Original Article Effect of Mesenchymal Stem Cell Derived Exosomes Carrying PDGFD on Lung Cancer

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 abolished 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 fibroblasts [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 buffer (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) group, MSC-Ex group were measured using ELISA kit (R& D Systems, USA) in accordance 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 membrane 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). ized and resuspended to a concentration of 1,000 cells/ 2 ml HG-DMEM with 10% FBS vimentin, anti-α-SMA (Abcam, USA); anti-N-cad- and were then incubated for two weeks. At the herin, anti-E-cadherin, anti-PDGFD (Santa Cruz end of the growth period, cells were fixed with Biotechnology, USA); PI3K, p-PI3K (phosphory- methanol for 30 min and stained with crystal lation of PI3K), proliferating cell nuclear antigen violet for 15 min. The cell colonies were photo-

226 Int J Clin Exp Pathol 2017;10(1):224-232 Effect of MSC-derived exosomes carrying PDGFD on lung cancer

After washed with PBS, the sections were then incubated with diluted secondary antibody for 20 min. Finally, sections were visualized with 3, 3’-diaminobenzidine (DAB) and then counterstained with hematoxylin for examination under a light microscope (Oly- mpus, Japan).

Animal model

Four to five-week-old BALB/c nude mice were purchased from Slac Laboratory Animal Center (Shanghai, China). Ani- mals were maintained in accordance with institutional Figure 2. MSC-exosome promoted lung tumor migration and growth. A. Rep- policies, and all studies were resentative images showing that MSC-exosomes improved A549 cell pen- performed with approval of etration through membrane (magnification 200 ×; scale bar = 50 μm); B. the University Committee on Cell colony formation assay showing that MSC-exosome improved the ability Use and Care of Animals of A549 cells to form colonies (**P < 0.01). of Jiangsu University. Animals were randomly divided into graphed and the number of colonies was 4 groups. The animals were injected subcuta- counted. neously with untreated A549 cells alone, A549 cells pretreated with MSC-exosome, A549 cells Immunofluorescence staining pretreated with MSC-exosome/NA-PDGFD and A549 cells pretreated with MSC-exosome/ Following hBM-MSCs at 3 passage cultured CtrlA-PDGFD into the backside of mice respec- for 48 h, the hBM-MSCs were washed 3 times tively. Tumor growth was evaluated by mea- with cold PBS, fixed with 4% paraformaldehyde suring the length and width of the tumor mass for 20 min, permeabilized with 0.1% Triton with calipers every 4 days. Tumor volumes were X-100 for 5 min, blocked with 5% BSA (Boster calculated by the modified ellipsoidal formu- Bioengineering, China) and incubated with anti- la: (length × width2)/2. Tumors were surgical- vimentin, anti-FAP, anti-α-SMA primary anti- ly removed, and photographed 40 days after body (Abcam, USA) at 4°C overnight and fol- injection. lowed by Cy3-conjugated anti-rabbit secondary antibody (Sigma, USA). The cells were then Statistical analyses stained with DAPI for nuclear staining, and All experiments were conducted at least in trip- images were acquired using a Nikon Eclipse licate. All data are expressed as the means ± Ti-S microscope. SD. SPSS 11.0 software was used to analyze Immunohistochemistry the data. The means of different treatment groups were compared by two-way ANOVA Formalin-fixed paraffin-embedded mouse tu- or the Student’s t-test. A P-value < 0.05 was mor tissue sections were first deparaffinized considered to indicate a statistically signifi- in xylene, rehydrated through graded ethanol. cant difference. The sections were boiled for 10 min in citrate Results buffer (pH 6.0, 10 mM) for antigen retrieval. The endogenous peroxidase activity was inhib- Isolation and identification of MSCs and MSC- ited with exposure to 3% hydrogen peroxide for exosomes 10 min. Then the sections were blocked with 5% BSA and incubated with proper diluted Human bone marrow MSCs were isolated and PCNA primary antibody (1:200) at 37°C for 1 h. confirmed positive for CD29, CD44, CD105,

