Cancer Genetics and Cytogenetics 178 (2007) 1e10

Identification of brain-derived neurotrophic factor as a novelangiogenic protein in multiple myeloma

Yu Hu1,*, Ya-dan Wang1, Tao Guo, Wen-ning Wei, Chun-yan Sun, Lu Zhang, Jin HuangInstitute of Hematology, Union Hospital, Tongji Medical College, Huanzhong University of Science and Technology,

1277 Jiefang Dadao, Wuhan 430022, P.R. China

Received 19 February 2007; received in revised form 13 May 2007; accepted 15 May 2007

Abstract Patients with multiple myeloma (MM) have increased bone marrow angiogenesis, but the angio-genic properties of myeloma cells and the mechanism of MM-induced angiogenesis have not beencompletely clarified. The brain-derived neurotrophic factor (BDNF) and its high-affinity receptor,TrkB, have been identified as critical factors in the regulation of vessel formation. In this study,we demonstrate that patients with MM had increased BDNF and vascular endothelial growth factor(VEGF) in their peripheral blood. We also found in particular that a decreased BDNF level wascorrelated with the remission of MM. BDNF was expressed by the human myeloma cell lineRPMI8226 and primary myeloma cells, and TrkB was expressed by human umbilical vein endothe-lial cells (HUVEC) at the protein levels. In a coculture system, we observed that both RPMI8226cells and primary myeloma cells induced the migration and formation of a net-like structure in HU-VEC. The anti-BDNF monoclonal antibody significantly but partially restrained the angiogenesiseffect of MM cells. Moreover, in an experimental model of angiogenesis in vivo, BDNF and VEGFsignificantly promoted vessel formation in Matrigel plug compared to the control. These effectswere also blocked by anti-BDNF monoclonal antibody. Finally, our in vitro results were supportedby the in vivo finding in human myeloma xenograft NOD/SCID models. Anti-BDNF mAb treat-ment resulted in inhibition of tumor growth, decreased vessel density, and tumor necrosis. Our studysuggested that the BDNF/TrkB signaling pathway could be involved, at least in part, in MM-induced angiogenesis. � 2007 Elsevier Inc. All rights reserved.

1. Introduction

Multiple myeloma (MM) is a B-cell malignancy charac-terized by the accumulation of malignant plasma cells inthe bone marrow [1]. The interaction of MM cells withthe microenvironment has been postulated to be critical inthe pathogenesis of MM [2]. In particular, recent evidenceunderscores the potential role of angiogenesis in the pro-gression of MM. It has been demonstrated that patients withMM with active disease have increased BM angiogenesiscompared with those in remission or with subjects withmonoclonal gammopathy of undetermined significance(MGUS) [3e5]. In addition, it has been shown that bonemarrow angiogenesis is correlated with the prognosis andsurvival of patients with MM [6]. A number of cytokines,

* Corresponding author. Tel.: þ86-27-85726335; fax: þ86-27-

85776343.

E-mail address: hillaryw27@yahoo.com.cn (Y. Hu).1 These authors contributed equally to this work.

0165-4608/07/$ e see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.cancergencyto.2007.05.028

including vascular endothelial growth factor (VEGF), basicfibroblast growth factor, and angiopoietin-1, were shown topromote MM angiogenesis in vitro and in vivo [7]. Themechanisms of MM-induced angiogenesis, however, havenot been completely clarified.

Brain-derived neurotrophic factor (BDNF) is a member ofthe neurotrophins superfamily that promotes the growth, sur-vival, and differentiation of developing neurons in the centraland peripheral nervous systems [8]. Despite the well-knowneffects on neurons, recent studies found that BDNF and itshigh-affinity receptor TrkB are also essential for the initiationof vascular development. BDNF deficiency results in a reduc-tion in endothelial cellecell contacts and in endothelial cellapoptosis [9]. Furthermore, BDNF was produced in numer-ous solid tumors and hematologic malignancies [10e14].In particular, it has been demonstrated that BDNF treatmentsignificantly induced the tube formation of human umbilicalvein endothelial cells (HUVEC) in vitro [15]. Plasma cells innormal human bone marrow express BDNF at low levels.These observations have raised the possibility that the

2 Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

BDNF/TrkB signaling pathway may modulate angiogenesisin MM.

In this study, we investigated the BDNF/TrkB signalingpathway in the human myeloma cell line RPMI8226, aswell as in patients with MM, and evaluated its relationshipwith MM-induced angiogenesis.

