
      Enrichr: a comprehensive gene set enrichment analysis web server 2016 update.
      ). Top significantly enriched pathways using Gene Ontology: Biological Process (GO: BP) were consistent with exosome enrichment and included extracellular matrix organization (Padj = 3.01×10-04), multivesicular body assembly (Padj = 4.63×10-04), and cell-matrix adhesion (Padj = 3.37×10-03) (Supplementary Fig. S10, a,c, Supplementary Table S3). Top significantly enriched pathways using Gene Ontology: Cellular Component (GO:CP) also included extracellular or cell membrane pathways, such as focal adhesion (Padj = 2.14×10-14), filopodium (Padj = 9.33×10-04), and phagocytic vesicle (Padj = 2.75×10-03) (Supplementary Fig. S10, b,d, Supplementary Table S3). GO: BP pathway analyses showed endosomal sorting complexes required for transport (ESCRT) complex disassembly and multivesicular body organization and assembly in HPSC sEVs, whereas HPaStec sEVs had elevated cell adhesion and extracellular organization pathways. The ESCRT pathway contained membrane proteins that ubiquitinate and promote the internalization of sEVs within the multivesicular endosome [59]. The GO: BP analysis of HPSC sEVs identified an abundance of differentially expressed proteins associated with the lysosomes, a major compartment of protein turnover in the cells. Additionally, GO: CP analysis identified focal adhesion pathway enrichment (Padj = 1.31×10-14), which has a central role in regulating cell signaling (
      • Electronic address a. a. d. h. e.
      • Cancer Genome Atlas Research N.
      Cancer Genome Atlas Research Network
      Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma.
      ), as well as a higher abundance of membrane proteins in both of the sEVs (Supplementary Fig. S11 a,b).
      A known shortcoming of pathway enrichment is that it does not consider interactions between pathway members (
      • Khatri P.
      • Sirota M.
      • Butte A.J.
      Ten years of pathway analysis: current approaches and outstanding challenges.
      ).Therefore, we hypothesized an interaction-based approach would capture additional relationships beyond what was found with pathway enrichment. To test this premise, we queried the 87 differentially expressed protein groups with the STRING protein-protein interaction database (
      • Szklarczyk D.
      • Gable A.L.
      • Nastou K.C.
      • Lyon D.
      • Kirsch R.
      • Pyysalo S.
      • Doncheva N.T.
      • Legeay M.
      • Fang T.
      • Bork P.
      • Jensen L.J.
      • von Mering C.
      The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets.
      ). We used the first protein of the protein group if there was more than one present, and we required at least medium confidence interactions (minimum score 0.400). The resulting network had significantly more interactions than expected (STRING protein-protein interaction enrichment p-value < 1.0E-16), and from this we found 69 protein nodes shared edges in a large sub-network (Figure 5) with ITGB1 having the highest degree or number of connections (n= 17; Supplementary Table S3). These results suggest a commonality between differentially expressed protein groups and may point to a deeper biological process driving the HPSC-HPaStec differences.
      Figure thumbnail gr5
      Fig. 5STRING protein-protein interactions: Eighty-seven differentially expressed were queried with the STRING protein-protein interaction database. Seventy-three proteins with a minimum interaction score of 0.400 were identified. Notably, 69 were connected in a large network with ITGB1 having the highest degree of interactions (n = 17).

      CSE1L is expressed in PDAC-associated pancreatic stellate cell sEVs and is associated with poor survival

