Article.

1. Biological and molecular characterization
of 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, USA
b
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 availability
of canine HSA-derived cell lines. We report the development of a canine HSA-derived cell line, DEN-HSA, which recapitulates features
of angiogenic endothelium. DEN-HSA cells were derived from a spontaneous HSA arising in the kidney of a dog. DEN-HSA displayed
surface 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 MatrigelÒ
, and proliferated in response to a variety of angiogenic growth factors. The
cells 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 of
angiogenic growth factor autocrine loops, and offers the potential to utilize DEN-HSA cells for the study of novel therapies that mod-
ulate endothelial proliferation.
Ó 2005 Published by Elsevier Ltd.
Keywords: Dog; Angiosarcoma; Endothelium; Angiogenesis
1. Introduction
Altered endothelial cell proliferation and survival is of
prime importance in many disease conditions, including
neoplasia, wound healing, and chronic inflammatory dis-
ease. Perhaps the most dramatic example of dysregulated
angiogenesis is seen in malignancy derived from vascular
endothelium. Investigation of the mechanisms responsible
for tumourigenesis in these neoplasms has the potential
to shed light onto basic concepts of angiogenesis, and
potentially 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% of
soft-tissue sarcomas in one evaluation (Fata et al., 1999).
When encountered, haemangiosarcomas of the human
breast, liver, spleen and scalp behave aggressively, metasta-
sizing commonly and yielding very poor long-term survival
rates (Falk et al., 1979; Holden et al., 1987; Neuhauser
et al., 2000; Silverman et al., 1994).
Haemangiosarcoma (HSA) is a relatively common,
spontaneous tumour in dogs, accounting for approximately
20% of all soft-tissue sarcomas (Dorn et al., 1968), and up
to 5% of all malignant canine neoplasms (Bastianello, 1983;
MacVean et al., 1978). Canine HSA can occur in any site,
0034-5288/$ - see front matter Ó 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, CO
80523-1620, USA. Tel.: +1 970 297 4075; fax: +1 970 297 1254.
E-mail address: dthamm@colostate.edu (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.
www.elsevier.com/locate/rvsc
Research in Veterinary Science 81 (2006) 76–86

2. however the spleen, skin, right atrium, and liver are the
most common primary sites (MacEwen, 2001). Canine
HSA is typified by very aggressive biological behavior, with
wide and rapid metastasis being common. Doxorubicin-
based chemotherapy has the potential to modestly improve
survival time following surgery, however, one-year survival
rates remain less than 10% (Sorenmo et al., 2000; Sorenmo
et al., 1993; Vail et al., 1995).
Canine HSA has potential utility as a spontaneously
occurring model of human endothelial tumours, such as
angiosarcoma/hemangiosarcoma, hemangioma of infancy,
and KaposiÕs sarcoma, a malignant tumour characterized
by the dysregulated proliferation of endothelial cells.
Moreover, canine HSA cells may also be useful as a tool
for the study of endothelial cell biology in malignancy.
For these purposes, we sought to establish a continuous
cell line derived from a canine HSA, and to characterize
this line with regard to its in vitro biological behavior, cell
surface 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 male
golden retriever. The primary tumour was mechanically
and 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 tissue
culture flasks in complete minimal essential medium (Cell-
gro Mediatech, Herndon, VA) supplemented with 5% fetal
bovine 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 with
0.25% trypsin in EDTA. Characterization was performed
on cells having undergone >60 passages.
The D17 (ATCC CRL-8468) cell line is a canine osteo-
sarcoma cell line obtained from American Type Culture
Collection (ATCC), Rockville, MD. The Eoma cell line is
a well-characterized murine endothelial-derived tumour
cell line (Obeso et al., 1990). Normal canine endothelial
cells (EC) were isolated from circulating precursors utiliz-
ing a modification of a published protocol (Solovey et al.,
1999). Briefly, normal dog peripheral blood mononuclear
cells were separated by density centrifugation, and plated
in EGM-2 medium (Clonetics, Temecula, CA). After 1
month of daily medium changes, a confluent population
of endothelial-appearing cells was present. These cells were
determined to be of endothelial origin by morphology on
plastic, CD34 and avb3 immunoreactivity by flow cytome-
try, and the expression of Flt-1, KDR and Tie2/Tek
mRNA by RT-PCR. Human umbilical vein EC (HU-
VECs) were purchased from ATCC, maintained in
EGM-2 medium, and utilized at passage 3–5.