2. Materials and methods

2.1. Patients

According to the classification of Salmon and Durie, allof 27 patients had stage III MM. There were 21 males and 6females with a median age of 61 years (range 5 41e81). Atotal of 5/27 patients were newly diagnosed. The other 22patients were treated previously with at least two lines ofchemotherapy [VAD (vincristine, doxorubicin, and dexa-methasone), MP (melphalan and prednisone), or M2 (mel-phalan, prednisone, vincristine, and cyclophosphamide)].The disease course ranged from 2 to 25 months. These 22patients were stratified on the basis of disease status (pro-gressive versus stable). The patients in the progressive stageincluded those whose M-protein level increased rapidly,whose tumor cells invaded out of bone marrow, or whoseserum creatinine was 176.8 mM or higher. The patients’characteristics are described in Table 1. A total of 20healthy volunteers, including 13 males and 7 females witha median age of 59 years (range 5 52e77), were used ascontrols (Table 2). Volunteers with history of cerebrovascu-lar diseases, malignant tumors, hepatic and renal dysfunc-tions, or diabetes were excluded. Peripheral blood (PB)and bone marrow (BM) aspirates were obtained from eachpatient, and PB was obtained from each volunteer after in-formed consent was given, according to the tenets of theDeclaration of Helsinki.

Before induction, complete blood count, differentialwhite blood cell count, hemoglobin, serum b2-microglobu-lin, serum albumin, serum calcium, skeletal radiography,and hepatic and renal functions were assessed.

2.2. MM cell lines and patient cells

The human myeloma cell line RPMI 8226 was obtainedfrom the Cell Culture Facility, Chinese Academy of Medi-cal Sciences (Beijing, China). The cells were cultured inRPMI 1640 medium supplemented with 10% fetal bovineserum, 100 U/mL penicillin, and 100 mg/mL streptomycin.Before inoculation to mice, cells were washed and resus-pended in sterile phosphate-buffered saline (PBS) ata concentration of 1 � 108 cells/mL.

Primary myeloma cells (MC) were isolated from BMaspirate samples by positive immunomagnetic bead selec-tion using anti-CD138 antibodies and magnet-assisted cellsorting (MACS; Miltenyi Biotech, Bergisch Gladbach,Germany). Purity of primary MC (O95%) was assessedby both flow cytometry (FACSort; Becton-Dickinson,

San Jose, CA) d monitoring the expression of CD38,CD45, and forward and side-scatter characteristics of MCd and morphologic examination.

2.3. HUVEC isolation and culture

HUVEC were isolated from human cord veins and usedin passages 3e6 [16]. In brief, the untraumatized umbilicalcord segments, at least 20 cm in length, were cannulatedand perfused with 200e400 mL PBS to remove all tracesof blood, then the vein lumen was filled with PBS contain-ing 1 mg/mL collagenase I (Invitrogen, Grand Island, NY),and incubated at 37�C for 10 minutes. The contents of thevein were gently flushed with an equal volume of PBS andcollected in a centrifuge tube, centrifuged at 1,000 rpm for10 minutes, and resuspended in M199 medium supple-mented with 20% fetal bovine serum (GIBCO Life

Table 1

Patients’ characteristics

Patient no. Age Gender Stage * Isotype Bone lesions Status

1 67 M IIIA G/l Y P

2 81 M IIIA G/k Y S

3 59 M IIIA A/l Y S

4 63 M IIIA G/k Y S

5 70 F IIIA G/l N P

6 69 F IIIA G/l Y S

7 53 F IIIA G/k Y U

8 70 M IIIA G/l Y S

9 47 M IIIA A/k Y S

10 52 M IIIB G/l Y P

11 71 M IIIB A/l N S

12 61 F IIIB A/l Y P

13 60 M IIIA G/k Y S

14 69 M IIIB G/l Y U

15 59 M IIIA G/k N S

16 41 F IIIB A/l Y U

17 44 M IIIA G/l Y U

18 65 M IIIB G/l Y S

19 43 M IIIB G/l N U

20 62 M IIIB A/l Y S

21 67 M IIIA G/l Y P

22 59 M IIIA A/l Y S

23 69 F IIIA G/l Y S

24 67 M IIIA G/l Y P

25 59 M IIIA A/l Y S

26 44 M IIIA G/l Y S

27 59 M IIIA A/l Y S

Abbreviations: F, female; M, male; N, no; Y, yes; P, progressive

disease; S, stable disease; U, untreatment before.

* Clinical stage at diagnosis was classified according to the Durie-

Salmon staging system.

