[CANCER RESEARCH 59, 728–733, February 1, 1999]

Expression of Vascular Endothelial Growth Factor and Its Receptors inHematopoietic Malignancies1

William T. Bellamy, 2 Lynne Richter, Yvette Frutiger, and Thomas M. GroganDepartment of Pathology, University of Arizona, Tucson, Arizona 85724

ABSTRACT

Vascular endothelial growth factor (VEGF) plays an important role inangiogenesis by acting as a potent inducer of vascular permeability as wellas serving as a specific endothelial cell mitogen. The importance of angio-genic factors such as VEGF, although clearly established in solid tumors,has not been fully elucidated in human hematopoietic neoplasms. Weexamined the expression of mRNA and protein for VEGF in 12 humanhematopoietic tumor cell lines, representing multiple lineages and dis-eases, including leukemia, lymphoma, and multiple myeloma. Our resultsrevealed that VEGF message was expressed in these cells and that thecorresponding protein was secreted into the extracellular environment.Five of the 12 cell lines were also found to express the Flt-1 receptor forVEGF at a moderate to strong level, suggesting an autocrine pathway.When human vascular endothelial cells were exposed to recombinanthuman VEGF, there was an increase in the mRNA for several hemato-poietic growth factors including macrophage colony-stimulating factor,granulocyte colony-stimulating factor and interleukin 6. Plasma cells inthe bone marrow from patients diagnosed with multiple myeloma werefound to express VEGF, whereas both the Flt-1 and KDR high affinityVEGF receptors were observed to be markedly elevated in the normalbone marrow myeloid and monocytic cells surrounding the tumor. Thesedata raise the possibility that VEGF may play a role in the growth ofhematopoietic neoplasms such as multiple myeloma through either aparacrine or an autocrine mechanism.

INTRODUCTION

For solid tumors to grow beyond 2 mm3 in size, there is a strictrequirement for angiogenesis (1, 2). Tumor angiogenesis is influencedby a number of regulatory factors and although a wide variety ofgrowth factors with known angiogenic activity could have been eval-uated, we chose to focus initially on the expression of VEGF3 andbFGF, two growth factors that appear to play key roles in angiogen-esis (1, 3, 4). Because of its effects on endothelial cell growth andmicrovascular permeability, VEGF is believed to be an importantmediator of angiogenesis (5). A variety of malignant human tumors,including breast, lung, and prostate carcinomas, are known to secreteVEGF (6–9). In general, the levels of VEGF mRNA and proteinexpression in nonhematopoietic human tumors often correlate posi-tively with malignant progression (7, 10). Clinical studies have dem-onstrated that both the number and density of microvessels in severalhuman solid cancers may be directly correlated with invasion andmetastasis and often predicts an unfavorable prognosis in many cases(11–15). A central role of VEGF in tumor growthin vivo was

demonstrated by Kimet al. (16), who showed that the addition ofneutralizing antibodies to VEGF resulted in a marked suppression oftumor growth. In addition to its expression in neoplastic tissues,VEGF is expressed in normal cells including activated macrophages(17, 18), renal glomerular epithelial and mesangial cells (19, 20),platelets (21), and keratinocytes (22).

Although the importance of angiogenesis in solid tumors is wellestablished, its role in hematopoietic tumors is not. In a study of 88patients with B-cell non-Hodgkin’s lymphoma, Ribattiet al. (23) foundan increase in the microvessel density in lymph nodes that correlated withthe severity of the disease. A similar study of childhood acute lympho-cytic leukemia also revealed an increase in the microvessel density, asassessed by factor VIII-related antigen staining, in the bone marrow ofleukemic patients compared with that found in normal controls (24). Anincrease in bone marrow microvessel density was also observed in aseries of patients diagnosed with multiple myeloma compared withpatients with monoclonal gammopathies of unknown significance (25).Such findings, although not conclusive, are suggestive of a role forangiogenic growth factors in hematopoietic malignancies.

Although its expression was demonstrated previously in the HL-60promyelocytic leukemia cell line (26), it is not known whether VEGFor other angiogenic factors play a role in human hematopoietic ma-lignancies. The present studies were undertaken to identify whetherVEGF and its receptors were expressed in other hematopoietic ma-lignancies in general and in multiple myeloma in particular.

MATERIALS AND METHODS

Cell Culture Conditions. Human tumor cell lines were obtained from theAmerican Type Culture Collection (Rockville, MD). All tumor cell lines weregrown in RPMI 1640 supplemented with 10% fetal bovine serum, 1% (v/v)penicillin (100 units/ml), streptomycin (100mg/ml), and 1% (v/v)L-glutamine(all from Grand Island Biological Supply Co, Grand Island, NY) and main-tained at 37°C in 5% CO2-95% air atmosphere. The HUVEC line was obtainedfrom Dr. S. Williams (University of Arizona) and was maintained in endothe-lial growth medium-MV medium (Clonetics, San Diego, CA).

Northern Blot Analysis. Total cellular RNA was isolated from the cell linesusing guanidinium isothiocyanate and cesium chloride gradient centrifugationaccording to the method of Chirgwinet al. (27). RNA (5mg) was denatured at65°C for 10 min in 50% formamide, 6.5% formaldehyde, 40 mM 3-(N-morpho-lino)propanesulfonic acid, and subjected to electrophoresis on a 1% formaldehyde-agarose gel. The RNA was transferred by capillary action to a nylon membrane(Nytran; Schleicher & Schuell, Keene, NH) and immobilized by exposure to UVlight. Membranes were prehybridized in Rapid-hyb buffer (Amersham, ArlingtonHeights, IL) according to the manufacturer’s instructions. Hybridization wascarried out at 42°C for 24 h with32P-labeled cDNA probes generated by RT-PCR.Following high stringency washes, the filters were exposed to X-ray film (Kodak;XAR-5) at 280°C with an intensifying screen.