      We have identified specific protein markers that were highly elevated in HPSC sEVs, and among them, CSE1L, a human homolog of CSE1 that is a yeast chromosome segregation protein (
      • Liao C.F.
      • Lin S.H.
      • Chen H.C.
      • Tai C.J.
      • Chang C.C.
      • Li L.T.
      • Yeh C.M.
      • Yeh K.T.
      • Chen Y.C.
      • Hsu T.H.
      • Shen S.C.
      • Lee W.R.
      • Chiou J.F.
      • Luo S.F.
      • Jiang M.C.
      CSE1L, a novel microvesicle membrane protein, mediates Ras-triggered microvesicle generation and metastasis of tumor cells.
      ), is preferentially accumulated in HPSC sEVs. CSE1L was dramatically increased in HPSC sEVs (log2 ratio = 11.25, Padj = 0.009; Table 2, Supplementary Table S3). CSE1L/XPO2_HUMAN was identified by the peptide, AADEEAFEDNSEEYIRR, with a mass measurement accuracy of 4.36 ppm (see sequence interpretation in Supplementary Fig. S12, a). For validation, relative quantitation with extracted ion chromatograms of the isotopes of the intact peptide and the sum of normalized signal intensity of all identified peptides of the CSE1L protein for each sample showed increased intensity in HPSC sEVs compared to HPaStec sEVs (Figure 6, a-c). CSE1L expression was also significantly higher in the HPSC sEV fractions compared to the HPaStec sEVs (Figure 2a) via Western blot analyses. CSE1L expression is correlated with poor overall survival in other cancer types (
      • Wellmann A.
      • Flemming P.
      • Behrens P.
      • Wuppermann K.
      • Lang H.
      • Oldhafer K.
      • Pastan I.
      • Brinkmann U.
      High expression of the proliferation and apoptosis associated CSE1L/CAS gene in hepatitis and liver neoplasms: correlation with tumor progression.
      ), but to our knowledge, it has not been investigated for PDAC. Given the dramatic increase of CSE1L in HPSCs, we hypothesized our findings may be relevant to outcomes of patients with PDAC. Therefore, we identified 174 patients with PDAC from the Cancer Genome Atlas (TCGA) (
      • Electronic address a. a. d. h. e.
      • Cancer Genome Atlas Research N.
      Cancer Genome Atlas Research Network
      Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma.
      ) with CSE1L gene expression measurements and clinical outcome data, and we stratified these patients by median CSE1L expression. Kaplan-Meier survival analysis revealed that higher expression of CSE1L was associated with a significant decrease in survival (log-rank P value = 9.95E-4; Supplementary Fig. S13).
      Figure thumbnail gr6
      Fig.6LC-MS/MS Identification of CSE1/XPO2: (a,b) Relative quantitation with Extracted Ion Chromatograms of the peptide, AADEEAFEDNSEEYIRR, from CSE1L_HUMAN protein shows increased intensity in HPSC sEVs compared to HPastec sEVs. (c) Relative Quantitation for CSE1L_human protein. Sum of normalized signal intensity of all identified peptides of CSE1L protein for each sample. (d) Panc1 or Miapaca2 cells were treated with 20 μg/ml HPSC-sEVs or HPaStec sEVs for 72 hrs. before Western blot analysis using antibodies against CSE1L, CD63, p-ERK and ERK. Actin was used as a loading control.

      CSE1L expression and ERK signaling in Panc1 cells upon HPSC sEVs treatments

      Next, to investigate the effect of HPSC sEVs and HPaStec sEVs on CSE1L expression, HPSC sEVs (20 μg/ml) were co-cultured with Panc1 or Miapaca2 cells for 72 hrs. prior to Western blot analyses using CSE1L, CD63, and β-actin antibodies. In addition, to determine if sEVs treatment to the cells induce the cell survival signaling we have assessed the phosphorylated ERK signaling after sEVs treatment. The Western blots revealed elevated CSE1L expression and increased ERK signaling (Figure 6d) in Panc1 cells but not in Miapaca2 cells upon treatment with HPSC sEVs. On the contrary, primary HPaStec sEVs did not modulate the expression of CSE1L or ERK signaling (Figure 6d) in any of the cancer cells.

