biological and molecular characterization of a canine hemangiosarcoma-derived cell line

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Research in Veterinary Science 81 (2006) 76–86

Biological and molecular characterizationof a canine hemangiosarcoma-derived cell line

Douglas H. Thamm a,b,*, Erin B. Dickerson a,1, Nasim Akhtar c, Rachel Lewis b,c,2,Robert Auerbach c, Stuart C. Helfand a,b, E. Gregory MacEwen a,b,z

a Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, USAb The Comprehensive Cancer Center, University of Wisconsin-Madison, USA

c Department of Zoology, University of Wisconsin-Madison, USA

Accepted 6 September 2005

Abstract

Canine hemangiosarcoma (HSA) is a devastating disease. Investigation of novel therapies has been limited by the limited availabilityof canine HSA-derived cell lines. We report the development of a canine HSA-derived cell line, DEN-HSA, which recapitulates featuresof angiogenic endothelium. DEN-HSA cells were derived from a spontaneous HSA arising in the kidney of a dog. DEN-HSA displayedsurface molecules distinctive of endothelial histogenesis, including factor VIII-related antigen, ICAM-1 and avb3 integrin. In vitro, DEN-HSA formed microvascular tube-like structures on Matrigel�, and proliferated in response to a variety of angiogenic growth factors. Thecells expressed mRNA and protein specific for bFGF and its receptors, and VEGF and its receptors, among others. DEN-HSA condi-tioned medium evoked a marked angiogenic response in a murine corneal pocket assay, indicating potent proangiogenic activity of sub-stances secreted by this cell line. This research confirms the DEN-HSA cell line as endothelial in origin, suggests the presence ofangiogenic growth factor autocrine loops, and offers the potential to utilize DEN-HSA cells for the study of novel therapies that mod-ulate endothelial proliferation.� 2005 Published by Elsevier Ltd.

Keywords: Dog; Angiosarcoma; Endothelium; Angiogenesis

1. Introduction

Altered endothelial cell proliferation and survival is ofprime importance in many disease conditions, includingneoplasia, wound healing, and chronic inflammatory dis-ease. Perhaps the most dramatic example of dysregulatedangiogenesis is seen in malignancy derived from vascularendothelium. Investigation of the mechanisms responsible

0034-5288/$ - see front matter � 2005 Published by Elsevier Ltd.

doi:10.1016/j.rvsc.2005.09.005

* Corresponding author. Present address: The Animal Cancer Center,Colorado State University, 300 West Drake Road, Fort Collins, CO80523-1620, USA. Tel.: +1 970 297 4075; fax: +1 970 297 1254.

E-mail address: [email protected] (D.H. Thamm).1 Present address: Georgia Tech/IBB, 315 Forest Drive, Box 160,

Atlanta, GA 30332, USA.2 Present address: Primate Research Center, University of Wisconsin-

Madison, 425 Henry Mall, Madison, WI 53706, USA.z MacEwen is deceased.

for tumourigenesis in these neoplasms has the potentialto shed light onto basic concepts of angiogenesis, andpotentially identify novel strategies for its modulation.

Despite the fact that vascular tissue is extremely abun-dant, malignant tumours derived from the vascular endo-thelium are rare in humans, accounting for only 2% ofsoft-tissue sarcomas in one evaluation (Fata et al., 1999).When encountered, haemangiosarcomas of the humanbreast, liver, spleen and scalp behave aggressively, metasta-sizing commonly and yielding very poor long-term survivalrates (Falk et al., 1979; Holden et al., 1987; Neuhauseret al., 2000; Silverman et al., 1994).

Haemangiosarcoma (HSA) is a relatively common,spontaneous tumour in dogs, accounting for approximately20% of all soft-tissue sarcomas (Dorn et al., 1968), and upto 5% of all malignant canine neoplasms (Bastianello, 1983;MacVean et al., 1978). Canine HSA can occur in any site,

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D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86 77

however the spleen, skin, right atrium, and liver are themost common primary sites (MacEwen, 2001). CanineHSA is typified by very aggressive biological behavior, withwide and rapid metastasis being common. Doxorubicin-based chemotherapy has the potential to modestly improvesurvival time following surgery, however, one-year survivalrates remain less than 10% (Sorenmo et al., 2000; Sorenmoet al., 1993; Vail et al., 1995).