Detection of Angiogenic Molecules and Growth Factor Cytokines byRT-PCR. Total RNA isolated from exponentially growing cells was treatedwith an enzyme reverse transcriptase (SuperScript II; Gibco BRL, Gaithers-burg, MD) to generate cDNA; the cDNA was then amplified by the PCR usingTaq DNA polymerase (Life Technologies, Inc., Gaithersburg, MD). The prim-ers were designed to span intron-exon borders to distinguish amplified cDNAfrom genomic DNA. PCR primers used to detect VEGF were: 59-GAA GTGGTG AAG TTC ATG GAT GTC (forward) and 59-CGA TCG TTC TGT ATCAGT CTT TCC (reverse); flt-1, 59- GAG AAT TCA CTA TGG AAG ATCTGA TTT CTT ACAGT-39 (forward) and 59- GAG CAT GCG GTA AAA

Received 7/16/98; accepted 12/2/98.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grant CA-32102 from the National CancerInstitute and Grant ESO6694 from the National Institute of Environmental Health Sci-ences.

2 To whom requests for reprints should be addressed, at Department of Pathology,University of Arizona, 1501 North Campbell Avenue, Tucson, AZ 85724. Phone:(520) 626-6032; Fax: (520) 626-6463; E-mail: wbellamy@u.arizona.edu.

3 The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, basicfibroblast growth factor; HUVEC, human umbilical vein endothelial cell; RT-PCR,reverse transcription-PCR; hIL, human interleukin; CSF, colony-stimulating factor; GM-CSF, granulocyte-macrophage CSF; NBT, nitroblue tetrazolium; VMS, Ventana MedicalSystems.

728

TAC ACA TGT GCT TCT AG-39(reverse); and KDR, 59-CAA-CAA-AGT-CGG-GAG-AGG-AG-39 (forward) and 59-ATG-ACG-ATG-GAC-AAG-TAG-CC-39 (reverse; Ref. 28). The VEGF primers detect the four splicedRNA transcripts. The primers used for bFGF were: 59- GTG TGT GCT AACCGT TAC CT (forward) and 59-GCT CTT AGC AGA CAT TGG AAG(reverse; Ref. 7). Amplification of the “housekeeping” gene, glyceraldehyde-3-phosphate dehydrogenase was used to verify mRNA isolation and RT-PCRtechniques. PCR-amplified products were electrophoresed in 1.0% agarose geland stained with ethidium bromide.

ELISA. Supernatants were obtained fromin vitro tissue culture studies andwere examined for expression of VEGF using a quantitative solid-phase ELISAassay (R&D Systems, Minneapolis, MN). Samples (200ml) or standards (200ml)were added to a coated microtiter plate and incubated for 2 h atroom temperature.The plates were then rinsed, and 200ml of recombinant anti-VEGF165 polyclonalantibody conjugated to horseradish peroxidase were added to the wells. ThisELISA assay will detect both theMr 165,000 andMr 121,000 VEGF isoforms.After an additional 2- incubation, the wells were again rinsed, and 200ml ofhydrogen peroxide and tetramethylbenzidine were added. The reaction wasstopped by the addition of 50ml of 2 N sulfuric acid. The absorbance of each wellwas measured spectrophotometrically at 450 nm and plotted against a standardcurve with VEGF levels expressed as ng/ml. The lower detection limit of theELISA assay is 5.0 pg/ml. Each cell line was analyzed in triplicate.

RNase Protection Assay.HUVECs were plated into T-75 tissue cultureflasks and allowed to grow to;60% confluence. The cells were then exposedto 10 ng/ml VEGF for 72 h. A parallel flask of HUVEC cells was set upwithout VEGF exposure and maintained under identical culture conditions asa control. After VEGF exposure, total cellular RNA was isolated, and anRNase protection assay was carried out using the Riboquant system accordingto the manufacturer’s instructions (PharMingen, Inc., San Diego, CA). Thehuman cytokine/chemokine set hCK-4, which included templates for thefollowing human cytokines: hIL-3, hIL-7, human GM-CSF, human granulo-cyte-CSF, human macrophage-CSF, hIL-6, leukemia inhibitory factor, humanstem cell factor, and human oncostatin M was used for probe generation. Probesynthesis was carried out with a reaction mixture containing [a-32P]UTP,GACU nucleotide pool, DTT, transcription buffer, RNase protection assaytemplate, and T7 DNA polymerase. The mixture was incubated at 37°C for 1 hand purified using phenol/chloroform extraction. Fivemg of total cellular RNAisolated from either the VEGF-exposed HUVEC cells or from unexposedcontrol HUVEC cells were pipetted into a microfuge tube and dried in avacuum concentrator. The samples were then reconstituted in hybridizationbuffer. Concomitantly, the probe was diluted with the same hybridizationbuffer to yield a concentration of 2.93 104 cpm/ml, with 2 ml added to the tubecontaining the RNA. Mineral oil was added to each tube to prevent evapora-tion. Samples were then denatured at 90°C and hybridized for 16 h at 56°C.After hybridization, the samples were placed at 37°C, and an RNase cocktailwas added to each tube. The RNase treated samples were purified usingphenol/chloroform extraction and resolved on a nondenaturing acrylamide gel.After electrophoresis, the gel was carefully removed from the glass plate anddried at 80°C for;1 h. The dried gel was then placed on X-ray film (KodakX-AR), and autoradiography was carried out. To control for differences insample loading, two housekeeping genes,glyceraldehyde-3-phosphate dehy-drogenaseand leukemia inhibitory factorwere included as controls.

