Int J Clin Exp Pathol 2017;10(1):224-232 www.ijcep.com /ISSN:1936-2625/IJCEP0041431 Original Article Effect of mesenchymal stem cell derived exosomes carrying PDGFD on lung cancer Feng Huang1, Yongliang Yao1, Jianhong Wu1, Liya Yu1, Shiyue Wu1, Xiongyong Pu1, Liang Xu1, Mei Wang2, Longfei Xia2 1Department of Clinical Laboratory, The First People’s Hospital of Kunshan Affiliated with Jiangsu University, Su- zhou, China; 2School of Medicine, Jiangsu University, Zhenjiang, China Received October 9, 2016; Accepted October 25, 2016; Epub January 1, 2017; Published January 15, 2017 Abstract: Mesenchymal stem cells (MSCs) are capable of promoting lung tumor progression, but the underlying mechanism remains unclear. In this study, we investigated the effect of bone marrow MSC-derived exosomes (MSC- exosomes) carrying PDGFD on in vitro and in vivo lung tumor growth and its underlying mechanism. MSC-exosomes carrying PDGFD promoted lung cancer cell migration, EMT, proliferation and PI3K signaling pathway. Recombinant PDGFD could mimic the effect of MSC-exosomes carrying PDGFD on lung cancer cells. These effects were abol- ished by a neutralizing antibody against PDGFD in vitro and in vivo. We found that MSC-exosomes were capable of regulating lung cancer cell progression through PDGFD MSC-exosomes carry. This study may pave the way for novel therapeutic strategy targeting the PDGFD pathway regulating MSC-exosome-mediated cell-cell interactions. Keywords: MSC, exosome, PDGFD, lung cancer cells Introduction and MSCs [10, 11]. Studies have shown that paracrine signals from MSCs significantly in- Non-small cell lung cancer (NSCLC) is the lead- fluence tumor cell behavior [12, 13]; however, ing cause of cancer-related mortality world- the mechanism underlying tumor progression wide. Due to rapid disease development, pa- mediated by MSCs is still not fully understood. tients with NSCLC have poor prognoses [1]. However, the molecular indicators for NSCLC Platelet-derived growth factors (PDGFs) are and its underlying mechanisms are still un- activated by two structurally related cell sur- clear. Recent studies have revealed that can- face receptors tyrosine kinases α-PDGF recep- cer associated fibroblasts (CAF) play important tor (PDGFR) and β-PDGFR, which regulate a roles in cancer microenvironment and malig- variety of cellular processes, including cell inva- nant transformation, and are closely related to sion, proliferation, migration and transforma- prognosis [2-4]. tion during pathogenesis [14]. PDGF-D is a newly identified isoform of PDGF and primarily Bone marrow derived mesenchymal stem cells interacts with β-PDGFR. It is up-regulated in (BM-MSCs) are multipotent progenitor cells various cancers, and plays an important role characterized by their capacity to self-renew in tumor metastasis, angiogenesis and growth and differentiate into various cell types such as [15, 16]. chondrocytes, osteocytes, adipocytes or fibro- blasts [5, 6]. MSCs are important sources of Exosomes are small, lipid bilayer membrane carcinoma-associated fibroblasts, and can be vesicles of endocytic origin. They are 50-100 recruited to tumor microenvironment, thereby nm in diameter and play an important role in differentiate into tumor-associated fibroblasts cell to cell communication. Exosomes consti- and constitute tumor stroma [7-9]. It has been tute an interaction network to synergistical- established that tumor progression partly de- ly promote cancer development [17, 18]. Exo- pends on the interaction between cancer cells somes from BM-MSCs contain a number of bio- Effect of MSC-derived exosomes carrying PDGFD on lung cancer active substances such as proteins, lipids, Isolation and identification of hBMMSC-Exo- mRNAs, and microRNAs (miRNA). It has been somes found that they are major players in intercellu- lar communication. However, the mechanisms When the MSCs reached 80%-90% confluence, involved in the promotional roles of exosomes they were cultured in conditioned medium for in tumor have not yet been fully elucidated. 48 h, and then the supernatants containing Therefore, the purpose of this study was to exosomes were harvested. The exosomes were determine whether exosomes carrying PDGFD isolated following the procedure described by mediated the malignant transformation of BM- Qu et al. with added modifications [20]. Briefly, MSC to lung cancer. the conditioned medium was collected and centrifuged at 1,000 g for 20 min to remove Materials and methods cell debris, followed by centrifugation at 2,000 g for 20 min and 10,000 g for 20 min. The Isolation and culture of hBM-MSC supernatant was collected and concentrated Human MSCs were isolated from the bone mar- using 100 KDa molecular weight cut off (MWCO) row of normal donors with informed consent (Millipore) at 1,000 g for 30 min. The concen- in accordance with the institutional guidelines trated supernatant was loaded upon 5 mL of under the approved protocol. hBM-MSCs were 30% sucrose/D2O cushions, and then ultracen- obtained and the