Table 2

Comparison of selected variables between patients with MM and healthy

controls

MM (n527) Healthy controls (n520) P

Gender (M/F) 21/6 13/7 0.333

Age (years, mean6SD) 60.4610.0 61.066.7 0.810

3Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

Technologies Paisley, UK), 50 ng/ml endothelial cellgrowth supplement (Becton-Dickinson, San Jose, CA),and 100 U/mL heparin, and then placed in a 25-cm2 plasticflask. After 12 hours, fresh medium was added. The me-dium was exchanged at 48-hour intervals thereafter. After6e8 days, the primary cultures formed uniform monolayersand 0.25% trypsin was used to harvest the cells. Factor VIIIstaining was performed using a labeled streptavidin-biotinperoxidase kit (Zhongshan Biotechnology, Beijing, China)to identify the specificity of endothelial cells.

2.4. Enzyme immunoassay for BDNF and VEGF

BDNF and VEGF concentrations in the culture superna-tant and plasma of MM patients were measured by a sandwichenzyme immunoassay kit (R&D Systems, Inc., Minneapolis,MN) according to the manufacturer’s instructions.

2.5. Western blot analysis for BDNF expression in MMcells and TrkB expression in HUVEC

The cells were washed twice with ice-cold PBS and har-vested using a cell scraper. The cells were lysed in 50mmol/L Tris-HCl with 1% sodium dodecyl sulfate and1% ß-mercaptoethanol. The lysate was then boiled for 1minute for protein denaturation. Glycerol was added to a fi-nal concentration of 10%, and bromphenol blue was addedbefore loading onto a 7.5% SDS-polyacrylamide gel. Afterelectrophoresis, the separated proteins were elecrotrans-ferred onto a nitrocellose membrane. The membrane wasblocked in 2% nonfat milk in tris buffered saline, pH 7.6,with 0.1% Tween 20 (TBS-T) for 1 hour at room tempera-ture with shaking. The membrane was then incubated for 1hour with mouse anti-BDNF monoclonal antibody (mAb)or with a mouse anti-TrkB mAb (Santa Cruz Biotech, SantaCruz, CA), washed extensively in TBS-T, and then incu-bated with horseradish peroxidaseelinked antibody (Amer-sham Biosciences, Freiburg, Germany). The signal wasvisualized by a chemiluminescent kit (Pierce Biotechnol-ogy, Inc., Rockford, IL).

2.6. Migration assay

Twenty-four-well Transwell inserts with a 5-mm poresize were coated with a thin layer of collagen (type I, rattail; BD Biosciences, San Jose, CA). MM cells and ECwere cultured in serum-free medium for 12 hours beforethe start of the experiment. MM cells or HUVEC werewashed twice with RPMI 1640 alone and plated into thelower chambers at a ratio of one cell (in insert) to one cell(in chamber cell number), except in the titration study.HUVEC were cultured for 6 hours before staining themembranes with hematoxylin and eosin. Cells were photo-graphed at �200 magnification, and four fields each fromthe duplicate samples were counted to quantify migration.In some experiments, the effect of anti-BDNF antibodyon HUVEC migration was examined. Anti-BDNF mAb

(Santa Cruz Biotech) or mouse IgG2b isotype controlmAb (Santa Cruz Biotech) was added to the coculture sys-tem at 250, 500, 750, and 1,000 ng/mL.

2.7. Matrigel network formation assay

The MM cells and HUVEC were cultured in serum-freemedium for 12 hours before the experiments. Cells werewashed two times with RPMI 1640 before coculturing inbasement membrane reconstruct gel (Matrigel). In thisstudy, we used growth factorereduced Matrigel (Becton-Dickinson) that had been depleted of a variety of growthfactors to minimize the effect of growth factors in thegel. The HUVEC and MM cells were adjusted at a ratioof 1:1, except in the titration study. The HUVEC were thencultured on Matrigel, whereas the MM cells were culturedwithin the Transwell inserts to prevent physical contact.The cells were cocultured and incubated with variousamounts of anti-BDNF mAb or control IgG for 20 hours.The number of net-like structures was analyzed in three dif-ferent fields at �100 magnification using Image-pro plus5.1 software.

2.8. Animals

Six-week-old male C57BL/6 mice and five-week-oldmale NOD/SCID mice were purchased from ShanghaiSLAC Laboratory Animal Co. Ltd. (Shanghai, China) andwere housed and monitored in a specific pathogen-free en-vironment with sterile food and water in our animal facility.All experiments were approved by the Committee on Ani-mals of Huazhong University of Science and Technology.