2.2. Morphology on matrigel
Tube formation on MatrigelÒ
was assessed by adapta-
tion of a published protocol (Gho et al., 1999). Briefly,
MatrigelÒ
(Becton Dickinson, Franklin Lakes, NJ) was di-
luted to a concentration of 6 mg/mL with EGM-2 medium
(Clonetics, Walkersville, MD). Diluted MatrigelÒ
(320 lL)
was added to wells of a 24-well plate and incubated at
37 °C for 1 h. DEN-HSA cells, at a concentration of
1 · 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 photographed
after 6 and 24 h of culture.
2.3. Immunohistochemistry/immunocytochemistry
Prediluted rabbit polyclonal antibody against human
Von 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 Beust
et al., 1988), were utilized. The DEN-HSA cells were de-
tached and cytospun onto glass slides, then air-dried for
24 h. The slides were washed and incubated in primary
antibody (vWF: prediluted; CD31: 1:100) for 12 h. Biotin-
ylated goat anti-rabbit or chicken anti-mouse (Rockland
Immunochemicals, Gilbertsville, PA) secondary antibodies
were then applied at a 1:100 dilution for 1 h. Finally, a
horseradish peroxidase–streptavidin conjugate (Vector
Laboratories, Burlingame, CA) was applied at 1:250 for
20 min, followed by application of a DAB substrate (Bio-
care, Concord, CA) and counterstaining with MayerÕs hae-
matoxylin, dehydrated and mounted in xylene-based
mounting medium. Slides were observed and photographed
using light microscopy. Omission of the primary antibody
served 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 antibody
CL18/6, 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 in
non-enzymatic cell dissociation solution (Sigma) and scrap-
ing. 2.5 · 105
DEN-HSA cells were incubated on ice with
primary antibody (0.5 lg for ICAM-1, 5 lg for avb3 and
D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86 77

3. CD34) for 30 min, washed in HankÕs buffered saline solu-
tion (HBSS) containing 0.1% bovine serum albumin, and
then incubated with a goat anti-mouse FITC conjugate
(Serotec, Raleigh, NC) on ice in the dark for 30 min for
detection 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 from
Molecular Probes (Eugene, OR). Subconfluent DEN-
HSA cells were tested for the expression of the acetylated
low density lipoprotein surface receptor by removing the
medium and incubating the cells overnight under standard
conditions in serum-free DMEM containing diIAcLDL
(1:300 dilution). After washing, the cells were observed
with an inverted fluorescent microscope (Nikon) with a
rhodamine filter. Eoma and D17 cells served as positive
and negative controls, respectively.
Angiotensin converting enzyme (ACE) activity was mea-
sured by using radiolabeled diglycyl hippurate ([3
H]-Hip-
Gly-Gly, Ventrex Laboratories, Portland, ME) as substrate.
Enzyme activity was assessed by the ability of the cells to
cleave the radioactive tripeptide, then separating the cleav-
age product on the basis of its differential solubility in ethyl
acetate. Equal numbers of Eoma and D17 cells, serving as
the positive and negative controls, were also tested. In addi-
tion, the ACE level of the furnished control serum and the
efficacy of the radioactive substrate were determined. Incu-
bations were carried out overnight to permit maximum
labeling without significant increase in background incorpo-
ration. Radioactivity in the aqueous fraction, corresponding
to amount of cleaved tripeptide, was measured using a gam-
ma counter. ACE expression was also investigated flow cyto-
metrically utilizing a monoclonal antibody known to
recognize human, bovine and murine ACE (Auerbach
et al., 1982), which was raised in Dr. AuerbachÕs 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 in
our laboratory have demonstrated that recombinant hu-
man HGF and IGF-1 have bioactivity in canine tumour
cells (MacEwen et al., 2003, 2004).
