Original Article DOI: 10.1111/vco.

12073

Wnt5a expression in canine osteosarcoma

N. Szigetvari1 , D. M. Imai2 , C. M. Piskun1 , L. C. S. Rodrigues1 , E. Chon1
and T. J. Stein1,3,4
1
Deparment of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison,
WI, USA
2 Deparment of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison,

Madison, WI, USA

3
Institute for Clinical & Translational Research, University of Wisconsin-Madison, Madison, WI, USA
4 Carbone Cancer Center, University of Wisconsin-Madison, Madison, WI, USA

Keywords
cell signalling, comparative
oncology, oncology, small
animal, tumour biology
Abstract
Osteosarcoma is the most common primary malignancy of bone in dogs and is associated with poor
long-term outcomes due to its highly metastatic nature. A better understanding of the signalling
pathways and proteins involved with osteosarcoma pathogenesis may aid in improved outcomes
through the use of targeted therapies. The Wnt5a protein, a ligand for the non-canonical Wnt
signalling pathway, is implicated in mediating the aggressiveness of cancer cell lines, including those
of human osteosarcoma origin. Given the close relationship between human and canine
osteosarcoma, the primary goal of this study was to characterize Wnt5a expression in canine
osteosarcoma. Second, if Wnt5a expression was present in canine osteosarcoma, the study aimed to
determine any potential association with clinical outcome and clinical variables in similarly treated
osteosarcoma-bearing dogs. Wnt5a expression was present in 26 of the 48 (54%) cases of canine
osteosarcoma. Wnt5a expression was not associated with progression-free survival (P = 0.4) or overall
survival (P = 0.1).

Introduction and how they contribute to the highly aggressive

Correspondence address:
T. J. Stein
2015 Linden Drive
Madison, WI 53706, USA
e-mail:
tjstein@wisc.edu
Osteosarcoma is the most common primary
malignancy of bone in dogs, accounting for 80%
of bone tumours.1 It is believed that this tumour
develops from cells of the osteoblast lineage and
typical lesions are often at the metaphysis of
appendicular bone with a radiographic proliferative
and lytic appearance.1 At diagnosis, only 10%
of cases will have evidence of metastatic disease.
Unfortunately, 90% of cases will develop metastatic
lesions despite control of the primary disease
through surgical resection. The most common sites
for the development of osteosarcoma metastases are
the lungs and less commonly to distant bone sites,
lymph nodes and soft tissues.1 An emphasis has
been placed on improving our understanding of the
molecular alterations occurring in osteosarcoma
behaviour of this cancer.
Recently, the results of in vitro assays and in
vivo murine models have implicated the Wnt-5a
protein in promoting a more aggressive phenotype
of human osteosarcoma cell lines.2 The Wnt-5a
protein serves as a ligand for the non-canonical
Wnt pathway, which encompasses Wnt protein
signalling independent of ß-catenin (i.e. canonical
signalling), including the Wnt-5a/calcium pathway
and Wnt-5a/c-Jun N-terminal kinase (JNK)
pathways.3,4 In bone development, this 38–45 kDa
glycoprotein is believed to be involved in planar
cell polarity and it is surmised that deregulation of
this pathway may enhance tumourigenesis and/or
metastasis.4,5 The activation of the non-canonical
Wnt pathway commences with binding of Wnt5a to

