Circulating Microvesicles from Pancreatic Cancer Accelerate the

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Circulating Exosomes from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC-1 Cells Mingrui An, Jianhui Zhu, Jing Wu, Kyle C. Cuneo, and David M. Lubman J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00014 • Publication Date (Web): 01 Mar 2018 Downloaded from http://pubs.acs.org on March 2, 2018

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Journal of Proteome Research

Figure 1: Schematic overview of migration and proliferation assays, the sample preparation and LC-MS/MS analysis of HMTCs and CMTCs. Microvesicles were isolated from serum of patients with pancreatic cancer or commercial healthy serum using several steps of differential centrifugation. Then cancer microvesicles or healthy microvesicles were exposed to PANC-1 cells and migration and proliferation assays were performed. Next, proteins from HMTCs and CMTCs were extracted, alkylated and digested in ultrafiltration tubes using the filter-aided sample preparation (FASP) method. After iTRAQ labeling, peptides were mixed and run on a mass spectrometer. Database search of raw data and statistical analysis identified the differentially expressed proteins. Finally, IPA database revealed the network of directly interacting proteins and possible pathways involved in cell migration and proliferation. 82x93mm (300 x 300 DPI)

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Figure 2: TEM images and the size distribution of the microvesicles. The TEM images showed that the circulating microvesicles from pancreatic cancer patients and healthy controls had similar morphology (a, b) and size distribution (c). 100x126mm (300 x 300 DPI)


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Figure 3: Comparison of protein coverage and identified peptide number by histograms. (a) Elution efficiency comparison of 500 mM NaCl and water on eluting 0.1 µg, 1 µg or 10 µg of BSA digests. There is no significant difference between NaCl and water on elution efficiency. (b) Different amounts of BSA (0.1 µg, 1 µg or 10 µg) were eluted 6 rounds; peptides still could be identified by mass spectrometry in the seventh eluent. (c) The former six eluents of digest (Sample A) and digest on the top of filter (Sample B) were collected and identified. We found 6% - 8% more peptides were identified in Sample B. There were 3 replicates for each assay. 149x276mm (300 x 300 DPI)



Figure 4: Microvesicles from pancreatic cancer patients increase the migration and proliferation of PANC-1 cells. PANC-1 cells were exposed to microvesicles isolated from patients with pancreatic cancer or healthy control for 24 hours before the migration and proliferation assay. We used pooled microvesicles in this assay due to limited amount of serum from each patient. (a) CMTCs migrated faster than HMTCs or PANC-1 cells without exposure. Error bars represent SD (n=3). (b) CMTCs have higher proliferation activity. (c) Compared with HMTCs, CMTCs were more resistant to treatment with gemcitabine. 94x68mm (300 x 300 DPI)


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Figure 5: Western blot verification and subcellular and functional analysis of differentially expressed proteins between CMTCs and HMTCs Among 4102 quantified proteins, 62 proteins had GO information. (a) The subcellular distribution shows that proteins from cytoplasm, nucleus, plasma membrane and extracellular space account for 53.23%, 25.81%, 9.68% and 6.45%, respectively. (b) Western blot verified the quantification results of CD44, TP53 and PPP2R1A by mass spectrometry. (c) The IPA analysis shows that 31 proteins have direct interactions. Red and green represent up- and down-regulation in CMTCs, respectively. 123x184mm (300 x 300 DPI)



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Circulating Microvesicles from Pancreatic Cancer Accelerate the Migration and Proliferation of PANC-1 Cells Mingrui An1, Jianhui Zhu1, Jing Wu1, Kyle C. Cuneo2, * and David M. Lubman1, *

1

Department of Surgery, University of Michigan Medical Center, Ann Arbor, Michigan 48109, United

States 2

Department of Radiation Oncology, University of Michigan, Ann Arbor, Michigan 48109, United

States

Author email address Mingrui An; [email protected] Jianhui Zhu; [email protected] Jing Wu; [email protected] Kyle C. Cuneo; [email protected] David M. Lubman; [email protected]

*

To whom correspondence should be addressed David M. Lubman, Department of Surgery,

University of Michigan Medical Center, 1150 West Medical Center Drive, Building MSRB1, Rm A510B, Ann Arbor, MI 48109-0656, United States. Phone: (734)-615-5081. Fax: (734)-615-2088; Email: [email protected] *

To whom correspondence should be addressed Kyle C. Cuneo, Department of Radiation Oncology,

University of Michigan, 1500 E. Medical Center Dr. SPC 5010, University Hospital Floor B2 Room

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C490, Ann Arbor, MI 48109-0656, United States. Phone: (734)-936-4300. Fax: (734)-763-7370; Email: [email protected]