Canine HSA has potential utility as a spontaneouslyoccurring model of human endothelial tumours, such asangiosarcoma/hemangiosarcoma, hemangioma of infancy,and Kaposi�s sarcoma, a malignant tumour characterizedby the dysregulated proliferation of endothelial cells.Moreover, canine HSA cells may also be useful as a toolfor the study of endothelial cell biology in malignancy.For these purposes, we sought to establish a continuouscell line derived from a canine HSA, and to characterizethis line with regard to its in vitro biological behavior, cellsurface phenotype, and growth factor/growth factor recep-tor expression.

2. Materials and methods

2.1. Cell lines

The DEN-HSA cell line was established from a sponta-neous renal HSA arising in an 11-year-old castrated malegolden retriever. The primary tumour was mechanicallyand enzymatically dissociated (collagenase III 50 U/mL,DNAse 100 U/mL, hyaluronidase 200 U/mL, Sigma, St.Louis, MO) (Kemmer et al., 1987), and passaged in tissueculture flasks in complete minimal essential medium (Cell-gro Mediatech, Herndon, VA) supplemented with 5% fetalbovine serum, 5% newborn bovine serum, 100 U/mL pen-icillin, 100 lg/mL streptomycin, and 0.25 lg/mL ampho-tericin B (Antibiotic Antimycotic Solution, Sigma, St.Louis, MO) (C/10) under standard conditions (37 �C, 5%CO2, humidified). DEN-HSA grew as an adherent mono-layer and was serially passaged following detachment with0.25% trypsin in EDTA. Characterization was performedon cells having undergone >60 passages.

The D17 (ATCC CRL-8468) cell line is a canine osteo-sarcoma cell line obtained from American Type CultureCollection (ATCC), Rockville, MD. The Eoma cell line isa well-characterized murine endothelial-derived tumourcell line (Obeso et al., 1990). Normal canine endothelialcells (EC) were isolated from circulating precursors utiliz-ing a modification of a published protocol (Solovey et al.,1999). Briefly, normal dog peripheral blood mononuclearcells were separated by density centrifugation, and platedin EGM-2 medium (Clonetics, Temecula, CA). After 1month of daily medium changes, a confluent populationof endothelial-appearing cells was present. These cells weredetermined to be of endothelial origin by morphology onplastic, CD34 and avb3 immunoreactivity by flow cytome-try, and the expression of Flt-1, KDR and Tie2/TekmRNA by RT-PCR. Human umbilical vein EC (HU-

VECs) were purchased from ATCC, maintained inEGM-2 medium, and utilized at passage 3–5.

2.2. Morphology on matrigel

Tube formation on Matrigel� was assessed by adapta-tion of a published protocol (Gho et al., 1999). Briefly,Matrigel� (Becton Dickinson, Franklin Lakes, NJ) was di-luted to a concentration of 6 mg/mL with EGM-2 medium(Clonetics, Walkersville, MD). Diluted Matrigel� (320 lL)was added to wells of a 24-well plate and incubated at37 �C for 1 h. DEN-HSA cells, at a concentration of1 · 105/mL, were added in 500 lL of EGM-2, and incu-bated under standard conditions. The plates were then ob-served with phase-contrast microscopy and photographedafter 6 and 24 h of culture.

2.3. Immunohistochemistry/immunocytochemistry

Prediluted rabbit polyclonal antibody against humanVon Willebrand factor (vWF) (Ventana Medical Systems,Tuscon, AZ) and murine monoclonal antibody against hu-man CD31 (Platelet Endothelial Cell Adhesion Molecule[PECAM], Dako, Carpintiera, CA), with known immuno-reactivity against canine EC (Ferrer et al., 1995; von Beustet al., 1988), were utilized. The DEN-HSA cells were de-tached and cytospun onto glass slides, then air-dried for24 h. The slides were washed and incubated in primaryantibody (vWF: prediluted; CD31: 1:100) for 12 h. Biotin-ylated goat anti-rabbit or chicken anti-mouse (RocklandImmunochemicals, Gilbertsville, PA) secondary antibodieswere then applied at a 1:100 dilution for 1 h. Finally, ahorseradish peroxidase–streptavidin conjugate (VectorLaboratories, Burlingame, CA) was applied at 1:250 for20 min, followed by application of a DAB substrate (Bio-care, Concord, CA) and counterstaining with Mayer�s hae-matoxylin, dehydrated and mounted in xylene-basedmounting medium. Slides were observed and photographedusing light microscopy. Omission of the primary antibodyserved as the negative control.