characteristics of the isolat- trifuged at 100,000 g for 60 min (optimal-90K; ed hBM-MSCs were investigated as previously Beckman Coulter). The exosome enriched frac- described [18, 19]. Osteogenic and adipogenic tion was diluted with PBS, and then centrifuged differentiation assays were also performed on thrice at 1,000 g for 30 min using 100 KDa the MSCs, respectively. hBM-MSCs at passage MWCO. Finally, the purified exosomes were sub- 3 were used for the experiments. jected to filtration on a 0.22 μm pore filter (Millipore) and stored at -80°C. The protein con- Lung cancer cell lines culture tent of the concentrated exosomes was deter- mined using a BCA protein assay kit (Pierce). Lung cancer cells A549 were obtained from hBMMSCs-Ex was identified by transmission cell banks of Shanghai Institutes of Biological electron microscopy (FEI Tecnai 12; Philips), Sciences, and maintained in Dulbecco’s modi- and CD9 (Bioworld Technology, Inc. Louis Park, fied Eagle’s medium with low glucose (LG- MN) and CD81 (Epitomics, Burlingame, CA) DMEM, Invitrogen, Carlsbad, CA) supplement- were measured by western blotting. ed with 10% fetal bovine serum (FBS, Invitro- gen, USA), at 37°C in a humidified atmosphere Western blot analysis of 5% CO2. The cells were passaged every 2-3 days to maintain exponential growth. The cells were collected and lysed in RIPA buf- fer (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 Osteogenic and adipogenic differentiation of mM EGTA, 0.1% SDS, 1 mM NaF, 1 mM Na VO , hBM-MSCs in vitro 3 4 1 mM PMSF, 1 mg/ml aprotinin and 1 g/ml leu- hBM-MSCs in passage 3 were cultured in a peptin) on ice. Aliquots containing identical medium that contained osteogenic (0.1 mM amounts of protein were fractionated by sodi- dexamethasone, 10 mM b-glycerophosphate, um dodecyl sulfate-polyacrylamide gel electro- and 50 mM ascorbate-phosphate) or adipo- phoresis (SDS-PAGE) and then transferred onto genic (0.5 mM isobutylmethylxanthine, 1 mM methanol pre-activated polyvinylidene difluo- dexamethasone, 10 mM insulin, and 200 mM ride (PVDF) membranes (Millipore, Billerica, indomethacin) reagents, respectively. The rea- MA, USA). The membranes were blocked by gents were from Sigma-Aldrich. Two weeks la- 5% w/v non-fat dry milk. Following sequential ter, osteogenic differentiation was assessed by incubation with the primary and secondary the examination of neutrophil alkaline phos- antibody (Santa Cruz Biotechnology, Inc., USA), phatase (NAP) with the NAP staining kit (Sun the signal was visualized using HRP substrate Bio, China) and adipogenic differentiation was (Millipore) and analyzed using MD ImageQuant examined via intracellular lipid accumulation, Software. GAPDH (Kangcheng, China) was used which was visualized using Oil-Red-O staining as the loading control. The sources of the pri- by an inverted microscope (Olympus, Japan). mary antibodies were as follows: anti-FAP, anti- 225 Int J Clin Exp Pathol 2017;10(1):224-232 Effect of MSC-derived exosomes carrying PDGFD on lung cancer (PCNA) (Signalway Antibody Co., Ltd.). Enzyme-linked immunosor- bent assay PDGF-D in the Dulbecco’s modified Eagle’s medium with low glucose (LG-DMEM) gro- up, MSC-Ex group were mea- sured using ELISA kit (R& D Systems, USA) in accor- dance with the manufactur- er’s instruction. Assays were performed in duplicate, and readings were compared with standard curves obtained with standard proteins pro- vided with the kits. Transwell migration assay Following A549 cells treated with MSC-exosome for 72 h, A549 (1 × 105/well) were put into the upper chamber (8 mm) (Corning, NY, USA) in serum-free medium. The complete medium was pla- ced into the lower chamber. Following incubation at 37°C in 5% CO2 for 8 h, cells re- maining at the bottom of the polycarbonate membrane were wiped off with cotton swabs. The cells migrating to the lower surface of the mem- brane were then fixed with methanol for 30 min. The migrated cells were stained with crystal violet and count- Figure 1. Isolation and identification of MSCs and MSC-exosomes. A. Human BM-MSCs (a) were spindle-shaped and plastic adherent when maintained in ed in six random fields un- Dulbecco’s modified Eagle’s medium with low glucose supplemented with der the microscope (Olympus, 15% fetal bovine serum (magnification 200 ×; scale bar = 50 μm); hBM- Japan) for each assay. MSCs (b, c, d) express high levels of fibroblast proteins such as vimentin (b), FAP (c) and α-SMA (d) (Magnification 200 ×; scale bar = 50 μm); B. Adipo- Cell colony formation assay genic and osteogenic differentiation of hBM-MSCs. Oil-Red-O staining (a) of lipids in hucMSCs; Neutrophil alkaline phosphatase (NAP) staining (b) of huc- Following A549 cells treated MSCs. Cells were cultured for 14 days before staining (magnification 200 ×); with MSC-exosome for 72 h, C. Identification of hBMMSCs-exosome. Transmission electronic micrograph the A549 cells were trypsin- (a) of hBM-MSC-Ex, scale bar = 100 nm; Detection of hBMMSC-exosome CD9 and CD81 expression by western blot analysis (b).