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Abstract Circulating microvesicles are able to mediate long-distance cell-cell communications. It is essential to understand how microvesicles from pancreatic cancer act on other cells in the body. In this work, serum-derived microvesicles were isolated from 10 patients with locally advanced pancreatic cancer and healthy controls. Using Cell Transwell and WST-1 reagents, we found that microvesicles from pancreatic cancer accelerated migration and proliferation of PANC-1 cells. Meanwhile, the proliferation of these cancer microvesicles treated cells (CMTCs) was affected less by 10 µM of gemcitabine relative to healthy microvesicles treated cells (HMTCs). Next, we optimized the filteraided sample preparation (FASP) method to increase the recovery of protein samples and then applied it to the quantification of the proteome of CMTCs and HMTCs. The peptides were labeled and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In total, 4102 proteins were identified, where 35 proteins were up-regulated with 27 down-regulated in CMTCs. We verified the quantitative results of three key proteins CD44, PPP2R1A and TP53 by Western blot. The Ingenuity Pathway Analysis (IPA) revealed pathways that cancer microvesicles might participate in to promote cell migration and proliferation. These findings may provide novel clues of treatment for tumorigenesis and metastasis. Keywords: microvesicles, migration, pancreatic cancer, PANC-1 cells, proliferation


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Introduction Microvesicles originate from the luminal membrane of multivesicular bodies (MVB) at the late stage of endocytosis in most cells.1 They are membrane-bound nano-size vesicles carrying proteins, metabolites and nucleic acids (mRNA, miRNA) from their original cells.2 Microvesicles that are secreted and circulate throughout the body in the bloodstream are termed “circulating microvesicles”. Circulating microvesicles can fuse with the plasma membrane of recipient cells and release their cargos.3 In this manner, they are able to mediate nearby or distant cell-cell communications.4-6 Previous studies have shown that tumor-derived microvesicles play critical roles in various physiological and pathologic processes including tumorigenesis and metastasis.7-9 For example, gastric cancer-derived microvesicles have been shown to promote peritoneal metastasis by destroying the mesothelial barrier10 and long noncoding RNA ZFAS1 in circulating microvesicles promotes gastric cancer progression.11 Also, EGFR in microvesicles regulates the liver microenvironment to promote gastric cancer liver metastasis.12 In breast cancer, the miRNAs in microvesicles released by cancerassociated fibroblasts cause an aggressive phenotype.13 Furthermore, microvesicles released by HT29 cells increase the proliferation and motility of colon cancer cells14 and microvesicles released by mesenchymal non-small cell lung cancer cells promote chemoresistance.15 Considering little improvement has been achieved in outcomes of patients with pancreatic cancer over the past couple decades, it is essential to understand how microvesicles from pancreatic cancer act on cells in order to discover novel drug targets and treatment strategies.5,16-18 Since cancer and healthy microvesicles are known to contain different protein cargo, it is important to study the impact of these cancer related microvesicles on other cells. Several pioneering studies found that many kinds of RNAs are differentially expressed between cancer and healthy microvesicles. For example, certain



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miRNAs are preferentially expressed in circulating microvesicles derived from esophageal and colorectal cancer.19,20 Proteomic cargo of microvesicles has also been found to be different between osteosarcoma and normal osteoblasts.21 In a previous study from our laboratory, we found dozens of proteins were differentially expressed between pancreatic cancer and healthy microvesicles.22 In the present study, we used a pancreatic cancer cell line to measure the potential of microvesicles derived from patients with pancreatic cancer to promote migration and proliferation. We collected sera from 10 patients with locally advanced pancreatic cancer and used commercial healthy sera as controls. Circulating microvesicles from cancer or healthy sera were isolated and exposed to PANC-1 cells. Also, these effects were evaluated using various concentrations of gemcitabine to treat PANC-1 cells. We have also studied changes in the proteome of PANC-1 cells due to exposure to microvesicles, where differentially expressed proteins were analyzed by the Ingenuity Pathway Analysis (IPA).


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Experimental Section Isolation of microvesicles from human serum Commercially available serum samples (Innovative Research, Novi, MI) were chosen as healthy controls. Serum samples from 10 patients with locally advanced pancreatic cancer were obtained as part of an Institutional Review Board approved protocol. Whole blood samples were centrifuged at 500×g for 10 min to isolate serum. All serum samples were stored at -80 oC before use. The initial volume of serum was 1 mL per sample. The serum samples were diluted with 3 mL of PBS (AppliChem, St. Louis, MO) to decrease the viscosity. The diluted serum samples were centrifuged at 2,000×g for 10 min and 10,000×g for 30 min at 4 °C to remove dead cells and cell debris. The supernatant was transferred into Ultra-ClearTM tubes (Beckman Coulter, Indianapolis, IN) and centrifuged at 100,000×g using a Beckman Optima XL-70 Ultracentrifuge for 70 min at 4 °C. The supernatant was discarded, and 2 mm of supernatant remained above the pellet to avoid the loss of sedimentary microvesicles. The microvesicles were suspended in 4 mL of PBS and centrifuged at 100,000×g for 60 min at 4 °C to clean the microvesicles. This clean up step was repeated 3 additional cycles to eliminate the serum protein contamination.23 Transmission electron microscopy (TEM) and NanoSight analysis The size of microvesicles was measured by TEM. Briefly, a carbon film (Hatfield, PA, USA) was placed in a vacuum environment for 1min. Glow discharge was then performed by turning on high voltage power to incubate the carbon film with electrons for 2 min. This step was performed to make the surface of the carbon film hydrophilic. Then, 5 μL of microvesicle sample (2.5×107) was loaded on the carbon film and incubated for 2 min. Next, the supernatant was removed by a piece of filter paper and 5 μL of 2.5% w/v glutaldehyde in PBS was loaded to fix the microvesicles for 5 min. After removing the supernatant, the carbon film was washed with water for 3 times and then negatively 6 ACS Paragon Plus Environment