Immunohistochemistry. Immunohistochemical analysis was carried outon formalin-fixed, paraffin-embedded bone marrow clots or cores as describedpreviously (29). Slides were stained for expression of factor VIII-relatedantigen (Dako-Patts, Santa Barbara, CA), VEGF, KDR, or Flt-1 (Santa CruzBiotechnology, Santa Cruz, CA). The anti-VEGF antibody used in thesestudies has been demonstrated to be specific for VEGF and does not cross-react with other known VEGF/placenta growth factor family members. Itsspecificity has been established through the use of VEGF-specific blockingpeptides. Both the KDR and Flt-1 antibodies are also specific and do notcross-react with each other or with other protein tyrosine kinase membranereceptors. All reactions were performed using an automated immunostainer(GenII; VMS, Tucson, AZ; Ref. 29). Detection of bound antibody was as-sessed through the use of immunoperoxidase methodologies with diaminoben-zidine serving as the color substrate or by alkaline phosphatase methodologiesusing a biotinylated goat-anti-rabbit antibody (Dako-Patts) in conjunction withalkaline phosphatase-conjugated streptavidin followed by NBT/5-bromo-4-

chloro-3-indolyl phosphate as the color substrate. The VMS antibody diluentwas used as a negative control. Nuclei were counterstained with methyl greenor hematoxylin, and sections were evaluated by light microscopy. Endogenousperoxidase was inhibited with methanol containing 0.01% H2O2.

In Situ Hybridization. Paraffin-embedded bone marrows (clots or cores)were sectioned 3mm thick and placed on glass slides with a “sausage” controlsection containing placenta, liver, spleen, colon, and pancreas. The slides werebaked 1 h at60°C and then deparaffinized in two changes of xylene for 10 mineach, two changes of 100% ethanol for 2 min each, followed by a graded seriesof alcohols (95%, 80%, and 70%), and finally, two changes of diethylpyro-carbamate-treated H2O. They were then placed in APK wash (VMS). Alladditional steps were carried out using an automatedin situ hybridizationinstrument (Gen II; VMS). The details of this procedure have been publishedpreviously (29–31). A 24-mer VEGF-specific oligonucleotide probe was de-signed based on published sequences and was synthesized with six biotins atthe 39end (Research Genetics, Inc., Huntsville, AL). Before the addition to theprobe, slides were treated with Protease 1 (VMS) for 4 min. A total of 100mlof probe, diluted to a concentration of 1 ng/ml in a hybridization solutioncomposed of 35% formamide, 53 Denhardt’s, 10% dextran sulfate, 100mg/mlof salmon sperm DNA, and 43 SSC (0.6 M NaCl; 0.06 M Na3 citrate, pH 7.0),was manually added to each slide. For a negative control, the hybridizationsolution alone was applied. The slides were then denatured at 65°C for 4 min,followed by hybridization at 45°C for 60 min. After hybridization, threestringency washes were performed for 4 min each at 50°C as follows: 13 SSC,0.53SSC, and 0.13SSC to remove unbound probe. Detection was carried outat 40°C by incubating the slides in streptavidin-alkaline phosphatase (Boeh-ringer Mannheim Biochemicals, Indianapolis, IN), followed by NBT and5-bromo-4-chloro-3-indolyl phosphate substrate. The slides were counter-stained off the instrument with contrast red (Kirkegaard & Perry Laboratories,Gaithersburg, MD). Hybridization with a d(T)30 oligonucleotide probe con-firmed the integrity of the RNA (not shown). To insure that the oligonucleotideprobe was recognizing mRNA and not genomic DNA, a subset of slides wasincubated in either DNase or RNase before the addition of the probe.

RESULTS

Studies were undertaken to examine the expression of angiogenicgrowth factors and their receptors in a series of human hematopoietictumor cell lines representing multiple cell lineages (Table 1).

Our data revealed the expression of either VEGF, bFGF, or both byall of the cell lines in our panel (Table 2). As shown in Fig. 1, RT-PCRanalysis revealed transcripts corresponding to the 121 and the 165isoforms of VEGF in the cell lines examined. Fig. 2 shows RT-PCRdata demonstrating the expression of bFGF transcripts. In contrast toVEGF, bFGF was found in 50% of the cell lines examined. Thesefindings suggest that, although different angioregulatory moleculesmay be operating in a given tumor, VEGF appears to be expressed inall hematopoietic tumors examined and therefore is likely playing animportant role in their pathogenesis (Table 2). Expression of VEGFwas confirmed using Northern blot analysis. As shown in Fig. 3, weobserved a 4.4-kb transcript in all cell lines examined and an addi-tional transcript of;3.7 (not shown).

Table 1 Human hematopoietic cell lines

Cell line Disease Cell phenotype

HUT 78 Cutaneous T-cell lymphoma T-cellJurkat Acute T-cell leukemia T-cellMolt-4 Acute lymphoblastic leukemia T-cellCCRF-CEM Acute lymphoblastic leukemia T-cellRaji Burkitt’s lymphoma B-cellRamos Burkitt’s lymphoma (EBV negative) B-cellNALM 16 Acute lymphocytic leukemia Non-T, non-B cellU937 Histiocytic lymphoma Monocytic cellHL-60 Promyelocytic leukemia Myeloid cellK-562 Chronic myelogenous leukemia Myeloid cell, blast crisis8226 Multiple myeloma Plasma cellARH-77 Plasma cell leukemia Plasma cell

729

EXPRESSION OF VEGF IN MYELOMA

On the basis of these findings, additional studies were initiated to examinethe expression of the two receptors for VEGF, KDR and Flt-1. UsingRT-PCR and Northern blot analysis, we found Flt-1 to be expressed not onlyin the HUVEC cell line as expected but also in several of the hematopoietictumor cell lines examined (Table 2 and Fig. 4). This was an unexpectedfinding in light of the literature reports of KDR and Flt-1 expression beingrestricted to vascular endothelial cells. Interestingly, the finding of VEGFreceptor expression in tumor cells may not be unique to hematopoietic celllines because we have observed expression of KDR, but not Flt-1, in a seriesof small cell and non-small cell lung cancers.4 The finding of Flt-1 in thesecell lines suggests the possibility of an autocrine pathway in which the tumorcells may stimulate their own growth after VEGF exposure. Studies arepresently under way to examine this possibility.