2.9. Matrigel plug assay

Matrigel plug assays were performed as described previ-ously, with modifications [17]. C57BL/6 mice were anes-thetized and injected subcutaneously at the abdominalmidline with 0.5 mL of the growth factorereduced Matrigel(Becton-Dickinson) supplemented with 400 ng/mL BDNF(R&D Systems) and 50 U/mL of heparin. As a positive con-trol, 100 ng/mL VEGF was added. Negative control micewere injected with Matrigel-added PBS. Anti-BDNF mAbor control mAb was administered intraperitoneally at 40mg/dose or 100 mg/dose every 3 days starting 24 hours afterMatrigel injection. Matrigel plugs were removed 14 daysafter implantation and prepared for histologic examination.

2.10. Human tumor xenograft models

RPMI 8226 cells (2 � 107 cells) were inoculated by sub-cutaneous injection in the left back of the mice. The micewere then divided into three groups: an untreated controlgroup (n55), an anti-BDNF mAb group (n55), and a con-trol mAb group (n55). The mAb were diluted into sterileendotoxin-free PBS at a concentration of 200 mg/mL. Theywere subsequently injected intraperitoneally at a dose of

4 Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

100 mg/mouse twice a week after 27 days. The tumor vol-ume was determined by measuring the longest (a) andshortest (b) diameter using a caliper and calculated usingthe formula: ab2p/6 (mm3). After 2 weeks of antibodytreatment, tumors were removed and prepared for histo-logic examination.

2.11. Assessment of microvascular density

Matrigel plugs and tumor tissue were fixed in 10% neu-tral buffered formalin overnight at 4�C, paraffin-embedded,and sectioned for examination of vessel density. Immunos-taining of microvessels was performed using the avidinebiotinylated peroxidase complex method, as describedpreviously in detail [18]. Vascular endothelial cells wereidentified with rat antiemouse CD34 (1:100; Biolegend,San Diego, CA) or with a rat antiemouse FVIII-RagmAb (1:400; Biolegend). Microvessels were counted fromthree separate, most highly vascularized areas (‘‘hot spots’’)according to Foss et al. [19]. The hot spots were identifiedby scanning tumor sections at a low-power magnification(�100) and then counted at a high-power magnification(�400). Any immunolabeled vessel that was clearly sepa-rate from an adjacent one and either totally inside the eye-piece or touching its border was counted as a microvessel.The microvascular density was analyzed by two investiga-tors in a blinded fashion by using double-headed lightmicroscopy and Image-pro plus 5.1 software. The area of�400 magnification is 0.216 mm2.

2.12. Statistical analysis

Experiments were performed in triplicate. All the con-tinuous data (e.g., age, BDNF, and VEGF) were presentedas the mean 6 standard deviation (SD) and analyzed by theStudent’s t test. Frequency data (e.g., gender) wereanalyzed by the chi-squared test. The associations were es-timated by multivariate linear regression models. Pearsoncorrelation analysis was used to evaluate the correlationvalue of PB-BDNF levels to PB-VEGF levels. The statisti-cal significance of the differences among the in vitro exper-iment data was determined using Student-Newman-Keulstest. P ! 0.05 was considered statistically significant.

3. Results

3.1. Cytokine measurements

To determine whether patients with progressive MM hadincreased levels of BDNF or VEGF in their PB, we mea-sured cytokine levels by ELISA from plasma and comparedthose with healthy controls. Myeloma patients had statisti-cally significant higher PB-BDNF (4.2260.64 ng/mL, P 5

0.010) and PB-VEGF (79.35613.25 pg/mL, P 50.006)levels, compared with healthy controls (BDNF, 2.0360.38ng/mL; VEGF, 34.4161.78 pg/mL). It showed decreased

levels of PB-BDNF (2.2960.67 vs. 5.1461.13 ng/mL,P 5 0.040) and PB-VEGF (46.2564.57 vs. 81.59635.33pg/mL, P 5 0.032) in 12/27 patients with remissive MMafter a 3-month-conventional treatment.

The analysis for variables (gender, age, and group) andPB-BDNF level was carried out by using a multivariate lin-ear regression model built with an enter selection proce-dure. The results in Table 3 showed a statisticallysignificant positive association between the level of PB-BDNF and group (P 5 0.014). However, no significantassociation of gender and age with the level of PB-BDNFwas found.

Further analysis for variables (gender, age, stage, status,albumin, hemoglobin) and PB-BDNF level was carried outby using a multivariate linear regression model built withan enter selection procedure. The results in Table 4 showedno statistically significant positive association between thelevel of PB-BDNF and variables.