© 2013 John Wiley & Sons Ltd 225

226 N. Szigetvari et al.
cell surface protein frizzled-2 (fzd-2), which in turn
activates Rho/Rac signalling to effect changes in cell
migration and perhaps potentiate the metastatic
capability of cells.3,6 In addition, Wnt-5a can
propagate invasive ability through the Wnt/calcium
pathway.3 This pathway begins with binding to
fzd-2 as well as co-receptor orphan tyrosine
kinase receptor (Ror2). This cell surface complex
subsequently activates intracellular dishevelled
protein (Dsh/Dsl), creating a release of intracellular
calcium that activates either Rho/Rac GTPases,
AP-1 or JNK signalling all of which can promote
matrix metalloproteinase-13 (MMP-13).3,7
Canine osteosarcoma has been previously
described as indistinguishable from human
osteosarcoma based on histologic features and pro-
tein expression.8,9 The clinical characteristics also
bear such strong resemblance to human osteosar-
coma that canine osteosarcoma has been suggested
as an ideal model for the study of spontaneous
osteosarcoma.10 Therefore, this study was set to
determine the expression of Wnt-5a in sponta-
neous canine osteosarcoma samples. Furthermore,
if differences were recognized in protein expression
amongst patient samples, then its association with
clinical variables and prognostic value would be
evaluated retrospectively by comparing disease-free
interval (DFI) and overall survival (OS) using a pop-
ulation of similarly treated dogs. We hypothesized
Wnt-5a would be expressed in canine osteosarcoma
and Wnt5a expression would be associated with a
more aggressive clinical behaviour as indicated by
shorter time-to-progression and survival times.
Materials and methods
Sample selection
Archived tissue blocks of canine osteosarcoma
from the Pathology Service at the University of
Wisconsin-Madison Veterinary Medical Teaching
Hospital (UWVMTH) were identified by medi-
cal records search. Canine osteosarcoma tissue
was collected at the time of diagnostic biopsy,
surgical amputation or necropsy. The formalin-
fixed paraffin-embedded osteosarcoma samples
were sectioned in 5 μm slices and transposed to
slides for either immunohistochemical or haema-
toxylin & eosin (HE) staining. The diagnosis of
© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
osteosarcoma was confirmed on HE-stained slides
by a single board-certified veterinary pathologist in
the Department of Pathobiological Sciences at the
University of Wisconsin-Madison (DI).
Medical records were reviewed for all cases
with a histopathologic diagnosis of osteosarcoma.
The following information was obtained from
the medical record of each patient: age, breed,
tumour location, sex, serum alkaline phosphatase
concentration, treatment modality elected (surgical
amputation, chemotherapy, radiation therapy),
specific chemotherapy protocol utilized, use of
rescue therapy and protocol, results of thoracic
radiographs, development of metastatic disease
and date of death. The presumptive diagnosis
of pulmonary metastasis was based on thoracic
radiographs and confirmed in cases undergoing
necropsy, however, this was not permitted in all
cases. The development of bone or soft tissue
metastasis was confirmed by aspiration cytology
or biopsy histology. Information regarding the
development of metastatic disease and patient
outcome that was not in the medical record was
gained by telephone communications with primary
care veterinarians and/or owners themselves.
Cell line and tissue lysates
The human (Saos-2 and MG-63) and canine
(Abrams and D17) osteosarcoma cell lines were
kindly provided by Dr David Vail at UW-Madison.
The UWKOS1, UWKOS2, UWKOS3, UWKOS6,
UWKOS7 and UWKOS8 canine osteosarcoma cell
lines were generated from canine osteosarcoma
tumour tissue collected from dogs undergoing
amputation for treatment of osteosarcoma at UW
Veterinary Care.11 Tumour samples were collected
following amputation and immediately flash-frozen
in liquid nitrogen and stored at −80 ◦ C until
use. The K7M2 murine osteosarcoma cell line
was kindly provided by Dr Chand Khanna of
the National Cancer Institute. Canine normal
osteoblasts were purchased from Cell Applications
(Catalog #Cn406-05, San Diego, CA, USA).
Evaluation of Wnt5a RNA expression
Total RNA was isolated from cell lines using
Trizol (Invitrogen, Carlsbad, CA, USA), and