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stained with 5 μL of 1% uranyl acetate for 1 min. After removing uranyl acetate, the samples on carbon films were imaged in a Philips CM-100 TEM instrument. Microvesicle concentration was measured using the NanoSight NS300 (Malvern, Worcestershire, UK) according to the standard protocol. Microvesicles isolated from 1 mL of patient or healthy serum were suspended in 1 mL of PBS and analyzed for 5 min at 25 °C. Culture of PANC-1 cells and microvesicle exposure PANC-1 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). These cells were cultured in a 96-well plate using Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 μg/mL of streptomycin (Gibco BRL, San Diego, CA) at 37°C in a 5 % CO2 atmosphere. Microvesicles (1×109) isolated from 1 mL of serum (from cancer patients or healthy controls) were added into 200 μL of media and exposed to PANC-1 cells (around 50,000) in a well of 96-well plate for 24 hours. Cell migration assay The migration ability of PANC-1 cells was examined using Transwell chambers (Cell Biolabs Inc, San Diego, CA). Cells were suspended in 200 μL of FBS-free media (DMEM containing 0.5% BSA, 2 mM CaCl2 and 2 mM MgCl2) and transferred into the upper compartment of 24-well Transwell chambers for 2 h. The lower compartment contained culture media (DMEM supplemented with 10% FBS). After 2 h, the medium on the upper compartment was removed carefully. The upper compartment was washed by PBS and swabbed gently by cotton-tipped swabs to remove nonmigratory cells. The upper compartment was then transferred to a clean well containing 400 μL of Cell Stain Solution and incubated for 10 min at room temperature. After staining, the upper compartment was washed and the cells that migrated through the membrane onto the bottom of the upper


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compartment were viewed under a microscope (SK-Advanced, Kadima Zoran, Israel) and quantified by a microplate reader (BioTek, Winooski, VT) at a wavelength of 560 nm. Cell proliferation and toxicity assay The cell proliferation24 and toxicity25,26 of PANC-1 cells were examined using Water-soluble Tetrazolium (WST)-1 reagent. Cells that had been exposed to 5×109/mL of cancer microvesicles or healthy microvesicles for 24 hours were treated with various concentrations of gemcitabine for 2 h. To each well was added 100 μL of WST-1 reagent and incubated for 4 h in a cell incubator. The 96-well plate was read by a microplate reader at 450 nm. Lysis of PANC-1 cells, tryptic digestion and iTRAQ labeling After three times washing with PBS, PANC-1 cells from each well (around 50,000) were lysed with 20 µL lysis buffer composed of 50 mM triethylammonium bicarbonate (TEAB), 4% sodium dodecyl sulfate (SDS) and 100 mM 1, 4-dithiothreitol (DTT) at 99 °C for 5 minutes. We prepared samples using an optimized filter-aided sample preparation (FASP) method. Briefly, the sample was cooled down and diluted with 800 µL of 8M urea buffer (containing 50 mM TEAB), to which was added 20 µL of 250 mM iodoacetamide (IAA). Next, the sample solution was transferred to a centrifugal spin YM-30 filter (Millipore, Billerica, MA), and centrifuged at 14,000×g until the sample on the filter was less than 50 µL. Two hundred microliters of 8 M Urea was then added to the sample and centrifuged at 14,000×g for 20 minutes. This step was repeated for an additional 2 times to remove SDS. In order to remove urea, the sample was washed by 200 µL of 50 mM TEAB for 3 times under the same centrifugation condition. The samples were then digested by 200 ng sequencing grade modified trypsin (Promega, Fitchburg, WI) at 37 °C for 12 hours. According to the standard protocol, the tryptic peptides would be filtered through the filter and collected. To efficiently elute the peptides, 2 cycles of elution with 50 mM ammonium bicarbonate solution and 1 cycle of elution with 500 mM sodium



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chloride solution should be performed.27 Here, the tryptic peptides on the filter were directly collected, acidified and cleaned by desalting with C18 tips.28 The eluted samples were dried by SpeedVac (Labconco, Kansas City, MO). The resulting tryptic peptide samples of cells were labeled by 4-plex iTRAQ reagent according to the instructions enclosed in the commercial kit. The labeled samples were acidified, mixed and then desalted using C18 tips. The eluted samples were dried by SpeedVac for mass spectrometry analysis. nanoLC-MS/MS and data analysis The tryptic digests of PANC-1 cells were separated on an EASY-nLC 1000 liquid chromatograph system (Thermo Fisher Scientific, San Jose, CA) with a 250 mm and 75 µm ID reverse-phase (RP) C18 column. The samples were eluted under a 120 min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid at a constant flow rate of 300 nL/min.29 Samples were analyzed by an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) in the positive ion mode. The capillary temperature and the spray voltage were set as 200 °C and 2.5kV. The data was acquired in a data-dependent mode; the 15 strongest MS1 peaks were selected for subsequent MS2 analysis. For every selected peak, higher-energy collisional dissociation (HCD) was performed. The MS1 spectra (m/z 350-1650) and the MS2 spectra were both acquired in the Orbitrap. The raw data were searched against the protein database by Proteome Discoverer 1.4 software (Thermo Fisher Scientific, San Jose, CA) with SEQUEST as the search engine. The parameters were set as follow: database, human UniProt; enzyme, trypsin; fixed modifications, carbamidomethyl (C) and 4-plex iTRAQ (N-term and K); variable modification, oxidation (M); up to 2 missed cleavages allowed; mass tolerance, 10 ppm for MS1 and mass tag, 0.05 Da for MS2; 1% false discovery rate allowed for peptides.