Furthermore, we analyzed the correlation of the PB-BDNF levels to the PB-VEGF levels. The estimatedcorrelation coefficient was statistically significant for thePB-BDNF levels compared with PB-VEGF levels (r 5

0.430; P 5 0.025).

3.2. The expression of BDNF and its receptor, TrkB,by MM cells and HUVEC

To demonstrate that the BDNF/TrkB signal pathway hasa potential correlation with MM-induced angiogenesis, wefirst studied BDNF production by MM cells and the TrkBproduction by HUVEC. BDNF was detected in the culturesupernatant of RPMI 8226 (14.7462.45 ng/mL, at 24hours) and primary MM cells (12.3564.87 ng/mL, at 24hours). The expression of BDNF by RPMI 8226 and BDNFreceptor (TrkB) by HUVEC were confirmed by Westernblotting (Figure 1). The neuroblastoma cell was used asa positive control of BDNF and TrkB expression [20].

3.3. Coculture of HUVEC with MM cells activatesHUVEC migration and net-like formation, which aresuppressed by anti-BDNF mAb

To investigate the effects of RPMI8226 cells on EC an-giogenesis in vitro, we first used Transwell cell culturechamber inserts to restrict interactions between cell types

Table 3

Multivariate linear regression analysis of the association between PB-

BDNF and MM patients with gender and age

Variable Standardized regression coefficients t P

Constant 6853.555 2.398 0.021

Gender �0.093 �0.670 0.506

Age �0.225 �1.635 0.109

Group 0.351 2.554 0.014

The dependent variable is the BDNF (ng/mL) level of MM patients and

control; the independent variables include gender (0 5 male and 1 5 female),

age (continuous variable in years), group (0 5 control and 1 5 MM).

5Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

to soluble factors. At a 1:1 RPMI 8226/HUVEC ratio, weobserved an 88% increase in HUVEC migration in cocul-ture (126.3616.1) compared with HUVEC monoculture(67.269.2, P!0.01; Fig. 2, A and B). To examine thequantitative effect of RPMI 8226 cells for migration, HU-VEC were cocultured with different ratios of RPMI 8226cells. By decreasing the ratio of RPMI 8226 cells toHUVEC, the EC migration was significantly reduced(Fig. 2C). Similar results were obtained in the primaryMCeEC coculture system (Fig. 2C).

Next, using Transwell inserts with a pore size of 0.4 mmand no collagen coating, HUVEC were cultured in thechamber beneath coated by Matrigel with RPMI 8226 cellsin the inserts at a RPMI8226/HUVEC ratio of 1:1. Thepresence of RPMI 8226 cells increased the formation ofnet-like structures in the HUVEC compartment (Fig. 3).We noted that the morphology of the endothelial cells in-volved in the formation of net-like structures changed(Fig. 3 AeD). When cultured alone in the growth factorereduced Matrigel, HUVEC barely formed capillary struc-tures 20 hours after plating (Fig. 3D). However, HUVECcocultured with RPMI 8226 cells displayed a dramaticallyincreased network formation (Fig. 3, B and C). Within6e12 hours, HUVEC began to align themselves end-to-end and became elongated. At 20 hours after plating,the HUVEC showed abundant networks of branching andanastomosing cords of cells. The difference between cocul-ture (2164) and monoculture (461) was statistically sig-nificant (P50.027, Fig. 3C). By decreasing the ratio of

Table 4

Multivariate linear regression analysis of the association between PB-

BDNF level and variables for MM

Variable Standardized regression coefficients t P

Constant 12999.810 1.962 0.064

Gender 0.271 1.062 0.301

Age �0.461 �2.021 0.057

Stage �0.121 �0.529 0.603

Hb (g/L) 0.153 0.575 0.572

Alb (g/L) �0.118 �0.443 0.662

Status 0.202 0.846 0.408

The dependent variable is the BDNF (ng/mL) level of MM patients; the

independent variables include gender (0 5 male and 1 5 female), age (con-

tinuous variable in years), stage (0 5 IIIA and 1 5 IIIB), Hb (hemoglobin,

continuous variable), Alb (serum albumin, continuous variable), and status

(0 5 untreatment, 1 5 stable disease, and 2 5 progressive disease).

BDNF TrkB

Con RPMI8226 Con HUVEC

α-actin α-actin

Fig. 1. BDNF is expressed in RPMI8226 and its receptor is expressed in

HUVECs confirmed by western blotting. The neuroblastoma cells were

used as a positive control (Con) of BDNF and TrkB expression. a-actin

was used as a loading control.