To assess whether the VEGF protein was being secreted, cell lineswere seeded into T-25 flasks at a concentration of 13 106 cells/flaskin RPMI 1640 with 1% FCS. After 48 h, the supernatants werecollected for VEGF determination by ELISA assay. Secreted VEGFwas detected in the conditioned medium from all cell lines examined,thereby indicating that these cells are capable of synthesizing andexporting VEGF (Fig. 5). The H-460 human large cell lung carcinomacell line was included for reference to compare the expression ob-served in the hematopoietic tumor cell lines with that of a knownproducer of VEGF. Values obtained from hematopoietic tumor celllines in the ELISA assay ranged from a high of 2.2 ng/ml per 106 cellsin the Hut 78 cell line to a low of 0.1 ng/ml in the ARH-77 cell lineand compared with 2.8 ng/ml observed in the H-460 cells.

We next examined whether exposure of human endothelial cells toVEGF could stimulate the expression of known hematopoietic cyto-kines. After exposure of HUVEC cells to VEGF for 72 h, an increase

in the message for several hematopoietic growth factors was observed(Fig. 6). mRNAs for human macrophage-CSF, human granulocyte-CSF, human IL-6, human stem cell factor, and human oncostatin Mwere observed in both the VEGF-stimulated and control HUVECcells. However, there was clearly an increase in these messages inresponse to VEGF exposure. We have repeated this experiment threetimes with similar results.

Expression of VEGF and its receptors was examined in clinicalbone marrow samples from patients diagnosed with multiple my-eloma. To date, a total of 16 myeloma patients have been assessedwith similar findings observed in all. Immunohistochemical analysiswas carried out on formalin-fixed, paraffin-embedded bone marrowclots or cores as described (29). In a series of 16 cases diagnosed withmultiple myeloma, plasma cell expression of VEGF was observed inthe bone marrows from 12 of the patients (Table 3). An additionalpatient demonstrated a heterogeneous pattern of plasma cell expres-sion. VEGF expression in plasma cells revealed a diffuse cytoplasmicpattern of staining that varied in intensity among the individualpatients. Although a gradient of VEGF expression is usually observedin solid tumors, with the strongest expression being in areas ofhypoxia, we did not observe such a finding in the bone marrow coresamples. This observation may reflect a relative lack of hypoxic areasin the samples chosen for analysis. Our findings in myeloma patientscontrasted with those from normal bone marrow specimens, where alow level of plasma cell VEGF expression was observed in only oneof six samples. In both normal and myeloma samples, strong VEGFstaining was observed in myeloid and monocytic as well as inmegakaryocytic cells. Such a pattern of expression is consistent withthe known expression of VEGF in macrophages and platelets. Stain-ing in erythroblastic or lymphoblastic cells was not observed in any of

4 W. T. Bellamy, L. Richter, and P. Mendibles, Expression of VEGF receptors innon-small cell lung carcinomas, manuscript in preparation.

Fig. 1. RT-PCR analysis of VEGF transcripts in human hematopoietic tumor cell lines.VEGF-121 (408 bp) and VEGF-165 (541 bp) were detected in all of the cell linesexamined.

Fig. 2. RT-PCR analysis of bFGF transcripts (237 bp) in human hematopoietic tumorcell lines revealed expression in 6 of 12 cell lines examined.

Fig. 3. Northern blot analysis of VEGF expression in human hematopoietic tumor celllines. Total cellular RNA (5mg) was loaded into each lane, fractionated in a 1%agarose-formaldehyde gel, and transferred to a Nytran (Schleicher & Schuell) nylonmembrane. Before transfer, the fractionated RNAs in the gel were stained with ethidiumbromide to confirm equal loading (bottom panel). The membrane was probed with a32P-labeled cDNA probe specific for VEGF. HUVECs were included as a positive controlfor VEGF expression.

Table 2 Expressiona of angiogenic molecules in hematopoietic cell lines

Cell line bFGF VEGF KDR Flt-1

HUT 78 11 11 0b 1Jurkat 0 11 0 111Molt-4 0 11 0 111CCRF-CEM 0 11 0 1Raji 0 11 0 111Ramos 11 11 0 1U937 11 11 0 1HL-60 0 11 0 1K-562 11 11 0 1NALM 16 0 11 0 118226 1 11 0 0ARH-77 11 11 0 11

a As evaluated by RT-PCR and described in the “Materials and Methods.” The relativeintensity of staining was determined from examination of a photograph of ethidiumbromide-stained gels containing the RT-PCR products.

b 0, not detected;1, weak;11, moderate;111, strong signal.

730

EXPRESSION OF VEGF IN MYELOMA

the specimens examined. Occasional staining of polymorphonuclearcells was also observed in both normal and myeloma samples. Fig. 7Ademonstrates cytoplasmic expression of VEGF, as evidenced bybrown staining, in the neoplastic plasma cells growing in the bonemarrow of one of these patients. Fig. 7B presents the same tumor massstained with an antibody against factor VIII-related antigen, revealingthe expression of positive cells within the tumor mass, which repre-sent the neovasculature associated with an angiogenic response. InFig. 7, panels CandD display the expression of the Flt-1 and KDRreceptors, respectively. Although Flt-1 and KDR were not examinedin all patient samples, neither of these two receptors were found to be

expressed in the tumor cells; however, the normal bone marrow cells,predominantly myeloid and monocytic cells, revealed expression ofboth of these receptors, supporting a possible paracrine role of VEGFin this disease. Expression of VEGF in the malignant plasma cells wasconfirmed usingin situ hybridization (Fig. 8).