2.4. Flow cytometry

The FITC-conjugated LM609 murine monoclonal anti-body to human avb3 integrin was purchased from Chem-icon (Temecula, CA). Murine monoclonal antibodyCL18/6, against canine intracellular adhesion molecule-1(ICAM-1), was generously provided by Dr. C.W. Smith,Baylor College of Medicine. Monoclonal antibody to ca-nine CD34 was purchased from Pharmingen (San Diego,CA). Murine IgG1 and IgG1-FITC conjugate isotype con-trols were purchased from Santa Cruz Biotechnology (San-ta Cruz, CA).

The DEN-HSA cells were detached by incubation innon-enzymatic cell dissociation solution (Sigma) and scrap-ing. 2.5 · 105 DEN-HSA cells were incubated on ice withprimary antibody (0.5 lg for ICAM-1, 5 lg for avb3 and

78 D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86

CD34) for 30 min, washed in Hank�s buffered saline solu-tion (HBSS) containing 0.1% bovine serum albumin, andthen incubated with a goat anti-mouse FITC conjugate(Serotec, Raleigh, NC) on ice in the dark for 30 min fordetection of ICAM-1 and CD34. Cells were washed again,and fluorescence intensity was measured using a Facscali-bur flow cytometer (Becton Dickinson, San Jose, CA).

2.5. DiI-acetylated LDL uptake, angiotensin-converting

enzyme activity

DiI-acetylated LDL (diIAcLDL) was purchased fromMolecular Probes (Eugene, OR). Subconfluent DEN-HSA cells were tested for the expression of the acetylatedlow density lipoprotein surface receptor by removing themedium and incubating the cells overnight under standardconditions in serum-free DMEM containing diIAcLDL(1:300 dilution). After washing, the cells were observedwith an inverted fluorescent microscope (Nikon) with arhodamine filter. Eoma and D17 cells served as positiveand negative controls, respectively.

Angiotensin converting enzyme (ACE) activity was mea-sured by using radiolabeled diglycyl hippurate ([3H]-Hip-Gly-Gly, Ventrex Laboratories, Portland, ME) as substrate.Enzyme activity was assessed by the ability of the cells tocleave the radioactive tripeptide, then separating the cleav-age product on the basis of its differential solubility in ethylacetate. Equal numbers of Eoma and D17 cells, serving asthe positive and negative controls, were also tested. In addi-tion, the ACE level of the furnished control serum and theefficacy of the radioactive substrate were determined. Incu-bations were carried out overnight to permit maximumlabeling without significant increase in background incorpo-ration. Radioactivity in the aqueous fraction, correspondingto amount of cleaved tripeptide, was measured using a gam-ma counter. ACE expression was also investigated flow cyto-metrically utilizing a monoclonal antibody known torecognize human, bovine and murine ACE (Auerbachet al., 1982), which was raised in Dr. Auerbach�s laboratory.

2.6. Cell proliferation assay

Recombinant human basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), insu-lin-like growth factor 1 (IGF-1), epidermal growth factor(EGF), hepatocyte growth factor (HGF) and platelet-de-rived growth factor (PDGF-bb) were purchased from R& D Systems (Minneapolis, MN). Prior investigations inour laboratory have demonstrated that recombinant hu-man HGF and IGF-1 have bioactivity in canine tumourcells (MacEwen et al., 2003, 2004).

The DEN-HSA cells were plated at 2 · 103 per well in200 lL C/10%, in 96-well flat-bottom plates (Falcon, Bec-ton Dickinson) and incubated overnight under standardconditions. The plates were then washed with HBSS, andthe media was replaced with fresh C/10, CMEM with 1%FBS (C/1), or C/1 supplemented with 1, 10, or 100 ng/

mL of human recombinant bFGF, VEGF, IGF-1, HGF,EGF or PDGF-bb. C/1 was shown to decrease cell prolif-eration by approximately 50% in preliminary experimentswhen compared to C/10 (not shown). After a 96-h incuba-tion, relative viable cell number was assessed by means of aone-step tetrazolium-based (MTS) colorimetric assay(CellTiter AQueous One, Promega, Madison, WI) accord-ing to the manufacturer�s directions. Quintuplicate wellswere run for each condition, and each experiment was re-peated three times. Human umbilical vein EC were utilizedin an identical cell proliferation assay to insure the bioac-tivity of the human recombinant VEGF. Differences in rel-ative viable cell number between experimental conditionswere assessed statistically using Student�s t test.