      DISCUSSION

      The PDAC tumor microenvironment is largely composed of stromal cells, including stellate cells. Although activated stellate cells, and sEVs secreted from them, are known to contribute to the aggressive biology of PDAC(2, 3) (
      • Melo S.A.
      • Luecke L.B.
      • Kahlert C.
      • Fernandez A.F.
      • Gammon S.T.
      • Kaye J.
      • LeBleu V.S.
      • Mittendorf E.A.
      • Weitz J.
      • Rahbari N.
      • Reissfelder C.
      • Pilarsky C.
      • Fraga M.F.
      • Piwnica-Worms D.
      • Kalluri R.
      Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.
      ,
      • Takikawa T.
      • Masamune A.
      • Yoshida N.
      • Hamada S.
      • Kogure T.
      • Shimosegawa T.
      Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.
      ) (
      • Takikawa T.
      • Masamune A.
      • Yoshida N.
      • Hamada S.
      • Kogure T.
      • Shimosegawa T.
      Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.
      ,
      • Hoffman R.M.
      Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche.
      ,
      • Lugea A.
      • Waldron R.T.
      Exosome-Mediated Intercellular Communication Between Stellate Cells and Cancer Cells in Pancreatic Ductal Adenocarcinoma.
      ) much remains to be learned regarding stromal-derived sEVs in pancreatic cancer. Here we have performed a unique qualitative and quantitative comparison of the proteome found in the sEVs derived from stellate cells isolated and cultured from normal pancreas (HPaStec) and PDAC tumors (HPSC). Key findings from this study include: 1) Overall trend in total EV concentration that increases in HPSC cells and a significantly higher mean sEVs sizes in HPSCs; 2) efficient uptake, that is dependent upon sEV surface proteins, of both HPaStec- and HPSC-derived sEVs into both normal and pancreatic cancer epithelial cells; and 3) the absence of significant in vitro effect of either normal and tumor derived sEVs on pancreatic cancer cell survival or growth and 4) Detailed comparative proteomics analysis comparing normal and tumor stellate cell derived sEVs identified 87 differentially expressed proteins suggesting that each is biologically distinct from each other. Together, these findings support sEVs as warranting further study as potential tools for drug delivery across different biological barriers and use as next generation therapeutics.
      In our study, we isolated and characterized sEVs from HPSC and HPaStec cells in terms of morphology, size distribution, and concentration. The biology of both cell types are different that may contribute towards differences in sEVs generations. Previous co-culture experiments have identified that soluble factors from HPSC cells stimulate signaling pathways involved in proliferation and survival of pancreatic cancer cells in vitro and in vivo (
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      ).
      Investigators have demonstrated crosstalk via sEVs between stromal and cancer cells with demonstration of miRNAs for chemokine ligands proposed as a potential driver of proliferation and survival of pancreatic cancer cells (
      • Takikawa T.
      • Masamune A.
      • Yoshida N.
      • Hamada S.
      • Kogure T.
      • Shimosegawa T.
      Exosomes Derived From Pancreatic Stellate Cells: MicroRNA Signature and Effects on Pancreatic Cancer Cells.
      ). Our results suggest that HPSC secreted sEVs, in distinction from the soluble factors in the conditioned medium, do not influence the in vitro proliferation, apoptosis, or motility of both tumor and stellate cells. Efficient uptake of sEVs was demonstrated in culture conditions suggesting that observed differences versus previous reports (
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      ) could be related to the unique stellate cells used in the assays or differences in the sEV isolation techniques.
      Nevertheless, stellate cell sEVs contain a robust proteome that harbors surface and cargo proteins critical to their biology and our investigation of proteome offers unique opportunities to exploit sEVs as biomarkers or drug delivery vehicles. Small extracellular vesicular proteins can be more specific than secretory proteins in cells (
      • Li W.
      • Li C.
      • Zhou T.
      • Liu X.
      • Liu X.
      • Li X.
      • Chen D.
      Role of exosomal proteins in cancer diagnosis.
      ,
      • Li A.
      • Zhang T.
      • Zheng M.
      • Liu Y.
      • Chen Z.
      Exosomal proteins as potential markers of tumor diagnosis.
      ) in terms of identifying diagnostic biomarkers related to cancers. As previously reported by Melo SA et al., (
      • Melo S.A.
      • Luecke L.B.
      • Kahlert C.
      • Fernandez A.F.
      • Gammon S.T.
      • Kaye J.
      • LeBleu V.S.
      • Mittendorf E.A.
      • Weitz J.
      • Rahbari N.
      • Reissfelder C.
      • Pilarsky C.
      • Fraga M.F.
      • Piwnica-Worms D.
      • Kalluri R.