RPMI8226 to HUVEC, the EC network formation was re-duced significantly (Fig. 3E). Similar results were obtainedin the primary MCeEC cocluture system (Fig. 3E). Thesedata suggest that tumor cells can induce endothelial cells todifferentiate into structures that resemble in vivoneovascularization.

In addition, the potential role of the BDNF in the MM-induced angiogenesis was investigated by added anti-BDNF mAb in the coculture system. As shown in Fig. 4,blocking BDNF by anti-BDNF mAb inhibited the EC mi-gration and formation of net-like structures induced byRPMI 8226 in a dose-dependent manner at lower concen-tration (! 750ng/mL). The significant but partial inhibitionof migration and net-like formation was observed after ex-posure to anti-BDNF antibody at 750 ng/mL, whereas noinhibition was detected after exposure to control antibody.These data demonstrate that BDNF are required for MM-induced endothelial angiogenesis.

3.4. Anti-BDNF mAb inhibits vascularizationof Matrigel plugs

To further demonstrate that BDNF can induce angio-genesis in vivo, we tested BDNF in Matrigel plugs. Ma-trigel supplemented with BDNF or VEGF was injectedsubcutaneously into C57BL/6 mice, forming semisolidplugs. Twenty-four hours later, mice were treated withanti-BDNF mAb or control IgG every 3 days for a totalof 14 days. Matrigel plug angiogenesis was quantitatedby histologic examination. Plugs without growth factorshad virtually no vascularization or vessel structures after14 days (Fig. 5A). In contrast, plugs supplemented with400 ng/mL BDNF had extensive vascularization andvessels throughout the plug (Fig. 5B), similar to whatthe plugs that had been supplemented with 100 ng/mLVEGF had (data not shown). Plugs taken from micetreated with 40 or 100 mg of anti-BDNF antibody hadmarkedly reduced vascularization of plugs (Fig. 5, Cand D). The anti-BDNF mAb treatment at 100 mg/dosesignificantly (74%; P ! 0.01) inhibited the amount ofmicrovessel in Matrigel plugs compared to control anti-body (Fig. 5E).

3.5. Anti-BDNF mAb treatment results in inhibitionof tumor growth, tumor necrosis, and decreasedvessel density

Finally, we supported our in vitro results by the humanmyeloma xenograft model in NOD/SCID mice. When theantibody treatment (100 mg/mouse) was started 27 days af-ter tumor inoculation for 50 days twice a week, as shown inFig. 6, the local tumor growth was partially inhibited. Thecontrol IgG preparation did not show any tumor inhibitoryeffect compared with untreated mice.

Histologic examination of human xenograft tumorstaken at certain time points during therapy demonstrated

6 Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

A

B

C

1.0

1.2

1.4

1.6

1.8

2.0

0.50 1 0.25

mig

rati

on in

dex

#

##

#

*

*

RPMI8226primary MC

Fig. 2. Effect of the MM cells on HUVECs migration in the co-culture systems. A, B. At a 1:1 RPMI8226:HUVEC ratio, an increase was observed in HU-

VECs migration in co-culture (B) compared with HUVEC monoculture(A). (�200 H&E). C. ECs were co-cultured with different ratios of MM cells for 6h.

The ratios of co-cultured MM cells to ECs were 0 (MM cells: EC 0:1), 1 (1:1), 0.5 (0.5:1) and 0.25 (0.25:1). Decreasing the ratio of MM cells to ECs reduced

EC migration. *P!0.01 vs. HUVECs alone; #P!0.05 vs. MM:EC 5 1:1.

dramatic differences in tumors from BDNF-Abetreatedversus control animals. Microvessel density was examinedin BDNF-Abetreated and control-treated tumors by immu-nostaining with anti-CD34 mAb. A marked decrease in

endothelial cells and vessel structures was observed after3 weeks of treatment in BDNF-Abetreated tumors com-pared to controls (Fig. 7, A and B). Quantitation of micro-vessels showed that density was reduced 88% (P 5 0.002)

C

BA

D

100

150

200

250

0.50 1 0.25

E

*

* #

#

#

N. o

f ne

tlik

e st

ruct

ure

(

of

cont

rol)

RPMI8226primary MC

Fig. 3. Effect of the MM cells on HUVECs network formation in the co-culture systems. A, B, C. RPMI8226 and ECs co-cultured for 6(A), 12 (B) and 20

h(C). ECs started to migrate and contacted each other at 6 h. Marked elongated EC network formation was observed at 20 h (�100). D. ECs cultured alone for

20 h. (�100) E. ECs were co-cultured with different ratios of MM cells for 20h. The ratios of co-cultured MM cells to ECs were 0 (MM cells: EC 0:1), 1

(1:1), 0.5 (0.5:1) and 0.25 (0.25:1). Decreasing the ratio of MM cells to ECs reduced EC network formation. *P!0.01 vs. HUVECs alone; #P!0.05 vs.