The DEN-HSA cells were plated at 2 · 103
per well in
200 lL C/10%, in 96-well flat-bottom plates (Falcon, Bec-
ton Dickinson) and incubated overnight under standard
conditions. The plates were then washed with HBSS, and
the 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 experiments
when compared to C/10 (not shown). After a 96-h incuba-
tion, relative viable cell number was assessed by means of a
one-step tetrazolium-based (MTS) colorimetric assay
(CellTiter AQueous One, Promega, Madison, WI) accord-
ing to the manufacturerÕs directions. Quintuplicate wells
were run for each condition, and each experiment was re-
peated three times. Human umbilical vein EC were utilized
in an identical cell proliferation assay to insure the bioac-
tivity of the human recombinant VEGF. Differences in rel-
ative viable cell number between experimental conditions
were assessed statistically using StudentÕs 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 medium
with 1% FBS, 2.5 · 103
DEN-HSA cells were strained
through a 40-lm cell strainer to eliminate clumps, plated
in 200 lL 0.3% low-melting agarose in RPMI 1640 with
10% 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 were
incubated under standard conditions for three weeks, and
the number of colonies containing greater than 20 cells
was counted per well, using light microscopy. Experiments
were repeated for three separate times.
2.8. Reverse transcriptase – polymerase chain reaction
Total RNA was isolated from confluent monolayers of
cell cultures using Trizol reagent (Life Technologies, Inc.)
according to the manufacturerÕs instructions. Reverse tran-
scription was carried out according to the manufacturerÕs
instructions using the Superscript Preamplification System
(Life Technologies, Inc.). Oligonucleotide primers directed
against the canine genes (Table 1) were used to amplify the
specific canine sequences from the cDNA generated. All of
the PCR reactions consisted of 35 cycles followed by a 7-
min final extension at 72 °C, followed by a final cooling
step of 4 °C (see Table 1). The PCR reactions were designed
to generate a single PCR product of interest. Taq polymer-
ase was purchased from Promega (Madison, WI). All PCR
products were verified by automated DNA sequencing
using Big Dye Reagent, carried out at the University of
Wisconsin-Madison Biotechnology Center.
2.9. ELISA
Conditioned media were generated by culturing DEN-
HSA cells under standard conditions in C/10 medium in
6-well plates. After 72 h, the plates were washed five times
with HBSS, and serum-free CMEM was added. The super-
natant was harvested after an additional 24 h of culture,
and frozen at À80 °C. Cells were trypsinized and counted
78 D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86

4. on a hemocytometer using Trypan Blue. VEGF and bFGF
concentrations in cell supernatant were determined using
commercial ELISA kits with known cross-reactivity with
the canine growth factors (Allen et al., 1996; Clifford
et al., 2001) (Quantikine, R & D Systems) according to
manufacturer 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 American
Association for Laboratory Animal Clinicians accredited
facility under a protocol approved by the University of
Wisconsin Animal Care and Use Committee. DEN-HSA
cells were grown to a confluent monolayer. Cells were
washed 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 or
2 mL using a Centricon filter (Amicon, Bedford, MA) with
a 10,000 MW cutoff.
Polyvinyl sponges preirradiated to 2000 Gy (157
Cs
source) 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 a
Hamilton syringe. Phosphate-buffered saline served as a
negative control in place of conditioned medium. The
loaded sponges were air-dried, covered with a layer of
12% Hydron S, and then dried under vacuum. Adult
BALB/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 for
neovascularization daily using an ophthalmic microscope.