2.7. Anchorage-independent growth

Over a base layer of 200 lL 0.6% low-melting agarose(Sigma, St. Louis, MO) diluted in RPMI 1640 mediumwith 1% FBS, 2.5 · 103 DEN-HSA cells were strainedthrough a 40-lm cell strainer to eliminate clumps, platedin 200 lL 0.3% low-melting agarose in RPMI 1640 with10% FBS, 1% FBS, or 1% FBS supplemented with 10 ng/mL of recombinant human bFGF, VEGF, IGF-1, HGF,EGF, or PDGF in triplicate in 24-well plates. Plates wereincubated under standard conditions for three weeks, andthe number of colonies containing greater than 20 cellswas counted per well, using light microscopy. Experimentswere repeated for three separate times.

2.8. Reverse transcriptase – polymerase chain reaction

Total RNA was isolated from confluent monolayers ofcell cultures using Trizol reagent (Life Technologies, Inc.)according to the manufacturer�s instructions. Reverse tran-scription was carried out according to the manufacturer�sinstructions using the Superscript Preamplification System(Life Technologies, Inc.). Oligonucleotide primers directedagainst the canine genes (Table 1) were used to amplify thespecific canine sequences from the cDNA generated. All ofthe PCR reactions consisted of 35 cycles followed by a 7-min final extension at 72 �C, followed by a final coolingstep of 4 �C (see Table 1). The PCR reactions were designedto generate a single PCR product of interest. Taq polymer-ase was purchased from Promega (Madison, WI). All PCRproducts were verified by automated DNA sequencingusing Big Dye Reagent, carried out at the University ofWisconsin-Madison Biotechnology Center.

2.9. ELISA

Conditioned media were generated by culturing DEN-HSA cells under standard conditions in C/10 medium in6-well plates. After 72 h, the plates were washed five timeswith HBSS, and serum-free CMEM was added. The super-natant was harvested after an additional 24 h of culture,and frozen at �80 �C. Cells were trypsinized and counted

Table 1PCR primers and conditions

PCR product Primers Annealing temp (�C) Size (bp)

VEGF For: 5 0-CCATGAACTTTCTGCTCTCTTG-30 60 418Rev: 5 0-TTGTCTTGCTCTATCTTTGTT-3 0

VEGFR-1 (Flt-1) For: 5 0-AACTGAGTTTAAAAGGCAC-30 50 505Rev: 5 0-TCTTTGTACGTTGCATTTG-30

VEGFR-2 (Flk-1) For: 5 0-CTRGCYGTCGCYCTGTGGYTCTGC-30 55 371Rev: 5 0-AGARGCRATRAATGGWGATCCTGTA-30

bFGF For: 5 0-CTTCAAGGACCCCAAGCGGC-30 55 390Rev: 5 0-GCTCTTAGCAGACATTGG-30

FGFR-1 For: 5 0-ACCACCTACTTCTCCGTCAAT-3 58 354Rev: 5 0- TAGTTGCCCTTGTCAGARGG-3 0

FGFR-2 For: 5 0-CCAGAAGAGCCACCAACCAAATA-3 0 58 501Rev: 5 0-TCCTGCTTAAACTCCTTCCCG-3 0

IGF-1 For: 5 0-AAGCAGCACTCATCCACGAT-30 52 281Rev: 5 0-CAGCAGTCTTCCAACCCAAT-3 0

IGF-1R For: 5 0-CCTCCACATCCTGCTCATCT-30 52 444Rev: 5 0-GGATRCAGTACATGCTCTGG-3 0

HGF For: 5 0-CAGACACCACACCGGCACAA-30 54 421Rev: 5 0-GAGCAGTAGCCAACTCGGA-30

c-Met For: 5 0-GATCTGGGCAGTGAATTAGT-30 52 417Rev: 5 0-GTCCAACAAAGTCCCATGAT-30 *

EGF-R For: 5 0-AGGAGAGGAGAACTGCCAGA-30 55 250Rev: 5 0-CAGGTGGCACCAAAGCTGTA-3 0

Primer sequences for canine VEGF were provided by Dr. R. Chun, and sequences for bFGF were provided by Dr. R. Jacobs. Primers for canine IGF-1,IGF-1R, HGF, c-met, and EGF-R were designed by J. Carew and J. Schmidt.


on a hemocytometer using Trypan Blue. VEGF and bFGFconcentrations in cell supernatant were determined usingcommercial ELISA kits with known cross-reactivity withthe canine growth factors (Allen et al., 1996; Cliffordet al., 2001) (Quantikine, R & D Systems) according tomanufacturer directions, and VEGF and bFGF produc-tion expressed as pg/106 cells/24 h.