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We normalized the quantification results to eliminate the difference of protein amounts from different samples using Perseus software. The database search results were saved as a .txt files which were then loaded into Perseus. There were 3 columns of ratios 115/114, 116/114 and 117/114. All these ratio values were log2 transformed. The log2ratio values in each column were normalized by subtracting “Tukey’s biweight”. We performed a t-test to filter out proteins with a large variation of expression level among different patients, such as very low-abundance proteins which were only identified in a couple of patients. A protein was considered significantly changed if it had a normalized ratio greater than 2 (or less than 0.5), where a normalized log2ratio was greater than 1 or less than -1, with p value less than 0.05. Western blot Proteins from PANC-1 were separated on a 4-15% SDS-PAGE gradient gel (Bio-Rad, Berkeley, CA) and transferred to a PVDF membrane (Bio-Rad, Berkeley, CA). After blocking, the membrane was incubated overnight with anti-PPP2R1A antibody, anti-TP53 antibody or anti-CD44 antibody (Abcam, San Francisco, CA), followed by incubation with HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA) and was visualized using a chemiluminescent method kit (Merck Millipore, Billerica, MA).



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Results Isolation of circulating microvesicles from serum and exposure to PANC-1 cells In this study, circulating microvesicles were isolated from the serum of cancer patients and healthy controls. In order to obtain microvesicles with a minimum contamination of serum proteins, we performed 5 cycles of ultracentrifugation in the microvesicle isolation step (Figure 1). To evaluate the loss of microvesicles by multiple cycles of ultracentrifugation, we used Nanosight to compare the amount of microvesicles acquired by 1 cycle and 5 cycles, and found that the microvesicle amounts from 1 mL of serum were 1.51×109 and 1.00×109, respectively. Although there was 33% loss compared to 1 cycle, 5 cycles removed almost all the contaminant serum proteins. We could acquire around 0.2 µg of microvesicle protein from 4 mL of serum after 5 cycles of ultracentrifugation. Thus, the serum microvesicles were around 2 × 1010 / µg. The TEM images showed that the circulating microvesicles isolated by the ultracentrifugation from pancreatic cancer patients and healthy controls had similar morphology (Figure 2a, b) and size distribution (Figure 2c). We further used a negative stain EM where we used antibody-conjugated gold beads (5 nm diameter) to capture microvesicles from both pancreatic cancer patients and healthy controls, where anti-CD9, anti-CD63 and anti-CD81 antibodies were employed, respectively. The microvesicles captured by each of the antibodyconjugated gold beads also had similar morphology and size distribution between pancreatic cancer patients and healthy controls (Supplemental Figure S-1). PANC-1 cells were then exposed to the cancer and healthy microvesicles. Cell assays and mass spectrometry analysis were performed on the PANC-1 cells to demonstrate the effects of microvesicles derived from pancreatic cancer on other cells. Optimization of FASP method


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FASP is a routine method to prepare proteomic samples.30-32 Briefly, the lysate was added on the top of the 30 KD filter in a centrifugal spin YM-30 filter. After centrifugation, small molecules were filtered out while proteins were trapped. Proteins were cleaned by several cycles of buffer replenishing and centrifugation and then digested by trypsin. The tryptic peptides are filtered through the filter by several cycles of elution under the centrifugation at the last step. However, peptides are lost due to the absorption by the filter. When the sample amount is small, as in this case, the peptide loss is substantial. In this study, the original protein yield of the PANC-1 cells (around 50,000 cells) in a well of 96-well plate was around 2.5 µg. The rate of sample loss would be significant after many steps. According to the standard protocol, the elution by 500 mM NaCl will help to elute more peptides at the last step. Our results indicate that there is no significant difference between 500 mM NaCl and water when eluting 0.1 μg, 1 μg or 10 μg of bovine serum albumin (BSA) digests (Figure 3a). In addition, we found BSA could be detected even after six rounds of elution, although only three rounds of elution are suggested by the standard protocol (Figure 3b). We then digested 2 μg of protein from PANC-1 cells in each of two centrifugal spin YM-30 filter tubes. We collected and combined the eluent of six rounds from a tube (Sample A) while we directly collected the digested sample from the top of the filter from the other tube (Sample B). We then desalted the two samples before the analysis by mass spectrometry. It should be noted that the desalting step is very crucial to remove contaminants that hamper the mass spectrometry analysis. We found 6% - 8% more peptides were identified in the Sample B (Figure 3c). The mass spectrometry analyses of peptides directly collected from the top of the filter are found not to be affected by potential contaminants such as nucleic acids. The results indicate that it is not necessary to filter the peptide sample. We used this optimized FASP method to prepare PANC-1 cells in the next set of experiments. Microvesicles from patients with pancreatic cancer accelerate the migration of PANC-1 cells