227 Int J Clin Exp Pathol 2017;10(1):224-232 Effect of MSC-derived exosomes carrying PDGFD on lung cancer

Influence of MSC-exosome on lung tumor migration and growth

The function of paracrine interactions between BM-MSCs and A549 was evaluated in vitro by treating A549 cells with MSC-exosomes for 14 days. The A549 cell migration ability was evaluated by the transwell migration array. Af- ter A549 cells were cultured in the presence of MSC-exosomes for 2 weeks, their migration ability was significantly enhanced compared with the control group (Figure 2A). Moreover, the proliferation capacity of A549 cells was evaluated by the cell colony formation assay. Similarly, the results showed that cell proliferation was also significantly strengthened compared Figure 3. MSC-exosomes carrying PDGFD and the increased growth and mi- with the control group after gration of PDGFD treated lung tumor cells. A. Western blot analysis (a) show- A549 cells were treated with ing the expression of PDGFD in MSC-exosome (a). ELISA analysis showing (b) MSC-exosome for 2 weeks PDGF-DD level was much higher in MSC-exosomes than in the control cul- (Figure 2B). ture medium. B. Representative images showing that recombinant PDGF-DD (100 ng/ml) improved SGC-7901 cell penetration through membrane (mag- MSC-exosomes carrying nification, 200 ×;scale bar = 50 μm). C. Cell colony formation assay showing PDGFD and increased growth that PDGF-DD could improve the cell colony forming ability of SGC-7901 (**P < 0.01, ***P < 0.001). and migration of PDGFD- treated lung tumor cell and HLA-I surface antigen expressions, and To examine the molecular mechanism underly- negative for CD34, CD38 and HLADR surface ing MSC-exosome mediated promotion of antigen expressions. BMMSCs presented a tumor migration and proliferation, we investi- homogeneous population of spindle fibroblast- gated the expression of PDGFD in three differ- like cells. They possessed spindle shape and ent batches of MSC-exosome sources. Western plastic adherent characteristics (Figure 1A). blot and ELISA analyses revealed that PDGFD They also expressed high levels of fibroblast was highly expressed in exosome (Figure 3A). proteins, such as vimentin, fibroblast activation Because of the high expression of PDGFD in protein, FAP and α-SMA (Figure 1B-D). Von MSC-exosome, we investigated whether PDGFD Kossa staining and Oil Red O staining were was important for exosome in promoting the used to evaluate their osteogenic differentia- malignant transformation of lung cancer. To tion and adipogenic differentiation potential, evaluate the effect of PDGFD on lung tumor respectively. They showed spontaneous adi- cells, we performed migration and proliferation pocyte or osteoblast formation (Figure 2B). experiments after A549 cells were treated with MSC-exomes were isolated and purified using recombinant PDGFD for one week. The results differential centrifugation on a sucrose cush- showed that the migration of cells was signifi- ion. By transmission electronic microscopy, it cantly increased compared with the control was found that MSC-exosomes were 30-100- group (Figure 3B). We also found that the cell nm microvesicles (MVs) (Figure 3A). MSC-exo- cloning ability was significantly enhanced. The somes showed high levels of CD9 and CD81 untreated A549 cells were served as control in expressions (Figure 3B). these experiments.

228 Int J Clin Exp Pathol 2017;10(1):224-232 Effect of MSC-derived exosomes carrying PDGFD on lung cancer

chymal markers N-cadherin, Vimentin and α-SMA (Figure 4B). Similarly, compared with the control group, the proliferation ability of cells was significantly decreased in the PDGFD blocking antibody group (Figure 4C). In order to study the signal proteins associated with cell proliferation, we tested p-PI3K, PI3K and PCNA. Western blot analysis showed that the expressions of p-PI3K and PCNA were weakened in the PDG- FD blocking antibody group (Figure 4D).

Neutralizing antibody against PDGFD abolished MSC- exosome growth-promoting effect on lung cancer cells tumor xenografts in vivo