2.10. Corneal neovascularization assay

All animal experiments were carried out in an AmericanAssociation for Laboratory Animal Clinicians accreditedfacility under a protocol approved by the University ofWisconsin Animal Care and Use Committee. DEN-HSAcells were grown to a confluent monolayer. Cells werewashed three times with HBSS, and 10 mL DMEM [sup-plemented with 2 mM L-glutamine, penicillin (100 U/mL),and streptomycin (100 lg/mL)] without FBS was added.Cells were incubated overnight in the serum free medium,and the conditioned medium was harvested after 18 h.The conditioned medium was then concentrated to 1 or2 mL using a Centricon filter (Amicon, Bedford, MA) witha 10,000 MW cutoff.

Polyvinyl sponges preirradiated to 2000 Gy (157Cssource) were cut into 0.4 · 0.4 · 0.2 mm pieces, and 0.2–0.5 lL of DEN-HSA conditioned medium or 5· or 10·concentrates were introduced into each sponge using aHamilton syringe. Phosphate-buffered saline served as a

negative control in place of conditioned medium. Theloaded sponges were air-dried, covered with a layer of12% Hydron S, and then dried under vacuum. AdultBALB/c mice were anesthetized, and sponges were intro-duced into a surgically created micropocket in an avascu-lar area of one cornea. Mouse eyes were examined forneovascularization daily using an ophthalmic microscope.Seven days after implantation, 200 ll of FITC-conjugatedhigh molecular weight dextran (3,000,000 MW; Sigma, St.Louis, MO) was injected into the tail vein, and the ani-mals were sacrificed after 5 min. The eye was enucleatedand fixed for 5 min in 4% paraformaldehyde. The corneawith the adjacent limbus was dissected, rinsed in phos-phate-buffered saline, and mounted on a glass slide in10% glycerol. Phase contrast and fluorescence microscopywere used to visualize the overall appearance of the cor-nea and the presence of perfused blood vessels,respectively.

3. Results

3.1. Morphology

The DEN-HSA cell line has been morphologically stablein culture through >150 passages. When subconfluent,DEN-HSA cells appeared elongated and spindleoid, butadopted a more rounded, ‘‘cobblestone-like’’ morphologycharacteristic of EC from many species, including dogs

Fig. 1. Morphology. (A) DEN-HSA cells adopt a ‘‘cobblestone-like’’morphology characteristic of endothelial cells when grown to confluencyin 10% serum-containing medium on plastic (100·). (B) DEN-HSA cellswere plated on Matrigel in EGM-2 medium at a density of 1 · 105 cells/mL. Four hours later, the cells formed tube-like interconnectionscharacteristic of endothelial cells (100·).


(Gerhart et al., 1988) when confluent (Fig. 1(A)). Uponplating on Matrigel�, DEN-HSA formed microvasculartube-like structures (Fig. 1(B)). However, after 24 h thesetube-like structures disappeared, and the cells appearedto form small clumps and invade into the Matrigel�.

3.2. Immunophenotyping

The DEN-HSA cells demonstrated immunocytochemi-cal reactivity for vWF (Fig. 2(A) and (B)) but minimal tono reactivity for CD31 (not shown). Flow cytometric anal-ysis revealed surface expression of avb3 integrin andICAM-1 (Fig. 2(C) and (D)), but lack of expression ofCD34. This is in contrast to normal canine EC, which dem-onstrated expression of avb3 and ICAM-1, but also expres-sion of CD34 (not shown). vWF and CD31 are expressedon most EC, and avb3 and ICAM-1 are primarily expressedon cells with an ‘‘activated’’ or ‘‘angiogenic’’ phenotype.Together, these immunophenotypic data suggest an endo-thelial derivation for DEN-HSA.

3.3. Acetylated LDL uptake, angiotensin converting enzyme

activity

In contrast to Eoma murine endothelioma cells, DEN-HSA failed to take up diI-acetylated LDL (not shown)and failed to cleave [3H]-Hip-Gly-Gly, a process requiringACE activity (Fig. 3). ACE immunoreactivity was likewisenegative when assessed flow cytometrically using a mono-clonal antibody (not shown). Thus, although DEN-HSAexpressed many surface markers consistent with EC, thereare some functional characteristics of EC, such as ACEexpression and AcLDL uptake, which DEN-HSA doesnot recapitulate.