      Glypican-1 identifies cancer exosomes and detects early pancreatic cancer.
      ) that Glypican-1(GPC1) was specifically enriched on cancer cell-derived exosomes and showed high specificity over CA-199 or serum-free GPC1 (100% vs79.49% vs 82.14%) that distinguish non-cancer patients from pancreatic cancer patients. With respect to drug delivery, we demonstrated that membrane proteins on HPSC sEVs may be essential for sufficient exosomal entry to normal and cancer cells, as documented by recent studies (
      • Hoshino A.
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      Tumour exosome integrins determine organotropic metastasis.
      ,
      • Hoffman R.M.
      Stromal-cell and cancer-cell exosomes leading the metastatic exodus for the promised niche.
      ). In other words, the role of membrane proteins in uptake of small extracellular vesicles may be relevant to ongoing studies using sEVs as vehicles to improve drug delivery efficacy and reduce toxicity of chemotherapeutic drugs (
      • Costa-Silva B.
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      Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver.
      ).
      To the best of our knowledge, this report is the first to compare biological and proteomic analysis between cancer-associated HPSC sEVs and normal pancreatic stellate cell sEVs. Consistent with exosomal enrichment, the protein groups we identified shared a large degree of overlap with ExoCarta (
      • Keerthikumar S.
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      ), a database of exosomal proteins, RNAs, and lipids. We identified 87 differentially expressed protein groups, including APNEP, MMP14, and CSE1L, between HPSC and HPaStec sEVs. Our results (Table 2, Supplementary Table S3) showed differential expression of several histone protein groups. Strikingly, many DNA binding proteins, such as H2A, H2B, H3, and H4, were enriched in HPSC sEVs, although histones are not commonly regarded as proteins associated with extracellular vesicles, histones were seen to be enriched in sEVs in a murine B16 melanoma model and in primary HPSC exosomes (
      • Muhsin-Sharafaldine M.R.
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      Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker.
      ). Several studies have also shown that sEVs contain chromosomal DNA fragments, indicating that exosome secretion maintains cellular homeostasis by removing harmful cytoplasmic DNA from cells (
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      DNA damage primes the type I interferon system via the cytosolic DNA sensor STING to promote anti-microbial innate immunity.
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      Dnase2a deficiency uncovers lysosomal clearance of damaged nuclear DNA via autophagy.
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      ). In the current study, elevated DNA binding proteins were observed in HPSC sEVs which may be indicative of cell growth and protein production. More detailed studies with pancreatic stellate cell sEVs isolated from PDAC tumors are needed to better understand the source of enriched histone proteins in sEVs and their pathobiological roles.
      Among annexin group of proteins, ANAX 7 and ANAX 11, are differentially expressed in HPSC sEVs when compared to those from HPaStec. Annexins have several cellular functions, including cell migration, proliferation, and apoptosis (
      • Lokman N.A.
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      The role of annexin A2 in tumorigenesis and cancer progression.
      ), and changes in the expression of individual annexins have been observed in cancers (
      • Mussunoor S.
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      The role of annexins in tumour development and progression.
      ), thus implicating that differential expression of annexins in HPSC sEVs may account for PDAC development. HPSC sEVs are also enriched with syntenin (SDCBP), and it was shown that microvesicle formation is regulated through the syndecan heparan sulfate proteoglycans and their cytoplasmic adaptor, syntenin (
      • Baietti M.F.
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      Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.
      ). Pathway enrichment of the differentially expressed protein groups was again consistent with exosome enrichment and included extracellular matrix and cellular membrane pathways. Integrin β1 (ITGB1) found in sEVs had highest protein-protein interactions, suggesting it may serve as a hub of exosome cargo delivery and organ specific intracellular signaling (
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      ).
      CSE1L, a microvesicle membrane protein also known as Exportin, is located on the 20q13 locus. Although primarily expressed in the nucleus, CSE1L is also found in sEVs (
      • Jiang M.C.
      CAS (CSE1L) signaling pathway in tumor progression and its potential as a biomarker and target for targeted therapy.