DISCUSSION

Although it is well established that growth in solid tumors isdependent on the formation of neovasculature, the role of angiogen-esis in hematopoietic neoplasms has not been determined. VEGF, akey angiogenic molecule, is a multifunctional cytokine that acts bothas a potent inducer of vascular permeability and as a specific endo-thelial cell mitogen (5, 6). Because of its effects on endothelial cellgrowth and microvascular permeability, VEGF is believed to be animportant mediator of tumor angiogenesis.

We have observed the expression of either VEGF, bFGF, or both by allof the hematopoietic tumor cell lines in our panel. In contrast to VEGF,

Fig. 4.A, RT-PCR analysis of Flt-1 transcripts in human hematopoietic tumor cell lines. A1098-bp band derived from Flt-1 mRNA was clearly detected in several of the cell linesexamined. The Raji, Jurkat, and Molt cell lines demonstrated a level of expression comparablewith the HUVEC cell line, whereas the ARH-77 and HUT-78 cell lines displayed a lower levelof expression. No evidence of Flt-1 expression was observed in the 8226 cell line.B,Northernblot analysis of Flt-1 transcript in human hematopoietic tumor cell lines. Total cellular RNA(5 mg) was loaded into each lane, fractionated in a 1% agarose-formaldehyde gel, andtransferred to a Nytran (Schleicher & Schuell) nylon membrane. Before transfer, the fraction-ated RNAs in the gel were stained with ethidium bromide to confirm equal loading (bottompanel). The membrane was probed with a32P-labeled cDNA probe specific for Flt-1.HUVECs were included as a positive control for Flt-1 expression.

Fig. 5. ELISA assay results demonstrating VEGF concentrations in conditioned mediumcollected from human tumor cell lines 48 h after being seeded into a T-25 culture flask.

Fig. 6. Induction of human hematopoietic cytokines after exposure of HUVEC cells toVEGF. HUVEC cells were exposed to 0 (A) or 10 ng/ml (B) recombinant human VEGFfor 72 h and total cellular RNA isolated. An RNase protection assay was carried out usinga human cytokine template set hCK-4.

Table 3 Plasma cell expressiona of VEGF in clinical specimens

Patient Sex Age Diagnosis VEGF

1 F 80 Normal 0b

2 M 68 Normal 03 F 45 Normal 04 F 58 Normal 05 M 49 Normal 16 F 61 Normal 07 M 73 MMc 08 F 56 MM 1/119 M 72 MM 11/111

10 M 53 MM 11111 F 59 MM 112 M 68 MM 113 M 42 MM 1114 M 63 MM 11115 M 66 MM 016 M 43 MM 117 M 68 MM 118 F 55 MM 119 M 52 MM 120 M 38 MM 021 F 57 MM 0/122 M 64 MM 11

a Data derived from immunohistochemical examination of bone marrow samples asdescribed in “Materials and Methods.”

b 0, not detected;1, weak;11, moderate;111, strong signal.c MM, multiple myeloma.

731

EXPRESSION OF VEGF IN MYELOMA

which was observed in all of the cell lines, bFGF expression wasobserved in only 50% of the cell lines. These findings suggest thatdifferent angioregulatory molecules may be operating in hematopoietictumors and that VEGF appears to be expressed in all hematopoietic celllines examined. Results from our ELISA assay demonstrated that VEGFwas secreted into the culture medium at concentrations that are within itsrange of biological activity. Concentrations of VEGF as low as 0.05ng/ml have been demonstrated to stimulate endothelial cellsin vitro (17).Thus, concentrations produced by the ARH-77 and Jurkat cell lines, thetwo cell lines with the lowest observable VEGF levels in our study,potentially have the ability to stimulate endothelial cells.

Although the requirement of tumor-stromal interactions is well estab-lished in multiple myeloma, the role of angiogenic factors, which actprimarily on the stromal elements, is not understood in relation to hema-topoietic malignancies. Most cancers may be considered as a two-com-partment system in which tumor cells and stromal cells interact in aparacrine fashion. The role of the stromal compartment is well estab-lished in multiple myeloma, involving interactions between myelomacells and the microenvironment of the bone marrow by means of cell-cellcontact, adhesion molecules, and cytokines (32–36). Endothelial cells arefound in the stromal layer of long-term bone marrow cultures in closecontact with hematopoietic cells and are known to produce IL-6, animportant growth factor in multiple myeloma (36, 37). They have alsobeen demonstrated to secrete several CSFs in response to cytokines suchas IL-1 (38). Thus, it is possible that endothelial cells may, in response toangiogenic factors, release cytokines capable of sustaining tumor growth.Indeed, porcine brain microvascular endothelial cells have been shown tosupport the expansion of human progenitor cellsin vitro (39). In additionto endothelial cells, macrophages are also believed to play an importantrole in tumor angiogenesis through their ability to release proteases,growth factors (including VEGF), and other cytokines (18). Although wedid not measure the release of growth factors into the cell culturemedium, others have reported an increased secretion of GM-CSF fromhuman endothelial cells after VEGF exposure (40). Such findings reveal

that VEGF can increase the level of message expression for severalgrowth factors with known stimulatory effects in hematopoietic malig-nancies including multiple myeloma. Interestingly, GM-CSF and G-CSFhave been shown to stimulate human endothelial cellsin vitro to migrateand proliferate (41).