3.4. In vitro cell proliferation

Various concentrations of recombinant human growthfactors were added to DEN-HSA cells in 1% serum-con-taining medium and incubated for 96 h. A significant(p < 0.05) increase in relative viable cell number was ob-served in cells incubated with bFGF, IGF-1, HGF andEGF, but not VEGF or PDGF-bb (Fig. 4).

When DEN-HSA was plated in semisolid medium, col-onies failed to form in high or low serum conditions. How-ever, supplementation of 1% serum-containing mediumwith 10 ng/mL of rhEGF strongly supported colony for-mation. Other growth factors had minimal effects(Fig. 5). Colony size/number was not affected by alteringthe concentration of rhEGF.

3.5. Growth factor/growth factor receptor expression

The DEN-HSA cells expressed mRNA specific forVEGF (the expected PCR product is from the 5 0 end ofthe mRNA, common to all isoforms), VEGFR-1 and -2(flt-1 and flk-1/KDR), bFGF, FGFR-1 and 2, HGF, c-met, IGF-1, IGF-1 receptor, and EGF receptor (Fig. 6).Omission of reverse transcriptase from the RT reaction re-sulted in an absence of all PCR products, excluding geno-mic DNA contamination. After incubation for 3 days in C/10 followed by overnight incubation in serum-free medium,the DEN-HSA cells produced approximately 35 pg bFGF/106 cells/24 h, and 254 pg VEGF/106 cells/24 h as deter-mined by ELISA. These data suggest that DEN-HSA cellsexpress a variety of growth factors and growth factorreceptors that are important in angiogenesis, and suggestthe possibility of multiple autocrine loops that could pro-mote the proliferation and survival of these cells.

3.6. In vivo angiogenesis

Conditioned media and concentrated supernatants fromDEN-HSA cultures induced marked angiogenic responsesin the murine corneal pocket angiogenesis assay. The re-sponse appeared maximal at a 5· supernatant concentra-tion, and was not augmented by use of a 10· concentrate(Fig. 7(A) and (B)). In contrast, no vessel ingrowth was

Fig. 2. Endothelial Marker immunoreactivity. DEN-HSA cells cytospun onto microscope slides (A and B) were immunostained for fVIII using standardmanual immunocytochemistry protocols. A represents fVIII reactivity and B represents fVIII negative control. C and D are flow cytometry histogramsdemonstrating reactivity with the LM609 antibody against alpha-v beta-3 integrin (C) and the CL18/6 antibody against canine ICAM-1 (D).


noted after 7 days in control corneas (Fig. 7(C)). This find-ing indicates that DEN-HSA cells elaborate functionallypro-angiogenic substances, consistent with the RT-PCRand ELISA results.

Fig. 3. Angiotensin-converting enzyme (ACE) activity. Eoma, DEN-HSA,and D17 cells were incubated with [3H]-Hip-Gly-Gly, and radioactivity ofthe cleaved peptide measured as an indicator of ACE activity. DEN-HSAfailed to cleave detectable amounts of the tripeptide.

4. Discussion

There are few endothelial-derived malignant tumour celllines available for study. Those available include the Eomamurine endothelioma cell line (Obeso et al., 1990), and sev-eral artificially transformed murine endothelial cell lines,with in vivo characteristics similar to that of hemangiomaor angiosarcoma (Arbiser et al., 2000, 1997; Montesanoet al., 1990; RayChaudhury et al., 1994). More recently,cell line derived from spontaneous human and canine angi-osarcoma have been described (Akhtar et al., 2004; Fos-mire et al., 2004; Masuzawa et al., 1999). For the mostpart, these cell lines have been differently or incompletelycharacterized, and growth factor/receptor expression andbiologic response to growth factors have not been evalu-ated to our knowledge. The aggressive natural biologicbehavior of canine HSA makes the study of its pathogene-sis exceptionally interesting, as it represents an extremeexample of angiogenic dysregulation.