To evaluate the effect of PD- GFD-carrying exosomes on Figure 4. Neutralizing antibody against PDGFD greatly alleviated MSC-ex- lung tumor growth, we per- some-induced lung tumor progression in vitro. A. Transwell migration assay formed an animal model ex- histogram of migration cell number in the exosome group, the NA-PDGFD (neutralizing antibody against PDGFD)/exosome group, and the CtrlA-PDGFD periment using a neutraliz- (isotype-matched normal antidody) group. B. Western blot analysis showing ing antibody against PDGFD. the expressions of E-cadherin, N-cadherin, α-SMA, and Vimentin in A549 A549 cells were treated with cells in different groups. C. Cell colony formation assay histogram of A549 exosome in the presence of cells in the exosome group, the NA-PDGFD/exosome group, and the CtrlA- either a neutralizing antibody PDGFD group. D. Western blot analysis showing the expressions of PI3k, p- PI3k and PCNA in A549 cells in different groups (**P < 0.01). against PDGFD or a control normal antibody for one week before injection. Five weeks Neutralizing antibody against PDGFD greatly later, the xenograft tumors were removed, pho- alleviated MSC-exosome-induced lung tumor tographed and weighed. Our data revealed that progression in vitro the size of the tumor from the neutralizing antibody group was significantly smaller than those In order to study whether PDGFD was a key of the exosome group and the control antibody molecule in exosome mediated lung cancer group; however, it was approximately equal development, we added either a neutralizing to that in the control group (Figure 5A and 5B). antibody against PDGFD (NA-PDGFD) or an iso- In addition, we performed PCNA immunohis- type-matched normal antibody (CtrlA-PDGFD) tochemical staining on tissue sections from in the exosome group. Notably, this migration tumor tissue in each group. As shown by the effect was abrogated by a PDGFD-specific blo- increased number and intensity of PCNA-po- cking antibody, but not by a control antibody sitive cells, the results revealed that the per- (Figure 4A). The results of western blot analy- centage of PCNA-positive cells in the neutraliz- sis also showed that the expression of E-cad- ing antibody group was markedly lower than herin, an important feature of epithelial cell, those in the exosome group and the control was increased after A549 cells was treated antibody group (Figure 5C). with exosome in the presence of neutralizing Discussion antibody against PDGFD. The increased expression of E-cadherin was accompanied by de- Although MSCs is believed to be critically increased expressions of the important mesen- volved in tumor development and progression

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row MSCs was by confirming their diameters (50-100 nm) and the protein expressions of CD9 and CD81. Carrying proteins, DNAs, mRNAs, and non-coding RNAs, exosomes play critical roles in tumori- genesis [25], growth [26], angiogenesis [27], metastasis [28] and drug resistance [29]. At present, there is few research on the effect of MSC-exsomes on lung cancer. Therefore, we investigated whether MSC-exosomes promoted lung tumor migration and growth in vitro. In order to study the mechanism, we evaluated the expression of PDGFD in MSC- exsome. PDGFD is an isoform of the PDGF family, and may play a key role in the development and progression of human cancers [30]. The results of western blot and EL- ISA analyses showed high expression of PDGFD in MSC- Figure 5. Neutralizing antibody against PDGFD abolished the growth pro- exosome. moting effect of MSC-exosome growth-promoting on lung cancer cell tumor xenografts in vivo. A. Representative photographs of tumor tissues in different groups. B. The tumor volume of mice in different groups. C. Immu- To address the question of nohistochemical analysis of PCNA expression in tumor tissues in different whether MSC-exosome is me- groups. (a) Representative photomicrograph; (b) percentage of proliferating diated by PDGFD to promo- cell nuclear antigen (PCNA)-positive cells (magnification, 200 ×; scale bar = te the development of lung 200 μm) (**P < 0.01). cancer, firstly, we treated lung tumor cells A549 with recom- [21, 22], the exact mechanism is still not very binant PDGFD for one week. The results showed clear and poorly documented. The paracrine that the effect of recombinant PDGFD on lung interactions that involve tumor growth between tumor progression was similar to that of MSC- MSCs and cancer cells may be related to those exosome. Afterwards, we treated A549 cells responses. In addition to soluble factors, exo- with an antibody against PDGFD in the MSC- somes have also been deemed as an important exsome group. The results demonstrated that mechanism in the paracrine action of MSCs. the effect of MSC-exosome on migration and Recent studies have found that exosomes con- proliferation of tumor was greatly weakened tain functional nucleic acids and proteins when compared to that that in the non-treated/con- transferred into recipient cells, which indicated trol-antibody group in vitro. It has been sug- signal transmission for tumor progression [23, gested that epithelial-mesenchymal transition 24]. Here, we report an important finding that (EMT) and migration are closely related [31]. MSC-exosomes carrying PDGFD play a signifi- Besides, PI3k signaling pathway is important cant role in lung tumor progression. for cell proliferation [32]. Western blot analysis demonstrated that EMT and PI3K signaling is In this study, we cultured BM-MSCs from hu- significantly attenuated in the antibody against man bone specimens. The successful isolation PDGFD group. Consistent with the in vitro and purification of exosomes from bone - mar results, our in vivo data showed that tumors