Paracrine control mechanisms are increasingly recognized as beingimportant for tumor growth. IL-6 is well established as a paracrinegrowth factor in myeloma, bothin vitro and in vivo (33, 36, 42, 43).In normal bone marrow as well as in patients, the stromal cells appearto be the major producers of IL-6 (42, 44). We have observed thatrecombinant human VEGF is capable of increasing the expression ofmRNA for IL-6 in a human vascular endothelial cell line. This pointsto the possibility that VEGF may also increase the expression of IL-6in the bone marrow, a hypothesis we are presently examining.

To date, two high-affinity receptors for VEGF have been identified:KDR (also referred to as flk-1) and Flt-1, both of which are class IIIreceptor tyrosine kinases (45, 46). Although it is generally held thatthe expression of these two receptors is restricted to endothelial cells,both human uterine smooth muscle cells and retinal pigment cellshave been demonstrated to express both KDR and flt-1, whereasmurine retinal progenitor cells have also been reported to express theKDR receptor (47–49). In our studies, the VEGF receptor Flt-1 wasfound to be expressed at moderate to high levels in 5 of the 12 tumorcell lines examined. This finding alludes to the possibility of anautocrine pathway involving VEGF operating in these cells.

The expression of VEGF in patient samples with multiple myelomasuggests that this growth factor, which has previously been believed toplay a role only in solid tumors, may also be playing a role in hemato-poietic tumors such as myeloma. In bone marrow from myeloma patients,VEGF expression was observed in the tumor cells both by immunohis-tochemistry andin situ hybridization, whereas the Flt-1 and KDR recep-tors were observed to be expressed in the normal marrow myeloid andmonocytic cells. These data raise the possibility that VEGF may play arole in the growth of multiple myeloma through a paracrine or an

Fig. 7. (Left panels.) Immunohistochemical analysis of VEGF expression in human multiple myeloma.A, VEGF expression within the tumor mass (brown staining,31000);B, tumormass revealing factor VIII-related, antigen-positive endothelial cells (brown staining,3400);C, expression of Flt-1 within normal marrow cells (31000);D, expression of KDR withinnormal marrow cells (31000).

Fig. 8. (Right panels.)In situ hybridization analysis of VEGF expression in a formalin-fixed, paraffin-embedded bone marrow sample from a patient with multiple myeloma.A,hybridization with a biotinylated RNA-specific antisense probe produced a specific signal for VEGF in the plasma cells (arrows) as revealed by the blue NBT staining located in thecytoplasm.B, negative control demonstrating a lack of staining in the plasma cells. All samples were counterstained with contrast red. (31000).

732

EXPRESSION OF VEGF IN MYELOMA

autocrine mechanism. Studies are presently under way in our laboratoryto determine whether VEGF is indeed playing a role in the pathophysi-ology of this disease. By identifying a role for VEGF in multiple my-eloma or other hematopoietic malignancies, the possibility of using newtreatment strategies in these patients is thus opened. Traditionally, treat-ment of myeloma has been hampered by the development of drugresistance in this generally incurable disease. By developing treatmentstrategies that target both the stromal and tumor compartments, drugresistance may be overcome, and the effect on therapeutic outcomeenhanced. Although such an approach appears to hold promise for solidtumors (50), it remains to be seen whether it will be effective in hema-topoietic neoplasms as well.

ACKNOWLEDGMENTS

We thank Dr. Sydney Salmon for critical reading of the manuscript andthoughtful comments.

REFERENCES

1. Folkman, J. Clinical applications of research on angiogenesis. N. Engl. J. Med,333:1757–1763, 1995.

2. Folkman, J. Tumor angiogenesis.In: J. Mendelssohn, P. M. Howely, M. A. Israel, andL. A.Liotta (eds.), The Molecular Basis of Cancer, pp. 206–235. Philadelphia: W. B.Saunders, 1995.

3. Folkman, J. and Klagsburn, M. Angiogenic factors. Science (Washington DC),235:442–447, 1987.

4. Madri, J. A., and Williams, S. K. Capillary endothelial cell cultures: phenotypicmodulation by matrix components. J. Cell Biol.,97: 153–156, 1983.

5. Brown, L. F, Detmar, M., Claffey, K., Nagy, J. A., Feng, D., Dvorak, A. M., andDvorak, H. F. Vascular permeability factor/vascular endothelial growth factor: amultifunctional angiogenic cytokine.In: I. D. Goldberg and E. M. Rosen (eds.),Regulation of Angiogenesis, pp.233–269. Basel: Birkhauser Verlag, 1997.

6. Olson, T. A., Mohanraj, D., Carson, L. F., and Ramakrishnan, S. Vascular perme-ability factor gene expression in normal and neoplastic human ovaries. Cancer Res.,54: 276–280, 1993.

7. Ohta, Y., Endo, Y., Tanaka, M., Shimizu, J., Oda, M., Hayashi, Y., Watanabe, Y., andSasaki, T. Significance of vascular endothelial growth factor messenger RNA ex-pression in primary lung cancer. Clin. Cancer Res.,2: 1411–1416, 1996.

8. Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Manseau, E. J., Senger, D. R.,and Dvorak, H. F. Expression of vascular permeability factor (vascular endothelialgrowth factor) and its receptors in adenocarcinomas of the gastrointestinal tract.Cancer Res.,53: 4727–4735, 1993.

9. Joseph, I., Nelson, J. B., Denmeade, S. R., and Isaacs, J. T. Androgens regulatevascular endothelial growth factor content in normal and malignant prostatic tissue.Clin. Cancer Res.,3: 2507–2511, 1997.