The morphologic and surface phenotypic characteristicsof DEN-HSA are strongly suggestive of an endothelial der-ivation. Specifically, a compact, cobblestone-like morphol-

Fig. 4. Anchorage-dependent growth. DEN-HSA cells were plated for 96 h in 96-well plates with 10% serum-containing medium or 1% serum-containingmedium supplemented with 0, 1, 10, or 100 ng/mL of various recombinant human growth factors. Relative viable cell number was determined after 96 h byMTS assay. The data above are a representative experiment of three performed. Error bars represent standard error measurement. *p < 0.05 versus C/1.**p < 0.001 versus C/1.


ogy and the formation of tube-like structures when platedon Matrigel� are commonly described characteristics ofnormal EC, including those isolated from canine tissues(Gerhart et al., 1988). However, other investigators havereported that Matrigel� may induce tumour cell lines ofnon-endothelial derivation to form similar networks ofconnections (Obeso et al., 1990). Interestingly, the transientformation of vascular-like networks, followed by their ra-pid disappearance presumably as a result of increased pro-teolytic activity, has been described as a characteristic oftransformed and phenotypically malignant murine EC,

whereas normal EC appear to maintain vascular-like con-nections for a longer time (Montesano et al., 1990).

The surface expression of Von Willebrand factor andCD31 have been previously described as attributes of nor-mal canine EC, and of canine HSA (Ferrer et al., 1995;Gerhart et al., 1988; von Beust et al., 1988). Their expres-sion on DEN-HSA cells is further evidence that the cell linedisplays an endothelial-like phenotype. The expression ofICAM-1 and avb3 integrin has not been evaluated in nor-mal canine EC or HSA to our knowledge. Their expressionon DEN-HSA cells and normal canine EC is not unex-

Fig. 5. Anchorage-independent growth. DEN-HSA cells were plated in asemisolid medium containing 10% serum, or 1% serum plus 10 ng/mL ofvarious recombinant human growth factors. Number of colonies contain-ing greater than 20 cells was determined by light microscopy after 21 days.


pected based on data generated in EC from other species.The relatively abundant expression of ICAM-1 on culturedHSA cells could help to explain the positive clinical resultsobtained utilizing immunotherapeutic approaches such asintralesional interleukin-2 (IL-2) in humans with HSA(Inadomi et al., 1992; Masuzawa et al., 1989; Takanoet al., 1991), and intravenous liposome muramyl tripep-tide-phosphatidylethanolamine in dogs with splenic HSA(Vail et al., 1995). Previous in vitro and in vivo work hasdemonstrated the ability of IL-2 and muramyl peptidesto up-regulate ICAM-1 expression on normal and neoplas-tic cells (Heinzelmann et al., 2000; Ihda et al., 1995). Fur-ther upregulation of ICAM-1 on HSA cells andupregulation of its ligand(s) on leukocytes by these ap-proaches may increase adhesion to and subsequent killingof HSA cells by activated leukocytes.

avb3 Integrin expression has been associated primarilywith proliferating EC (Varner and Cheresh, 1996), but ithas also been detected on the surface of some non-endothe-lial tumour cells (Hofmann et al., 2000). Although impor-tant for the adhesion of cells to the extracellular matrix,avb3 integrin has also been shown to transduce signals reg-ulating cell proliferation/survival and invasion (Hofmannet al., 2000). Solovey et al. (1999) have demonstrated thatthe autocrine secretion of VEGF by circulating EC may re-place the survival signal transduced by avb3, allowing thecells to persist in circulation. The autocrine secretion of

Fig. 6. Angiogenic growth factor/receptor expression. Total RNA was extractedand conditions are listed in Table 1. The sequences for all PCR products wer

VEGF by HSA cells may likewise promote their persistencein circulation and eventual metastasis. Therapeutic strate-gies designed to inhibit avb3 and/or VEGF signaling arebeing investigated, and may be extremely useful in thetreatment of vascular neoplasms such as HSA.

In contrast to the Eoma murine endothelioma cells andnormal canine EC (Thomas et al., 1995), DEN-HSA failedto take up diI-acetylated LDL or express detectable angio-tensin-convering enzyme activity. The DEN-HSA cells, un-like the normal canine EC, also failed to express detectableCD31 or CD34. Thus, DEN-HSA does not recapitulate thebehavior of normal EC in every respect. It is relativelycommon for some phenotypic characteristics of the tissueof origin to be lost in tumour cells, especially after pro-longed passage in tissue culture.