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The effect of cancer-derived microvesicles on the migration ability of PANC-1 cells was evaluated using Transwell chambers. In a preliminary experiment, we isolated microvesicles from 1 mL of cancer serum or healthy control and added them into 5 mL of media to culture cells in a 10 cm plate, respectively. No difference in migration was observed between them. We then increased the concentration of microvesicles significantly. We added microvesicles isolated from 1 mL of serum (1×109) into 200 μL of media to culture cells in a well of a 96-well plate. We quantified the migrating cells by staining and measuring the absorbance at 560 nm and found that the migration ability of cancer microvesicles treated cells (CMTCs) was 21.5% higher than healthy microvesicles treated cells (HMTCs) or cells without microvesicle exposure (Figure 4a). It should be noted that the majority of circulating microvesicles from cancer patients were not from carcinoma tissues.33 We speculate that this was why the migration ability of CMTCs did not increase substantially. Effect of cancer microvesicles and gemcitabine on the proliferation of PANC-1 cells The WST-1 reagent has been broadly applied to assessing cell metabolic activity. Here, we used this reagent to further evaluate the effect of cancer-derived microvesicles on the proliferation ability of PANC-1 cells. We thus treated cells with various concentrations of gemcitabine, a widely used chemotherapy drug for pancreatic cancer. The results showed that CMTCs had higher proliferation activity than HMTCs independent of the gemcitabine concentration (Figure 4b). Compared to HMTCs, the proliferation of CMTCs was inhibited more significantly by low concentration (0.1 µM) of gemcitabine, but less significantly by high concentration (10 µM) (Figure 4c). HMTCs versus PANC-1 cells without microvesicle exposure With the iTRAQ-based mass spectrometry technique, we compared HMTCs with PANC-1 cells without exposure to microvesicles. In the quantitative proteomics results, few proteins were found


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changed between the above two groups, but 23 proteins (Supplemental Table S-1) were identified only in cells without exposure to serum derived microvesicles. We used the IPA database to analyze these “unique” proteins. The results did not show any interactions among these proteins and no canonical pathways related to these proteins were found. Next, we checked the signals of these proteins manually and found they were all low abundance proteins. Even in the group where they were identified, they were usually in only one or two samples. Thus, we speculated that these “unique” proteins most likely exist in both groups but were not identified in one of the groups due to their lowabundance. Based on the results above, one should be very careful to consider proteins only identified in one group as unique proteins unless the protein signals are high in one group and not detected in the other. HMTCs versus CMTCs We compared HMTCs with CMTCs exposed to microvesicles from 10 patients with locally advanced pancreatic cancer. We estimated the amount of peptides injected into the mass spectrometer was less than 1 µg according to the signal intensity in mass spectrometry. In total, 4102 proteins were identified (Supplemental Table S-2), where 35 proteins were up-regulated with 27 down-regulated in CMTCs (see Table 1) based on the consistent results of all samples. Since a Venn diagram cannot accommodate 10 groups, we have included a Venn diagram of 5 groups (Supplemental Figure S-2), where every group consists of 2 different patients. More than 80% of total proteins were identified in all these 5 groups. The differentially expressed proteins from the cytoplasm, nucleus, plasma membrane and extracellular space account for 53.23%, 25.81%, 9.68% and 6.45%, respectively (Figure 5a). There were three key proteins CD44, PPP2R1A and TP53 (see Table 1) that attracted our attention. The Western blot results of 2 samples looked similar and verified the quantitative results of mass spectrometry (Figure 5b). Among these differentially expressed proteins, the IPA database 14 ACS Paragon Plus Environment


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indicated that 31 proteins had direct interactions (Figure 5c). Among the differentially expressed proteins, PL53, NCOR2, PPP2R1A, MAPRE1, CTTN, CD44, TP53, PA2G4 and TXN were found to be involved in cell proliferation. In addition, the canonical pathway of Integrin Linked Kinase (ILK) was found to be involved in the cell proliferation process (Supplemental Figure S-3).

Interaction of differentially expressed proteins in PANC-1 cells and those in microvesicles In our previous study, circulating exosomal proteomes from 10 patients with locally advanced pancreatic cancer were compared with those from healthy controls. In total, 99 differentially expressed proteins were identified between cancer microvesicles and healthy microvesicles (Supplemental Table S-3) in all patients.22 Based on the fusion of microvesicles and recipient cells, these differentially expressed proteins entered PANC-1 cells and remodeled them. As a result, cell migration and proliferation of PANC-1 cells were accelerated by cancer microvesicles. We found 62 differentially expressed proteins (35 up-regulated and 27 down-regulated proteins) in CMTCs compared to HMTCs. The 62 differentially expressed proteins are different from those 99 proteins except for one protein namely Apolipoprotein B-100 (APOB) which is down-regulated in both cancer groups. The question is how the exosomal protein cargos influenced the recipient cells. We investigated the relationship of the differentially expressed proteins in different microvesicles and those in their recipient cells. We combined the two groups of proteins (99 that were in microvesicles and 62 that were in recipient cells) and analyzed using the IPA database to reveal the possible pathways in which these proteins participated. In addition to the ILK pathway, we also found that the Remodeling of Epithelial Adherens Junctions (REAJ) pathway (Supplemental Figure S-4) was likely involved in cell migration.