Seven days after implantation, 200 ll of FITC-conjugated
high 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 enucleated
and fixed for 5 min in 4% paraformaldehyde. The cornea
with the adjacent limbus was dissected, rinsed in phos-
phate-buffered saline, and mounted on a glass slide in
10% glycerol. Phase contrast and fluorescence microscopy
were 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 stable
in culture through >150 passages. When subconfluent,
DEN-HSA cells appeared elongated and spindleoid, but
adopted a more rounded, ‘‘cobblestone-like’’ morphology
characteristic of EC from many species, including dogs
Table 1
PCR primers and conditions
PCR product Primers Annealing temp (°C) Size (bp)
VEGF For: 50
-CCATGAACTTTCTGCTCTCTTG-30
60 418
Rev: 50
-TTGTCTTGCTCTATCTTTGTT-30
VEGFR-1 (Flt-1) For: 50
-AACTGAGTTTAAAAGGCAC-30
50 505
Rev: 50
-TCTTTGTACGTTGCATTTG-30
VEGFR-2 (Flk-1) For: 50
-CTRGCYGTCGCYCTGTGGYTCTGC-30
55 371
Rev: 50
-AGARGCRATRAATGGWGATCCTGTA-30
bFGF For: 50
-CTTCAAGGACCCCAAGCGGC-30
55 390
Rev: 50
-GCTCTTAGCAGACATTGG-30
FGFR-1 For: 50
-ACCACCTACTTCTCCGTCAAT-3 58 354
Rev: 50
- TAGTTGCCCTTGTCAGARGG-30
FGFR-2 For: 50
-CCAGAAGAGCCACCAACCAAATA-30
58 501
Rev: 50
-TCCTGCTTAAACTCCTTCCCG-30
IGF-1 For: 50
-AAGCAGCACTCATCCACGAT-30
52 281
Rev: 50
-CAGCAGTCTTCCAACCCAAT-30
IGF-1R For: 50
-CCTCCACATCCTGCTCATCT-30
52 444
Rev: 50
-GGATRCAGTACATGCTCTGG-30
HGF For: 50
-CAGACACCACACCGGCACAA-30
54 421
Rev: 50
-GAGCAGTAGCCAACTCGGA-30
c-Met For: 50
-GATCTGGGCAGTGAATTAGT-30
52 417
Rev: 50
-GTCCAACAAAGTCCCATGAT-30
*
EGF-R For: 50
-AGGAGAGGAGAACTGCCAGA-30
55 250
Rev: 50
-CAGGTGGCACCAAAGCTGTA-30
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.
D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86 79

5. (Gerhart et al., 1988) when confluent (Fig. 1(A)). Upon
plating on MatrigelÒ
, DEN-HSA formed microvascular
tube-like structures (Fig. 1(B)). However, after 24 h these
tube-like structures disappeared, and the cells appeared
to form small clumps and invade into the MatrigelÒ
.
3.2. Immunophenotyping
The DEN-HSA cells demonstrated immunocytochemi-
cal reactivity for vWF (Fig. 2(A) and (B)) but minimal to
no reactivity for CD31 (not shown). Flow cytometric anal-
ysis revealed surface expression of avb3 integrin and
ICAM-1 (Fig. 2(C) and (D)), but lack of expression of
CD34. 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 expressed
on most EC, and avb3 and ICAM-1 are primarily expressed
on 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 [3
H]-Hip-Gly-Gly, a process requiring
ACE activity (Fig. 3). ACE immunoreactivity was likewise
negative when assessed flow cytometrically using a mono-
clonal antibody (not shown). Thus, although DEN-HSA
expressed many surface markers consistent with EC, there
are some functional characteristics of EC, such as ACE
expression and AcLDL uptake, which DEN-HSA does
not recapitulate.
3.4. In vitro cell proliferation
Various concentrations of recombinant human growth
factors 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 and
EGF, 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 medium
with 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 altering
the concentration of rhEGF.
3.5. Growth factor/growth factor receptor expression
The DEN-HSA cells expressed mRNA specific for
VEGF (the expected PCR product is from the 50
end of
the 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 cells
express a variety of growth factors and growth factor
receptors that are important in angiogenesis, and suggest
the 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 from
DEN-HSA cultures induced marked angiogenic responses
in 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. 1. Morphology. (A) DEN-HSA cells adopt a ‘‘cobblestone-like’’
morphology characteristic of endothelial cells when grown to confluency
in 10% serum-containing medium on plastic (100·). (B) DEN-HSA cells
were plated on Matrigel in EGM-2 medium at a density of 1 · 105
cells/
mL. Four hours later, the cells formed tube-like interconnections
characteristic of endothelial cells (100·).
80 D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86

6. noted after 7 days in control corneas (Fig. 7(C)). This find-
ing indicates that DEN-HSA cells elaborate functionally
pro-angiogenic substances, consistent with the RT-PCR
and ELISA results.