The DEN-HSA cells were capable of enhanced anchor-age-dependent growth in the presence of recombinant hu-man IGF-1, EGF, bFGF and HGF but not PDGF-bb orVEGF. All of these growth factors are known mitogensfor normal human EC. It is possible that DEN-HSA failedto proliferate in response to VEGF and PDGF-bb owing toinsufficient amino acid sequence homology between the ca-nine and human molecules. However, canine VEGF andVEGFR-1 and -2 have recently been cloned and sequenced,and their binding regions found to be 100% homologous tothe human binding regions (Scheidegger et al., 1999). Addi-tionally, canine VEGF has been shown to stimulate thegrowth of human EC (Scheidegger et al., 1999). HumanVEGF did not appear to stimulate the growth of normalcanine EC in our system, despite the fact that mRNAencoding VEGFR-1 and -2 were expressed and that humanVEGF was capable of stimulating the growth of HUVECsin a similar assay. It is possible that the VEGF receptorsmay be saturated due to the autocrine secretion of VEGFin DEN-HSA, or that the tertiary structure of humanVEGF is sufficiently different from canine VEGF to pre-clude its entry into the binding site of the canine VEGFreceptors. Other measures of VEGF activity in EC, suchas enhancement of cell migration, may demonstrate thebioactivity of human VEGF in canine EC and HSA.

Interestingly, EGF was the only growth factor evaluatedthat strongly supported anchorage-independent growthand colony formation by DEN-HSA. Enhanced colony

, reverse transcribed, and utilized in PCR reactions. PCR primer sequencese confirmed using automated DNA sequencing.

Fig. 7. In vivo angiogenic response. Serum-free conditioned medium fromDEN-HSA was concentrated 5· using 10,000 MW Centricon filters, andutilized in a murine corneal pocket assay. Seven days following spongeimplantation, mice were injected IV with 3,000,000-MW dextran-FITCand sacrificed. (A) Cornea viewed under light microscopy. (B) Dissectedcornea viewed with fluorescence microcopy. (C) Control cornea, withPBS-soaked sponge, viewed under fluorescence microscopy.


formation is typically associated with a more aggressiveand metastatic phenotype. The inhibition of EGFR signal-ing could prove an important strategy for the in vivo con-trol of HSA growth and metastasis.

The DEN-HSA cells produced the angiogenic growthfactors VEGF and bFGF at the mRNA and protein level,and mRNA corresponding to several other growth factorswith angiogenic potential was detected. It is important tonote that a significant amount of bFGF and VEGF may re-main intracellular or at the cell surface, and thus assayingcell culture supernatant only rather than cell lysates may

underestimate the quantity of growth factors produced(Bikfalvi et al., 1997; Zhang et al., 2000). The vigorousangiogenic response produced by cell culture supernatantsin the mouse corneal pocket assay provides functional evi-dence for the production of active angiogenic growth fac-tors by DEN-HSA, making these cells useful for thestudy of various in vivo anti-angiogenic approaches wherea less artificial angiogenic stimulus may be preferable.

The presence of mRNAs coding for VEGF and itsreceptors, bFGF and its receptors, HGF and c-met, andIGF-1 and IGF receptor suggests the presence of severalputative autocrine growth factor signaling loops. Insulin-like growth factor and HGF autocrine loops have beendemonstrated in a number of human and canine non-endo-thelial tumour cell lines (Burrow et al., 1998; MacEwenet al., 2003; Parry et al., 1999), however autocrine loopsinvolving the prototypical angiogenic growth factors(e.g., bFGF, VEGF) are less well described. Retrospectivestudies have suggested the presence of both VEGF andVEGFR-1 in human angiosarcoma tissues (Hashimotoet al., 1995), and functional evidence of autocrine stimula-tion through the VEGF–VEGFR pathway exists for Kapo-si�s sarcoma cells and certain human leukaemias (Diaset al., 2000; Masood et al., 1997). Studies are in progressevaluating the effect of VEGF and bFGF signaling inhibi-tion in DEN-HSA cells.

In conclusion, we have described a unique cell line,DEN-HSA, derived from a malignant canine endothelialtumour. The cell line recapitulates many properties consis-tent with an endothelial lineage. Importantly, the cell line iscapable of eliciting in vivo angiogenic responses in mice,thereby offering novel opportunities to investigate the reg-ulation of neoangiogenic responses that may be importantfor cancer therapy. Furthermore, the tumour of origin ofDEN-HSA, canine HSA, is a relatively common spontane-ous cancer of dogs that may offer additional opportunitiesto translate information derived from our cell line to a nat-ural angiogenic tumour in a relevant, large animal model.

Acknowledgments

The authors dedicate this manuscript to their esteemedcolleague and co-author, the late Dr. E. Gregory MacE-wen, and to acknowledge the expert technical assistanceof A.K. Marr, J.M. Schmidt, B. Charles and B. Shinners.This work was supported by the University of WisconsinAnimal Cancer Treatment Fund, and by a generous dona-tion from Mr. A. Rolfe.

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