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Discussion Recent studies have shown that microvesicles derived from several types of cancer cells such as colorectal carcinoma, breast carcinoma, and nasopharyngeal carcinoma, are able to promote cell migration or/and proliferation.34-37 In this study, we tested the effects of circulating microvesicles from pancreatic cancer patients on these cellular processes. Circulating microvesicles from pancreatic cancer patients may only contain a small percentage of carcinoma-derived microvesicles. The effects of migration or proliferation were not significant when we exposed PANC-1 cells to a concentration of 2×108/mL of circulating microvesicles. However, after we increased the microvesicle concentration to 5×109/mL, we found the migration and proliferation of PANC-1 cells was significantly enhanced. We performed iTRAQ-based quantitative proteome analysis of HMTCs and CMTCs. We first optimized the FASP procedure to increase the recovery of proteins. Although it was a minor modification, it did increase the number of identified proteins and simplified the procedure. The quantitative proteomics analysis identified 62 differentially expressed proteins between HMTCs and CMTCs, where the IPA database indicated that PL53, NCOR2, PPP2R1A, MAPRE1, CTTN, CD44, TP53, PA2G4 and TXN were involved in cell proliferation. TP53 is a well-known tumor suppressor. It suppresses tumorigenesis mainly through the cell cycle arrest pathway and apoptosis pathway. CD44 is a multifunctional cell surface protein. In pancreatic cancers, a higher level of CD44 has been associated with more aggressive disease. It has been reported as a marker of pancreatic cancer stem cells (CSCs).38 Thus, down-regulated TP53 and up-regulated CD44 in CMTCs in the present study are concordant with the increase of cell proliferation. PPP2R1A is a scaffolding subunit of protein phosphatase 2A (PP2A). Inhibiting PP2A makes pancreatic cancer sensitive to radiotherapy both in vitro and in vivo by activation of CDC25C/CDK1 and inhibition of HRR,39 suggesting the up-



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regulation of PP2A has a positive relation with the progression of pancreatic cancer. Thus the upregulation of PPP2R1A in CMTCs in the present study is consistent with the cell proliferation result. Senescent cell-derived microvesicles have been shown to promote cancer cell proliferation, where EphA2 activated the Erk pathway through EphA2/ephrin-A1 reverse signaling.40 In the current study, we found cancer cell-derived microvesicles might promote cell proliferation by the ILK pathway. Integrin linked kinase (ILK) is a signaling protein, where various domains of ILK interact with different proteins. It connects integrins to the cytoskeleton and transduces signals from the extracellular matrix and growth factors. Here up-regulated PP2A in CMTCs enhanced the activation of ILK. Overexpression of ILK in vivo promotes tumorigenesis.41 Silencing of ILK decreases the abnormal proliferation and migration of human Tenon’s capsule fibroblasts.42 It has important roles in cancer progression, and has emerged as a therapeutic target in cancer.43 Gemcitabine is an anti-metabolic chemotherapy drug used to treat several types of cancer including pancreatic cancer. It attenuates DNA synthesis in rapidly dividing cells.44 Our cell proliferation assay showed that the effects of cancer microvesicles and gemcitabine on PANC-1 cells are antagonistic. Cargos in cancer microvesicles promoted cell proliferation and division by the ILK pathway, while gemcitabine hampered this procedure. However, blocking the creation of new DNA results in unwanted adverse effects.45 Normal tissues, especially bowel, bone marrow, liver, and kidneys can be adversely affected. In the 99 changed microvesicle proteins, ACTB, ACTC1, CFL1 might drive target proteins MYL6 and PPP2R1A to change in the ILK pathway in PANC-1 cells. The suppression of the ILK pathway, such as inhibiting PPP2R1A may be an alternative choice to block cell proliferation with less toxicity.


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In the Remodeling of Epithelial Adherens Junctions (REAJ) pathway, cadherins mediate homotypic cell-cell adhesion.46 The E-cadherins in the membrane of adjacent cells are connected together by calcium. In the inner side of the membrane, E-cadherin binds to catenins providing anchorage to the actin cytoskeleton to form stable cell-cell contacts. It also associates with zyxin, vinculin and αactinin, which in turn associates with F-actin. The cadherin-catenin mediated cell-cell adhesion leads to directional cell migration.47 In our results, zyxin and F-actin were both up-regulated in the CMTCs suggesting the cell migration was accelerated by the treatment of cancer microvesicles. In the 99 changed microvesicle proteins, ACTB, ARPC3, ARPC4, APRC18, ACTC1, MAPRE2, TUBA4A and ZYX might drive target proteins ACTR3 and MAPRE1 to change in the REAJ pathway in PANC-1 cells.



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Conclusions In this work, we found that microvesicles derived from patients with pancreatic cancer accelerate cellular processes such as migration and proliferation. Compared with HMTCs, the proliferation of CMTCs was inhibited more significantly by a low concentration (0.1 µM) of gemcitabine but less significantly by high concentration (10 µM). The results may suggest that CMTCs are more sensitive to gemcitabine but more difficult to kill than HMTCs. We optimized the filter-aided sample preparation (FASP) method to increase the protein recovery and used it to prepare PANC-1 cells for mass spectrometry analysis. In total, 4102 proteins were identified by LC-MS/MS, where 35 proteins were up-regulated with 27 down-regulated in CMTCs relative to HMTCs. We verified the quantitative mass spectrometry results of three key proteins CD44, PPP2R1A and TP53 in cell proliferation by Western blot. Ingenuity Pathway Analysis (IPA) suggests that the cancer microvesicles might participate in the pathways of REAJ and ILK in PANC-1 cells to promote cell migration and proliferation.