MM:EC 5 1:1.

7Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

0

50

100

150

200

250

250 500 750 1000

concentration of Ab(ng/ml)

0.0

0.5

1.0

1.5

2.0

2.5

250 500 750 1000

concentration of Ab(ng/ml)

mig

rati

on in

dex

control IgGanti-BDNF mAb

control IgGanti-BDNF mAb

A B

**

**

*

N. o

f ne

tlik

e st

ruct

ure

(

of

cont

rol)

Fig. 4. Anti-BDNF mAb inhibited the EC migration and network formation induced by RPMI 8226, *P!0.05 vs. control IgG.

by BDNF mAb treatment after 3 weeks. Similar resultswere obtained by immunostaining with anti-FVIII-RagmAb (data not shown). Accurate quantitation of tumor vas-culature was not possible beyond 3 weeks of treatment

because of the extensive tumor necrosis in BDNF-Abetreated tumors. A marked decrease in cellularity andlarge areas of necrosis replaced by fibrous tissue wasobserved in BDNF-Abetreated tumors (Fig. 7D).

0

5

10

15

20

25

30

35

Con

trol

BD

NF

BD

NF+

40µg

anti-

BD

NF

mA

b

BD

NF+

100µ

gan

ti-B

DN

Fm

Ab

BD

NF+

100µ

gco

ntro

l IgG

Num

bers

of

vess

el s

truc

ture

s(p

er s

ight

)

A B C D E

F*

*

Fig. 5. Anti-BDNF mAb inhibits vascularization of Matrigel plugs. C57BL/6 mice were injected s.c. at the abdominal midline with 0.5 ml of Matrigel with or

without BDNF. Anti-BDNF mAb or control antibody was administered i.p. every 3 days starting 24 h after Matrigel injection. Matrigel plugs were removed 14

days after implantation and prepared for histological examination. A-E, immunohistochemical staining of sections of Matrigel plugs with anti-CD34 antibody

shows reduced numbers of endothelial cells and vessel structures (arrows).�200. F, Matrigel plug vascularization was quantitated as described in ‘‘Materials and

Methods.’’ Anti-BDNF mAb significantly (P ! 0.01 at 100 mg compared to control IgG) reduced plug vascularization. *P!0.05 vs. control IgG.

8 Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

4. Discussion

It is known that patients with MM have increased BMangiogenesis because of the capacity of myeloma cells tostimulate vessel formation [3e6]. Several molecules havebeen identified as angiogenic factors in tumor cells. MMcells are known to produce VEGF, basic fibroblast growthfactor, angiopoietin-1, and matrix metalloproteinase [7,15],

0

100

200

300

400

500

tum

or v

olum

e (m

m3)

untreatedanti-BDNF IgGcontrol IgG

*

#

0 1 2 3

Weeks after mAb treatment

Fig. 6. Growth of s.c. RPMI8226 tumor xenografts in NOD/SCID mice is

inhibited by anti-BDNF mAb. Mice were injected subcutaneously with

RPMI 8226 cells at day 0. The anti-BDNF mAb or control IgG treatment

(100mg/dose) was started 27 days after tumor inoculation for 50 days twice

a week. There were five animals per group. *P ! 0.05 vs. the untreated

group. #P O0.05 vs. the untreated group.

but the entire spectrum of angiogenic properties is unknownand the mechanisms of MM-induced angiogenesis are yet tobe completely elucidated.

In this study, we focused our attention on BDNF, whichhad been detected in normal human bone marrow and haddemonstrated its angiogenic ability [9,15,21]. BDNF in-duces vessel sprouting and supports a long-lasting augmen-tation in vessel integrity and stability [9]. It also supportsneoangiogenesis in part through direct effects on localTrkB-expressing endothelial cells in skeletal muscle [22].Our study demonstrated for the first time that patients withprogressive MM have increased BDNF levels in their pe-ripheral blood, which is decreased in remissive patients.Moreover, both freshly purified MM cells and humanMM cell line RPMI 8226 produced BDNF as well as theHUVEC-expressed BDNF receptor TrkB. On the basis ofthis evidence, we propose the hypothesis that the produc-tion of BDNF by MM cells, together with the inductionof TrkB in endothelial cells, may contribute to the angio-genic properties of myeloma cells, in accordance with thephysiological role of BDNF/TrkB as a promoter ofangiogenesis.