4. Discussion
There are few endothelial-derived malignant tumour cell
lines available for study. Those available include the Eoma
murine endothelioma cell line (Obeso et al., 1990), and sev-
eral artificially transformed murine endothelial cell lines,
with in vivo characteristics similar to that of hemangioma
or angiosarcoma (Arbiser et al., 2000, 1997; Montesano
et 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 most
part, these cell lines have been differently or incompletely
characterized, and growth factor/receptor expression and
biologic response to growth factors have not been evalu-
ated to our knowledge. The aggressive natural biologic
behavior of canine HSA makes the study of its pathogene-
sis exceptionally interesting, as it represents an extreme
example of angiogenic dysregulation.
The morphologic and surface phenotypic characteristics
of DEN-HSA are strongly suggestive of an endothelial der-
ivation. Specifically, a compact, cobblestone-like morphol-
Fig. 2. Endothelial Marker immunoreactivity. DEN-HSA cells cytospun onto microscope slides (A and B) were immunostained for fVIII using standard
manual immunocytochemistry protocols. A represents fVIII reactivity and B represents fVIII negative control. C and D are flow cytometry histograms
demonstrating reactivity with the LM609 antibody against alpha-v beta-3 integrin (C) and the CL18/6 antibody against canine ICAM-1 (D).
Fig. 3. Angiotensin-converting enzyme (ACE) activity. Eoma, DEN-HSA,
and D17 cells were incubated with [3
H]-Hip-Gly-Gly, and radioactivity of
the cleaved peptide measured as an indicator of ACE activity. DEN-HSA
failed to cleave detectable amounts of the tripeptide.
D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86 81

7. ogy and the formation of tube-like structures when plated
on MatrigelÒ
are commonly described characteristics of
normal EC, including those isolated from canine tissues
(Gerhart et al., 1988). However, other investigators have
reported that MatrigelÒ
may induce tumour cell lines of
non-endothelial derivation to form similar networks of
connections (Obeso et al., 1990). Interestingly, the transient
formation 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 of
transformed 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 and
CD31 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 line
displays an endothelial-like phenotype. The expression of
ICAM-1 and avb3 integrin has not been evaluated in nor-
mal canine EC or HSA to our knowledge. Their expression
on DEN-HSA cells and normal canine EC is not unex-
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-containing
medium supplemented with 0, 1, 10, or 100 ng/mL of various recombinant human growth factors. Relative viable cell number was determined after 96 h by
MTS 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.
82 D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86

8. pected based on data generated in EC from other species.
The relatively abundant expression of ICAM-1 on cultured
HSA cells could help to explain the positive clinical results
obtained utilizing immunotherapeutic approaches such as
intralesional interleukin-2 (IL-2) in humans with HSA
(Inadomi et al., 1992; Masuzawa et al., 1989; Takano
et 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 has
demonstrated the ability of IL-2 and muramyl peptides
to 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 and
upregulation of its ligand(s) on leukocytes by these ap-
proaches may increase adhesion to and subsequent killing
of HSA cells by activated leukocytes.
avb3 Integrin expression has been associated primarily
with proliferating EC (Varner and Cheresh, 1996), but it
has 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 (Hofmann
et al., 2000). Solovey et al. (1999) have demonstrated that
the autocrine secretion of VEGF by circulating EC may re-
place the survival signal transduced by avb3, allowing the
cells to persist in circulation. The autocrine secretion of
VEGF by HSA cells may likewise promote their persistence
in circulation and eventual metastasis. Therapeutic strate-
gies designed to inhibit avb3 and/or VEGF signaling are
being investigated, and may be extremely useful in the
treatment of vascular neoplasms such as HSA.