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Supporting Information The following files are available free of charge at ACS website http://pubs.acs.org: Table S-1: Proteins not identified in HMTCs but in PANC-1 cells without exposure. (PDF) Table S-2: Quantitative proteome analysis of HMTCs and CMTCs. (XLSX) Table S-3: Differentially expressed proteins between cancer microvesicles and healthy microvesicles. (PDF) Figure S-1: Morphology and size distribution comparison of microvesicles captured by antibodyconjugated gold beads from pancreatic cancer patients and healthy controls. (PDF) Figure S-2: A Venn diagram of 5 groups, where each group consists of 2 patients. (PDF) Figure S-3: The canonical Integrin Linked Kinase (ILK) pathway. (PDF) Figure S-4: The Remodeling of Epithelial Adherens Junctions (REAJ) pathway. (PDF)



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Acknowledgments This work was supported by the National Institutes of Health under grant R01GM49500 (DML) and the National Cancer Institute under grants P5OCA130810 (TSL) and R21CA189775 (DML). We acknowledge the assistance of the Wayne State University Proteomics Core that is supported through National Institutes of Health grants P30 ES020957, P30 CA 022453 and S10 OD010700.


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Figures and Tables Figure 1: Schematic overview of migration and proliferation assays, the sample preparation and LC-MS/MS analysis of HMTCs and CMTCs. Microvesicles were isolated from serum of patients with pancreatic cancer or commercial healthy serum using several steps of differential centrifugation. Then cancer microvesicles or healthy microvesicles were exposed to PANC-1 cells and migration and proliferation assays were performed. Next, proteins from HMTCs and CMTCs were extracted, alkylated and digested in ultrafiltration tubes using the filter-aided sample preparation (FASP) method. After iTRAQ labeling, peptides were mixed and run on a mass spectrometer. Database search of raw data and statistical analysis identified the differentially expressed proteins. Finally, IPA database revealed the network of directly interacting proteins and possible pathways involved in cell migration and proliferation.

Figure 2: TEM images and the size distribution of the microvesicles. The TEM images showed that the circulating microvesicles from pancreatic cancer patients and healthy controls had similar morphology (a, b) and size distribution (c).

Figure 3: Comparison of protein coverage and identified peptide number by histograms. (a) Elution efficiency comparison of 500 mM NaCl and water on eluting 0.1 μg, 1 μg or 10 μg of BSA digests. There is no significant difference between NaCl and water on elution efficiency. (b) Different amounts of BSA (0.1 μg, 1 μg or 10 μg) were eluted 6 rounds; peptides still could be identified by mass spectrometry in the seventh eluent. (c) The former six eluents of digest (Sample A) and digest on the top of filter (Sample B) were collected and identified. We found 6% - 8% more peptides were identified in Sample B. There were 3 replicates for each assay.


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Figure 4: Microvesicles from pancreatic cancer patients increase the migration and proliferation of PANC-1 cells. PANC-1 cells were exposed to microvesicles isolated from patients with pancreatic cancer or healthy control for 24 hours before the migration and proliferation assay. We used pooled microvesicles in this assay due to limited amount of serum from each patient. (a) CMTCs migrated faster than HMTCs or PANC-1 cells without exposure. Error bars represent SD (n=3). (b) CMTCs have higher proliferation activity. (c) Compared with HMTCs, CMTCs were more resistant to treatment with gemcitabine.

Figure 5: Western blot verification and subcellular and functional analysis of differentially expressed proteins between CMTCs and HMTCs Among 4102 quantified proteins, 62 proteins had GO information. (a) The subcellular distribution shows that proteins from cytoplasm, nucleus, plasma membrane and extracellular space account for 53.23%, 25.81%, 9.68% and 6.45%, respectively. (b) Western blot verified the quantification results of CD44, TP53 and PPP2R1A by mass spectrometry. (c) The IPA analysis shows that 31 proteins have direct interactions. Red and green represent up- and down-regulation in CMTCs, respectively.



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Table 1. The differentially expressed proteins between CMTCs and HMTCs. Compared with HMTCs, 35 proteins were up-regulated with 27 down-regulated in CMTCs. The blue and red represent down-regulated and up-regulated, respectively. Accession P07237 P31939 P10599 Q9UQ80 Q00839 Q16881 O95994 P17987 P49411 P51571 P42704 Q16629 Q14112 P05187 Q9Y224 Q8IVM0 P04114 P61158 O95202 Q8N715 Q9UMZ3 Q8N9N8 Q96FQ6 Q96I13 P05204 P20340 P04637 P20042 Q13838 P11586 P17844 Q9UHD1 P23526 P43243 P16070 P55084 P30044 Q14247 P17812 Q9H0H5 O76003 Q15691 Q14204 Q1KMD3 P50454 O00410 P62158 P30153 Q16891 Q04917