10. Larcher, F., Robles, A. I., Duran, H., Murillas, R., Quintanilla, M., Cano, A., Conti,C. J., and Jorcano, J. L. Up-regulation of vascular endothelial growth factor/vascularpermeability factor in mouse skin carcinogenesis correlates with malignant progres-sion state and activated H-ras expression levels. Cancer Res.,56: 5391–5396, 1996.

11. Weidner, N., Semple, J. P., Welch, W. R., and Folkman, J. Tumor angiogenesis andmetastasis–correlation in invasive breast carcinoma. N. Engl. J. Med.,324: 1–8, 1991.

12. Weidner, N., Carroll, P. R., Flax, J., Blumenfeld, W., and Folkman, J. Tumorangiogenesis correlates with metastasis in invasive prostate carcinoma. Am. J.Pathol.,143: 401–409, 1993.

13. Giatromanlaki, A., Koukourakis, M., O’Byrne, K., Fox, S., Whitehouse, R., Talbot,D. C., Harris, A. L., and Gatter, K. C. Prognostic value of angiogenesis in operablenon-small cell lung cancer. J. Pathol.,179: 80–88, 1996.

14. Fontanini, G., Bigini, D., Vignati, S., Basolo, F., Mussi, A., Lucchi, M., Chine, S.,Angeletti, C. A., Harris, A. L., and Bevilacqua, G. Microvessel count predicts metastaticdisease and survival in non-small cell lung cancer. J. Pathol.,177: 57–63, 1995.

15. Meiter, D., Crawford, S. E., Rademaker, A. W., and Cohn, S. Tumor angiogenesiscorrelates with metastatic disease, n-myc amplification, and poor outcome in humanneuroblastoma. J. Clin. Oncol.,14: 405–414, 1996.

16. Kim, J. K., Li, B., Winer, J., Armanini, M., Gillett, N., Phillips, H. S., and Ferrara, N.Inhibition of vascular endothelial growth factor-induced angiogenesis suppressestumor growthin vivo. Nature (Lond.),362: 841–844, 1993.

17. Fava, R. A., Olsen, N. J., Spencer-Green, G., Yeo, K., Yeo, T., Berse, B., Jackman,R. W., Senger, D. R., Dvorak, H. F., and Brown, L. F. Vascular permeabilityfactor/endothelial growth factor (VPF/VEGF): accumulation and expression in humansynovial fluids and rheumatoid synovial tissue. J. Exp. Med.,180: 341–346, 1994.

18. Sunderkotter, C., Steinbrink, K., Goebeler, M., Bhardwaj, R., and Sorg, C. Macro-phages and angiogenesis. J. Leukocyte Biol.,55: 410–422, 1994.

19. Brown, L. F., Berse, B., Tognazzi, K., Manseau, E. J., van de Water, L., Senger,D. R., Dvorak, H. F., and Rosen, S. Vascular permeability factor mRNA and proteinexpression in human kidney. Kidney Int.,42: 1457–1461, 1992.

20. Iijima, K., Yoshikawa, N., Connolly, D. T., and Nakamura, H. Human mesangial cellsand peripheral blood mononuclear cells produce vascular permeability. factor. KidneyInt., 44: 959–966, 1993.

21. Verheul, H., Hoekman, K., Luykx-de Baker, S., Eekman, C., Folman, C., Broxterman,H., and Pinedo, H. Platelet: transporter of vascular endothelial cell growth factor.Clin. Cancer Res.,3: 2187–2190, 1997.

22. Frank, S., Hubner, G., Breier, G., Longaker, M. T., Greenhalgh, D. G., and Werner,S. Regulation of vascular endothelial growth factor expression in cultured keratino-cytes. J. Biol. Chem.,270: 12607–12613, 1995.

23. Ribatti, D., Vacca, R. D., Nico, B., Fanelli, M., Roncali, L., and Dammacco, F.Angiogenesis spectrum in the stroma of B-cell non-Hodgkin’s lymphomas. Animmunohistochemical and ultrastructural study. Eur. J. Haematol.,56: 45–53, 1996.

24. Perez-Atayde, A. R., Sallan, S. E., Tedrow, U., Connors, S., Allred, E., and Folkman,J. Spectrum of tumor angiogenesis in the bone marrow of children with acutelymphoblastic leukemia. Am. J. Pathol.,150: 815–821, 1997.

25. Vacca, A., Ribatti, D., Roncali, L., Ranieri, G., Serio, G., Silvestris, F., and Dam-macco, F. Bone marrow angiogenesis and progression in multiple myeloma. Br. J.Haematol.,87: 503–508, 1994.

26. Leung, D. W., Cachianes, G., Kuang, W-J., Goeddel, D. V., and Ferrara, N. Vascularendothelial growth factor is a secreted angiogenic mitogen. Science (WashingtonDC), 246: 1306–1309, 1989.

27. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. Isolation ofbiologically active ribonucleic acid from sources enriched in ribonuclease. Biochem-istry, 18: 5294–5299, 1979.

28. Brown, L. F., Berse, B., Jackman, R. W., Tognazzi, K., Manseau, E. J., Senger, D. R.,and Dvorak, H. F. Expression of vascular permeability factor (vascular endothelialgrowth factor) and its receptors in adenocarcinomas of the gastrointestinal tract.Cancer Res.,53: 4727–4735, 1993.

29. Grogan, T. M., Rangel, C., Rimsza, L., Bellamy, W., Martel, R., McDaniel, D., McGraw,B., Richards, W., Richter, L., Rodgers, P., Rybski, J., Showalter, W., Vela, E., and Zehab,R. Kinetic-mode, automated double-labeled immunohistochemistry andin situ hybrid-ization in diagnostic pathology. Adv. Pathol. Lab. Med.,8: 79–100, 1995.