This hypothesis was supported in two experimentalmodels of angiogenesis in vitro, by which we found thatBDNF blocking blunted the stimulatory effect of MM cellson EC migration and vessel formation. But blocking BDNFby anti-BDNF neutralizing antibody could not inhibit ECangiogenesis completely induced by either RPMI 8226 orprimary MC. This finding suggests that other angiogenic

Fig. 7. Tumors treated with anti-BDNF mAb have reduces vessel density in tumors and increased tumor cell necrosis. RPMI8226 tumors from mice treated

with control IgG (A, C) or anti-BDNF mAb (B, D) were resected after 3 week of antibody treatment, fixed, paraffin-embedded and sectioned at 5 mm. A, B.

Vessels were stained with anti-CD34 antibody. (� 400) C, D. H&E staining of paraffin sections showed extensive areas of necrosis (D) fibrosis in tumors from

animals of anti-BDNF mAb treatment.(� 400).

9Y. Hu et al. / Cancer Genetics and Cytogenetics 178 (2007) 1e10

factors from MM cells stimulate endothelial differentiation,in agreement with what previous studies have shown [3e6].Moreover, in an experimental model of angiogenesis invivo, the BDNF significantly stimulated vessel formationin the Matrigel plug compared with control as well asVEGF treatment, in agreement with evidence that deliveryof BDNF to adult mice can induce the recruitment of endo-thelial cells and the formation of rudimentary vascularchannels in the Matrigel model system [22]. These resultsalso confirmed that BDNF functions as an alternate angio-genic factor to modulate new vessel formation.

Our in vitro results were supported by the in vivo findingin human myeloma xenograft NOD/SCID models, whichhas high efficacy for growth of RPMI 8226 subcutaneoustumors and presents several pathologic features of plasma-cytomas (data not shown). Histologic examination of tu-mors from BDNF-mAbetreated animals show decreasedmicrovessel density as well as an increased necrosis inthe tumor cell fraction within the tumor. This finding isconsistent with previous studies showing that tumors be-come necrotic or apoptotic and/or fail to grow beyond2e3 mm3 in size in the absence of neovascularization [23].

Besides its role in angiogenesis, the BDNF/TrkB systemis involved in various tumor cell proliferations [24e26]. Infact, it was demonstrated that BDNF promotes TrkB-posi-tive MM cells growth and migration in vitro [27]. In addi-tion, the inhibition of tumor growth in BDNF-mAbetreatedmice has also been described in our study. It suggests thatBDNF might be a potential myeloma growth and chemo-tactic factor.

In this study, we used HUVEC to establish the coculturesystem. There are many different properties between thebone marrow EC of active MM and HUVEC, such as anti-gen profile (including that related to neovascularization),angiogenic ability in vitro and in vivo, genetic markers rel-evant for neovascularization, and ultrastructural morphol-ogy [28]. Therefore, the effect of BDNF on angiogenesisshould be confirmed with a coculture system with humanBM endothelial cell lines. In addition, our in vitro resultsshould be extended to patients with MM. Giuliani et al.[7] found that BM angiogenesis is significantly increasedin Ang1-positive patients compared with its occurrence inAng1-negative patients. It remains unknown whetherBDNF expression by MM cells is correlated with higherBM angiogenesis. We are currently conducting studies toaddress these questions. Weston et al. [29] used cDNA geneexpression microarray to analyze changes in gene expres-sion after stimulation of myometrial microvascular endo-thelial cells with VEGF. The BDNF gene was identifiedas one of 110 genes up-regulated by VEGF. We also ob-served that higher PB-BDNF levels are correlated withhigher PB-VEGF levels in MM patients. Further studiesmay be conduced to demonstrate the correlation at the genelevels between BDNF and VEGF.

In conclusion, our study demonstrates that myelomacells produce the trophic factor BDNF, and it underscores

the potential role of the BDNF/TrkB signalling pathwayin MM-induced angiogenesis. This pathway may contributeto the angiogenic effect of myeloma cells and could bea potential target for antiangiogenic therapy.

Acknowledgments

This work was supported by the National NaturalSciences Foundation of P.R. and the China Youth TalentFoundation of Hubei Province (Y.H.). We thank Dr. ZhongChen (University of Utah, Salt Lake City, UT) for assis-tance in manuscript preparation.

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