Protein name Protein disulfide-isomerase Bifunctional purine biosynthesis protein PURH Thioredoxin Proliferation-associated protein 2G4 Heterogeneous nuclear ribonucleoprotein U Thioredoxin reductase 1, cytoplasmic Anterior gradient protein 2 homolog T-complex protein 1 subunit alpha Elongation factor Tu, mitochondrial Translocon-associated protein subunit delta Leucine-rich PPR motif-containing protein, mitochondrial Serine/arginine-rich splicing factor 7 Nidogen-2 Alkaline phosphatase, placental type UPF0568 protein C14orf166 Coiled-coil domain-containing protein 50 Apolipoprotein B-100 Actin-related protein 3 LETM1 and EF-hand domain-containing protein 1, mitochondrial Coiled-coil domain-containing protein 185 Phosphatidylinositol phosphatase PTPRQ Probable RNA-binding protein EIF1AD Protein S100-A16 Abhydrolase domain-containing protein 8 Non-histone chromosomal protein HMG-17 Ras-related protein Rab-6A Cellular tumor antigen p53 Eukaryotic translation initiation factor 2 subunit 2 Spliceosome RNA helicase DDX39B C-1-tetrahydrofolate synthase, cytoplasmic Probable ATP-dependent RNA helicase DDX5 Cysteine and histidine-rich domain-containing protein 1 Adenosylhomocysteinase Matrin-3 CD44 antigen Trifunctional enzyme subunit beta, mitochondrial Peroxiredoxin-5, mitochondrial Src substrate cortactin CTP synthase 1 Rac GTPase-activating protein 1 Glutaredoxin-3 Microtubule-associated protein RP/EB family member 1 Cytoplasmic dynein 1 heavy chain 1 Heterogeneous nuclear ribonucleoprotein U-like protein 2 Serpin H1 Importin-5 Calmodulin Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha isoform Mitochondrial inner membrane protein 14-3-3 protein eta

Abbreviation

p value

log2(CMTCs/HMTCs)

P4HB ATIC TXN PA2G4 HNRNPU TXNRD1 AGR2 TCP1 TUFM SSR4 LRPPRC SRSF7 NID2 ALPP C14orf166 CCDC50 APOB ACTR3

0.006176 0.000673 0.001671 0.00191 0.000424 0.002643 0.000738 0.003758 9.83E-05 0.000953 0.000225 1.39E-06 0.000129 0.004714 0.008743 0.005208 0.002248 0.007959

-5.46123 -5.39358 -3.81445 -3.78786 -3.78086 -3.44095 -3.06049 -2.98979 -2.96518 -2.95912 -2.45097 -2.39883 -2.25182 -2.17463 -1.83512 -1.70429 -1.47771 -1.47413

LETM1 CCDC185 PTPRQ EIF1AD S100A16 ABHD8

0.004901 0.009593 0.006533 0.00411 0.003489 0.001347

-1.30823 -1.30689 -1.27531 -1.1958 -1.11726 -1.1119

HMGN2 RAB6A TP53 EIF2S2 DDX39B MTHFD1 DDX5 CHORDC1 AHCY MATR3 CD44 HADHB PRDX5 CTTN CTPS1 RACGAP1 GLRX3 MAPRE1 DYNC1H1 HNRNPUL2 SERPINH1 IPO5 CALM1

0.000283 0.007328 9.45E-06 0.003331 0.00506 1.64E-05 0.009201 0.001193 0.000506 4.21E-05 0.000256 0.000465 0.004756 0.005807 0.000178 0.000123 3.17E-06 0.003605 0.003087 0.000377 0.000736 0.007701 0.004363

-1.07958 -1.0464 -1.02266 1.062531 1.08455 1.101048 1.10227 1.110927 1.1153 1.119641 1.130534 1.147197 1.164929 1.177613 1.388005 1.522765 1.661114 1.70147 1.701699 1.712421 1.726279 2.071047 2.12305

PPP2R1A IMMT YWHAH

7.52E-05 0.002204 0.000902

2.129178 2.174018 2.192749


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Q9UBQ7 P14868 Q6PIU2 Q01082 P31946 P31947 P60660 P13667 Q9Y618 P34932 P13797 P52272

Glyoxylate reductase/hydroxypyruvate reductase Aspartate--tRNA ligase, cytoplasmic Neutral cholesterol ester hydrolase 1 Spectrin beta chain, non-erythrocytic 1 14-3-3 protein beta/alpha 14-3-3 protein sigma Myosin light polypeptide 6 Protein disulfide-isomerase A4 Nuclear receptor corepressor 2 Heat shock 70 kDa protein 4 Plastin-3 Heterogeneous nuclear ribonucleoprotein M

GRHPR DARS NCEH1 SPTBN1 YWHAB SFN MYL6 PDIA4 NCOR2 HSPA4 PLS3 HNRNPM


0.003872 0.00541 0.008315 0.001524 0.001868 2.44E-07 0.003148 0.006502 0.002516 0.007072 0.000567 0.005324

2.199724 2.218161 2.237665 2.244113 2.411062 2.485867 2.707021 2.844721 2.887301 3.339028 3.353014 3.824637


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For TOC Only HMTCs and CMTCs were compared with respect to cell migration and proliferation. Differentially expressed proteins were identified. The IPA database revealed the network of directly interacting proteins and possible pathways involved in cell migration and proliferation.


Circulating Microvesicles from Pancreatic Cancer Accelerate the

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