      ). CSE1L is involved in forming microtubule assembly and apoptosis, and it functions as a nuclear transporter and transcriptional regulator (
      • Tanaka T.
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      hCAS/CSE1L associates with chromatin and regulates expression of select p53 target genes.
      ,
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      • Shen S.C.
      • Lee W.R.
      • Jiang M.C.
      Cellular apoptosis susceptibility (CSE1L/CAS) protein in cancer metastasis and chemotherapeutic drug-induced apoptosis.
      ). CSE1L regulates Ras-induced ERK phosphorylation, microvesicle generation, and tumor metastasis (
      • Liao C.F.
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      • Chen H.C.
      • Tai C.J.
      • Chang C.C.
      • Li L.T.
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      • Hsu T.H.
      • Shen S.C.
      • Lee W.R.
      • Chiou J.F.
      • Luo S.F.
      • Jiang M.C.
      CSE1L, a novel microvesicle membrane protein, mediates Ras-triggered microvesicle generation and metastasis of tumor cells.
      ). It is amplified or highly expressed in pancreatic cancers (
      • Holzmann K.
      • Kohlhammer H.
      • Schwaenen C.
      • Wessendorf S.
      • Kestler H.A.
      • Schwoerer A.
      • Rau B.
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      • Lichter P.
      • Gress T.
      • Bentz M.
      Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.
      ) and a variety of other cancers (
      • Jiang M.C.
      CAS (CSE1L) signaling pathway in tumor progression and its potential as a biomarker and target for targeted therapy.
      ). Knockdown of CSE1L has been shown to inhibit tumorigenesis and induce apoptosis (
      • Pimiento J.M.
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      • Eschrich S.A.
      • Chen D.T.
      • Husain K.
      • Shibata D.
      • Coppola D.
      • Malafa M.P.
      Knockdown of CSE1L Gene in Colorectal Cancer Reduces Tumorigenesis in Vitro.
      ). Here, we report that CSE1L was significantly elevated in HPSC sEVs and may contribute towards the cancer signaling pathway in PDAC. Moreover, co-culture of HPSC sEVs with Panc1 cells increased ERK phosphorylation and CSE1L expression. CSE1L overexpression triggered microvesicle generation and tumor progression, thereby regulating the Ras-ERK signaling pathway (
      • Liao C.F.
      • Lin S.H.
      • Chen H.C.
      • Tai C.J.
      • Chang C.C.
      • Li L.T.
      • Yeh C.M.
      • Yeh K.T.
      • Chen Y.C.
      • Hsu T.H.
      • Shen S.C.
      • Lee W.R.
      • Chiou J.F.
      • Luo S.F.
      • Jiang M.C.
      CSE1L, a novel microvesicle membrane protein, mediates Ras-triggered microvesicle generation and metastasis of tumor cells.
      ). Our secondary analysis of the Cancer Genome Atlas (TCGA) data revealed that higher gene expression of CSE1L is significantly associated with reduced survival in PDAC (log-rank P value = 9.95E-4). This result is consistent with findings with other cancer types and hints at a translational avenue for our results. CSE1L-triggered microvesicle generation might explain the higher exosome content in CSE1L enriched HPSC sEVs compared to HPaStec sEVs (
      • Liao C.F.
      • Lin S.H.
      • Chen H.C.
      • Tai C.J.
      • Chang C.C.
      • Li L.T.
      • Yeh C.M.
      • Yeh K.T.
      • Chen Y.C.
      • Hsu T.H.
      • Shen S.C.
      • Lee W.R.
      • Chiou J.F.
      • Luo S.F.
      • Jiang M.C.
      CSE1L, a novel microvesicle membrane protein, mediates Ras-triggered microvesicle generation and metastasis of tumor cells.
      ).
      The limitations of our present study include reliance on sEV isolation exclusively from cultured cell lines, not from direct patient samples. Therefore, further studies are needed to understand the source, association, and secretion of elevated levels of CSE1L from stellate cell sEVs adjacent to a PDAC tumor and the relevant clinical importance in cancer therapy and diagnosis. Future work is planned to further investigate the relationship between exosomal CSE1L protein expression and PDAC patient outcomes. In future, it would be also interesting to determine whether stellate cells associated sEVs can be used as prognostic markers of PDAC to validate the current findings.
      In summary, we have successfully identified differentially expressed proteins in HPSC sEVs isolated from PDAC-associated stellate cells and in sEVs isolated from normal pancreases. Our results suggested that HPSC sEVs are biologically different from HPaStec sEVs and may provide an advantageous microenvironment to the pancreatic cancer cells and may potentially be targeted as a cargo vehicle for the safe delivery of drugs or other biological materials to the cancer cells. Additional studies are required to elucidate the role of cancer-associated stellate cell sEVs in PDAC progression, which would lead the way to therapeutic intervention targeting tumor stroma.