In contrast to the Eoma murine endothelioma cells and
normal canine EC (Thomas et al., 1995), DEN-HSA failed
to 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 detectable
CD31 or CD34. Thus, DEN-HSA does not recapitulate the
behavior of normal EC in every respect. It is relatively
common for some phenotypic characteristics of the tissue
of 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 or
VEGF. All of these growth factors are known mitogens
for normal human EC. It is possible that DEN-HSA failed
to proliferate in response to VEGF and PDGF-bb owing to
insufficient amino acid sequence homology between the ca-
nine and human molecules. However, canine VEGF and
VEGFR-1 and -2 have recently been cloned and sequenced,
and their binding regions found to be 100% homologous to
the human binding regions (Scheidegger et al., 1999). Addi-
tionally, canine VEGF has been shown to stimulate the
growth of human EC (Scheidegger et al., 1999). Human
VEGF did not appear to stimulate the growth of normal
canine EC in our system, despite the fact that mRNA
encoding VEGFR-1 and -2 were expressed and that human
VEGF was capable of stimulating the growth of HUVECs
in a similar assay. It is possible that the VEGF receptors
may be saturated due to the autocrine secretion of VEGF
in DEN-HSA, or that the tertiary structure of human
VEGF is sufficiently different from canine VEGF to pre-
clude its entry into the binding site of the canine VEGF
receptors. Other measures of VEGF activity in EC, such
as enhancement of cell migration, may demonstrate the
bioactivity of human VEGF in canine EC and HSA.
Interestingly, EGF was the only growth factor evaluated
that strongly supported anchorage-independent growth
and colony formation by DEN-HSA. Enhanced colony
Fig. 5. Anchorage-independent growth. DEN-HSA cells were plated in a
semisolid medium containing 10% serum, or 1% serum plus 10 ng/mL of
various recombinant human growth factors. Number of colonies contain-
ing greater than 20 cells was determined by light microscopy after 21 days.
Fig. 6. Angiogenic growth factor/receptor expression. Total RNA was extracted, reverse transcribed, and utilized in PCR reactions. PCR primer sequences
and conditions are listed in Table 1. The sequences for all PCR products were confirmed using automated DNA sequencing.
D.H. Thamm et al. / Research in Veterinary Science 81 (2006) 76–86 83

9. formation is typically associated with a more aggressive
and 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 growth
factors VEGF and bFGF at the mRNA and protein level,
and mRNA corresponding to several other growth factors
with angiogenic potential was detected. It is important to
note that a significant amount of bFGF and VEGF may re-
main intracellular or at the cell surface, and thus assaying
cell culture supernatant only rather than cell lysates may
underestimate the quantity of growth factors produced
(Bikfalvi et al., 1997; Zhang et al., 2000). The vigorous
angiogenic response produced by cell culture supernatants
in 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 the
study of various in vivo anti-angiogenic approaches where
a less artificial angiogenic stimulus may be preferable.
The presence of mRNAs coding for VEGF and its
receptors, bFGF and its receptors, HGF and c-met, and
IGF-1 and IGF receptor suggests the presence of several
putative autocrine growth factor signaling loops. Insulin-
like growth factor and HGF autocrine loops have been
demonstrated in a number of human and canine non-endo-
thelial tumour cell lines (Burrow et al., 1998; MacEwen
et al., 2003; Parry et al., 1999), however autocrine loops
involving the prototypical angiogenic growth factors
(e.g., bFGF, VEGF) are less well described. Retrospective
studies have suggested the presence of both VEGF and
VEGFR-1 in human angiosarcoma tissues (Hashimoto
et al., 1995), and functional evidence of autocrine stimula-
tion through the VEGF–VEGFR pathway exists for Kapo-
siÕs sarcoma cells and certain human leukaemias (Dias
et al., 2000; Masood et al., 1997). Studies are in progress
evaluating 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 endothelial
tumour. The cell line recapitulates many properties consis-
tent with an endothelial lineage. Importantly, the cell line is
capable of eliciting in vivo angiogenic responses in mice,
thereby offering novel opportunities to investigate the reg-
ulation of neoangiogenic responses that may be important
for cancer therapy. Furthermore, the tumour of origin of
DEN-HSA, canine HSA, is a relatively common spontane-
ous cancer of dogs that may offer additional opportunities
to 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 esteemed
colleague and co-author, the late Dr. E. Gregory MacE-
wen, and to acknowledge the expert technical assistance
of A.K. Marr, J.M. Schmidt, B. Charles and B. Shinners.
This work was supported by the University of Wisconsin
Animal Cancer Treatment Fund, and by a generous dona-
tion from Mr. A. Rolfe.
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