30. Rimsza, L. M., Vela, E., Frutiger, Y., Rangel, C., Solano, M., Richter, L., Grogan,T. M., and Bellamy, W. T. Rapid automated combinedin situ hybridization andimmunohistochemistry for sensitive detection of cytomegalovirus RNA and protein inparaffin embedded tissue biopsies. Am. J. Clin. Pathol.,106: 544–548, 1996.

31. Rimsza, L. M., Vela, E., Frutiger, Y. M., Richter, L. C., Grogan, T. M., and Bellamy,W. T. Combinedin situhybridization for automated detection of cytomegalovirus andp53. Mol. Diagnostics,1: 291–296, 1996.

32. Salmon, S. E., and Cassady, J. R. Plasma cell neoplasms.In: V. T. DeVita, S.Hellman, and S. A. Rosenberg (eds.)., Cancer: Principles and Practice of Oncology,Ed. 5, pp. 2344–2387. Philadelphia: J. P. Lippincott Co., 1997.

33. Bataille, R., and Harousseau, J. L. Multiple myeloma. N. Engl. J. Med.,336:1657–1664, 1997.

34. Klein, B., and Bataille, R. Cytokine network of human multiple myeloma. Hematol.Oncol. Clin. N. Am.,6: 273–284, 1992.

35. Barille, S., Collette, M., Bataille, R., and Amiot, M. Myeloma cells up-regulateinterleukin-6 secretion in osteoblastic cells through cell-to-cell contact but downregulate osteocalcin. Blood,86: 3151–3159, 1995.

36. Klein, B., Zhang, X., Lu, Z., and Bataille, R. Interleukin-6 in human multiplemyeloma. Blood,85: 863–872, 1995.

37. van Snick, J. Interleukin-6: an overview. Annu. Rev. Immunol.,8: 253–278, 1990.38. Griffin, J. D., Rambaldi, A., Vellenga, E., Young, D. C., Ostapovicz, D., and Cannistra,

S. A. Secretion of interleukin-1 by acute myeloblastic leukemia cellsin vitro inducesendothelial cells to secrete colony stimulating factors. Blood,70: 1218–1221, 1987.

39. Davis, T., Robinson, D., Lee, K., and Kessler, S. Porcine brain microvascularendothelial cells support thein vitro expansion of human primitive hematopoieticbone marrow progenitor cells with a high replicating potential: requirement forcell-to-cell interactions and colony- stimulating factors. Blood,85: 1751–1761, 1995.

40. Fiedler, W., Graeven, U., Ergun, S., Verago, S., Kilic, N., Stockschlader, M., andHossfeld, D. K. Vascular endothelial growth factor, a possible paracrine growth factorin human acute myeloid leukemia. Blood,89: 1870–1875, 1997.

41. Bussolino, F., Wang, J., Defilippi, P., Turrini, F., Sanavio, F., Edgell, C., Aglietta, M.,Arese, P., and Mantovani, A. Granulocyte- and granulocyte-macrophage-colonystimulating factors induce human endothelial cells to migrate and proliferate. Nature(Lond.), 337: 471–473, 1989.

42. Klein, B., Zhang, X. G., Jordan, M., Content, J., Houssiau, F., Aarden, L., Piechaczyk,M., and Bataille, R. Paracrine rather than autocrine regulation of myeloma cell growthand differentiation by interleukin-6. Blood,73: 517–526, 1989.

43. Kawano, M., Hirano, T., Matsuda, T., Taga, T., Horii, Y., Iwato, K., Asaoku, H., Tang,B., Tanabe, O., Tanaka, H., Kuramoto, A., and Kishimoto, T. Autocrine generation andrequirement of BSF/IL-6 for multiple myelomas. Nature (Lond.),332: 83–85, 1988.

44. Carter, A., Merchav, S., Silvian-Draxler, I., and Tatarsky, I. The role of interleukin-1 andtumor necrosis factor-a in human multiple myeloma. Br. J. Haematol.,74: 424–431, 1990.

45. Terman, B. I., Dougher-Vermazen, M., Carrion, M., Dimitrov, D., Armellino, D. C.,Gospodarowicz, D., and Bohlen, P. Identification of the KDR tyrosine kinase as areceptor for vascular endothelial cell growth factor. Biochem. Biophys. Res. Com-mun.,187: 1579–1586, 1992.

46. De Vries, C., Escobedo, J., Ueno, H., Houck, K., Ferrara, N., and Williams, L. T. Thefms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science(Washington DC),255: 989–991, 1992.

47. Brown, L. F., Detmar, M., Tognazzi, K., Abu-Jawdeh, G., and Iruela-Arispe, M. L.Uterine smooth muscle cells express functional receptors (flt-1 and KDR) for vascularpermeability factor/vascular endothelial growth factor. Lab. Investig.,76: 245–255, 1997.

48. Yang, X., and Cepko, C. L. Flk-1, a receptor for vascular endothelial growth factor(VEGF), is expressed by retinal progenitor cells. J. Neurosci.,16: 6089–6099, 1996.

49. Guerrin, M., Moukadiri, H., Chollet, P., Moro, F., Dutt, K., Malecaze, F., and Poulet, J.Vasculotropin/vascular endothelial growth factor is an autocrine growth factor for humanretinal pigment epithelial cells culturedin vitro. J. Cell Physiol.,164: 385–394, 1995.

50. Teicher, B. A., Holden, S. A., Dupuis, N. P., Kakeji, Y., Ikebe, M., Emi, Y., and Goff,D. Potentiation of cytotoxic therapies by TNP-470 and minocycline in mice bearingEMT-6 mammary carcinoma. Breast Cancer Res. Treat.,36: 227–236, 1995.

733

EXPRESSION OF VEGF IN MYELOMA