      DATA AVAILABILITY

      The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (
      • Perez-Riverol Y.
      • Csordas A.
      • Bai J.
      • Bernal-Llinares M.
      • Hewapathirana S.
      • Kundu D.J.
      • Inuganti A.
      • Griss J.
      • Mayer G.
      • Eisenacher M.
      • Perez E.
      • Uszkoreit J.
      • Pfeuffer J.
      • Sachsenberg T.
      • Yilmaz S.
      • Tiwary S.
      • Cox J.
      • Audain E.
      • Walzer M.
      • Jarnuczak A.F.
      • Ternent T.
      • Brazma A.
      • Vizcaino J.A.
      The PRIDE database and related tools and resources in 2019: improving support for quantification data.
      ) partner repository with the dataset identifier PXD030077 and 10.6019/PXD030077.
      This article contains supplementary figures and Tables.

      Declaration of Interest

      Dr. John M. Koomen is partially funded by BMS for a different project unrelated to this work. Dr. Jason B. Fleming is a scientific advisor/consultant in the following companies: Glycosbio Food Sciences, Inc., Biopath Holdings Inc., Natera, Panther Therapeutics Inc., and an independent contractor in MyCareGorithm. –Other Authors have no conflict of interests.

      ACKNOWLEDGEMENTS

      We wish to acknowledge Amanda Garces at the Lisa Muma Weitz Advanced Microscopy core laboratory of the USF College of Medicine for technical assistance in obtaining TEM micrographs. This work has been supported in part by the Biostatistics and Bioinformatics, Proteomics and Metabolomics, Moffitt Analytical Microscopy core, and Flow Cytometry Shared Resources at the H. Lee Moffitt Cancer Center & Research Institute, an NCI designated Comprehensive Cancer Center (P30-CA076292). Editorial assistance was provided by the Moffitt Cancer Center’s Scientific Editing Department by Daley Drucker. No compensation was given beyond her regular salary. The study was financially supported by Center for Immunotherapeutic Transport Oncophysics grant (awarded to JBF, U54 CA210181-02), Shirley E. Noland Foundation (awarded to JBF), Hoenle Foundation Fund (awarded to JBF) and a Moffitt-Advent Health Collaboration Award (awarded to JBP, JBF, and RP).

      Supplementary data

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