230 Int J Clin Exp Pathol 2017;10(1):224-232 Effect of MSC-derived exosomes carrying PDGFD on lung cancer grew slower in the control and the antibody Foidart JM, Jerusalem G, Cataldo D and Noel against PDGFD group than in the other two A. Mesenchymal stem cells shed amphiregulin groups. Moreover, immunohistochemical analy- at the surface of lung carcinoma cells in a sis of PCNA expression in tumor tissues indi- juxtacrine manners. Neoplasia 2015; 17: 552- cated that percentage of PCNA-positive cells 563. [4] Bremnes RM, Donnem T, Al-Saad S, Al-Shibli K, was lower in the control and the antibody Andersen S, Sirera R, Camps C, Marinez I and against PDGFD group than in the other two Busund LT. The role of tumor stroma in cancer groups. These results suggest that PDGFD progression and prognosis: emphasis on carci- might play a key role in MSC-exosome medi- noma-associated fibroblasts and non-small ated lung tumor progression. Further studies cell lung cancer. J Thorac Oncol 2011; 6: 209- are needed to investigate whether there are 217. other mechanisms involved. [5] Huang J, Sha H, Wang G, Bao G, Lu S, Luo Q and Tan Q. Isolation and characterization of ex In summary, it was revealed in this study that vivo expanded mesenchymal stem cells ob- MSC-exosomes promote lung tumor progre- tained from a surgical patient. Mol Med Rep ssion through exosome carrying PDGFD. No- 2015; 11: 1777-1783. vel features of the complex dialogue between [6] Zhao K, Hao H, Liu J, Tong C, Cheng Y, Xie Z, Zang L, Mu Y and Han W. Bone marrow-derived MSC-exosome and tumor cells were discover- mesenchymal stem cells ameliorate chronic ed in this study. These finding establishes an high glucose-induced β-cell injury through unprecedented link between both. This could modulation of autophagy. Cell Death Dis 2015; be a lung tumor diagnostic biomarker and a 6: e1885. novel selective therapeutic strategy targeting [7] Quante M, Tu SP, Tomita H, Gonda T, Wang SS, PDGFD exosomes carrying. Takashi S, Baik GH, Shibata W, DiPrete B, Betz KS, Friedman R, Varro A, Tycko B and Wang TC. Acknowledgements Bone marrow-derived myofibroblasts contrib- ute to the mesenchymal stem cell niche and This work was supported by the Clinical Me- promote tumor growth. Cancer Cell 2011; 19: dicine Science and Technology Development 257-272. Fund of Jiangsu University (grant no. JLY- [8] Kidd S, Spaeth E, Dembinski JL, Dietrich M, Watson K, Kloop A, Battula VL, Weil M, Andreeff 20140055) and the Develop the Health th- M and Marini FC. Direct evidence of mesenchy- rough Science and Education of Suzhou You- mal stem cell tropism for tumor and wounding th Science and Technology Project (grant no. microenvironments using in vivo biolumines- kjxw2015052). cent imaging. Stem Cells 2009; 27: 2614- 2623. Disclosure of conflict of interest [9] Kidd S, Spaeth E, Watson K, Burks J, Lu H, Klopp A, Andreeff M and Marini FC. Origins None. of the tumor microenvironment: quantita- tive assessment of adipose-derived and bone Address correspondence to: Longfei Xia, School marrow-derived stroma. PLoS One 2012; 7: of Medicine, Jiangsu University, 301 Xuefu Road, e30563. Zhenjiang 212013, China. Tel: 86-15850327201; [10] McAndrews KM, McGrail DJ, Ravikumar N and E-mail: [email protected] Dawson MR. Mesenchymal stem cells induce directional migration of invasive breast can- References cer cells through TGF-β. Sci Rep 2015; 20: 16941-16943. [1] Guo S, Reddy CA, Chao ST, Suh JH, Barnett [11] Wang J, Wang Y, Wang S, Cai J, Shi J, Sui X, Cao GH, Vogelbaum MA and Videtic GM. Impact Y, Huang W, Chen X, Cai Z, Li H, Bardeesi AS, of non small cell lung cancer histology on sur- Zhang B, Liu M, Song W, Wang M and Xiang AP. vival predicted from the graded prognostic as- Bone marrow-derived mesenchymal stem cell- sessment for patients with brain metastases. secreted IL-8 promotes the angiogenesis and Lung Cancer 2012; 77: 389-393. growth of colorectal cancer. Oncotargel 2015; [2] Quail DF and Joyce JA. Microenvironmental 6: 42825-42837. regulation of tumor progression and metasta- [12] De Boeck A, Pauwels P, Hensen K, Rummens sis. Nat Med 2013; 19: 1423-1437. JL, Westbroek W, Hendrix A, Maynard D, Denys [3] Carnet O, Lecomte J, Masset A, Primac I, Maer- H, Lambein K, Braems G, Gespach C, Bracke tens L, Detry B, Blacher S, Gilles C, Paupert J, M and de Wever O. Bone marrow-derived mes-

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