purified by PureLink RNA Mini Kit (Ambion; Life
Technologies, Carlsbad, CA, USA) according to the
manufacturer’s instructions. cDNA was synthesized
from 250 ng of total RNA using the High
Capacity cDNA Reverse Transcription Kit (Applied
Biosystems, Life Technologies, Carlsbad, CA, USA)
according to the manufacturers protocol. qPCR
was performed using TaqMan Gene Expression
Master Mix with TaqMan Gene Expression Assays
(Applied Biosystems, Life Technologies) according
to the manufacturers’ protocol on a Bio-Rad iCycler
machine with a Bio-Rad iQ5 Multicolor Real-
Time PCR Detection System. The canine Wnt5a
(Cf02676898_m1) assay was utilized (Applied
Biosystems, Life Technologies). Ct values were
normalized to 18S expression (4352930E). Normal
canine osteoblasts were used to determine relative
expression of Wnt5a in canine osteosarcoma cell
lines using the Ct method. Gene expression of
samples was measured in triplicate.
Western blot analysis with Wnt5a antibody
To determine the specificity of the Wnt5a antibody
(sc-30224, Santa Cruz Biotechnology, Santa Cruz,
CA, USA) used in this study, western blot analysis
was performed on cell lysates from osteosarcoma
cell lines of human (Saos-2, MG-63), canine (D17,
UWKOS6, UWKOS7 and UWKOS8) and murine
(K7M2) origin. In addition, osteosarcoma tissue
lysate from patient tumour samples (UWKOS6,
UWKOS7 and UWKOS8) were assessed for Wnt5a
expression. Lysate from the Saos-2 cell line served
as a positive control, as Wnt5a has been detected
in this cell line previously (16). Briefly, cells were
lysed using a mammalian protein extraction reagent
(Pierce, Rockford, IL, USA) and protein lysates
collected. Protein lysates were separated by elec-
trophoresis on a 7.5% Mini-PROTEAN TGX Gel
(BioRad, Hercules, CA, USA) at 150 V for ∼45 min,
transferred to nitrocellulose membranes (What-
man, Dassel, Germany) at 100 V for 1 h, then
blocked with tris-buffered saline/0.05% Tween20
(TBST) containing 5% non-fat dry milk and 1%
bovine serum albumin for 1 h (all reagents from
Fisher Scientific). The membranes were probed
overnight at 4 ◦ C with either (1) rabbit anti-Wnt5a
antibody (sc-30224, Santa Cruz Biotechnology)
© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
Wnt5a expression in canine osteosarcoma 227
diluted 1:200 in blocking solution or (2) rab-
bit anti-Laminin (ab11575, Abcam, Cambridge,
MA, USA) diluted 1:1000 in blocking solution.
Excess primary antibody was removed by wash-
ing three times for 5 min with TBST. Mem-
branes were incubated with 50 ng mL−1 horseradish
peroxidase-conjugated anti-rabbit secondary anti-
body (Thermo Scientific, Waltham, MA, USA)
diluted in blocking solution for 1 h at room tem-
perature, then washed three times for 5 min with
TBST, and treated with SuperSignal West Dura
Chemiluminescent Substrate (Thermo Scientific).
Blots were exposed to film, developed, and then
imaged using a Gel Logic 100 Imaging System
(Kodak, Rochester, NY, USA).
Wnt-5a detection by immunohistochemistry
Immunohistochemistry was performed to deter-
mine the presence of the Wnt-5a protein in canine
osteosarcoma. Slides were first deparaffinized and
rehydrated by soaking in CitriSolv for 5 min fol-
lowed by a series of decreasing concentrations of
ethanol (100, 95, 70 and 50%) and finally tap water.
Endogenous peroxidase activity was quenched with
hydrogen peroxide 0.5% in methanol for 20 min
and then rinsed in tap water. Antigen retrieval was
performed by incubation in boiling 0.1 M citrate
buffer solution 0.1 M (pH 6.0) for 10 min. Non-
specific binding was prevented by soaking slides in
a solution of phosphate-buffered saline containing
non-fat dry milk (PBS/milk, 0.5 g/100 mL). Vec-
tor Vectastain Elite ABC Kit rabbit IgG (PK-6101,
Vector Laboratories, Burlingame, CA, USA) was
utilized to provide serum for additional blocking
and indirect antibody detection methods. Normal
horse serum mixed with PBS/milk solution was
applied to tissue of each slide for 20 min with excess
serum subsequently being blotted from slides. Slides
were then incubated in a humidifying chamber with
rabbit polyclonal anti-Wnt-5a antibody for either
60 min at room temperature (37 ◦ C) or at 4 ◦ C for
8 h. A set of identical tissue sample slides served
as negative controls, and were incubated in the
same chamber with the primary antibody omitted.
All slides were washed in PBS for 5 min and incu-
bated for 30 min with Vectastain Elite anti-rabbit
biotinylated antibody. Following incubation with
228 N. Szigetvari et al.
the secondary antibody, slides were again washed
in PBS for 5 min and then Vectastain Elite ABC
reagent was applied for 5 min, before rinsing in
PBS for a further 5 min. A peroxidase substrate
solution (3,3 -diaminobenzidine) was applied to
all slides for 5 min, after which all slides were
rinsed and washed in PBS for 5 min. Finally, coun-
terstaining was performed with haematoxylin and
slide tissues were dehydrated by being soaked in
ethanol series in the reverse order as described
above.
Evaluation of Wnt5a IHC staining
Wnt-5a immunohistochemistry, appropriate con-
trols and HE-stained sections were evaluated simul-
taneously for each canine osteosarcoma by one
board-certified veterinary pathologist (D. I.). Pos-
itive Wnt-5a staining was defined as strong to
moderate uniform cytoplasmic immunoreactivity
in 90–100% of the neoplastic cell population.
Negative Wnt-5a staining was defined as weak, non-
uniform or absent cytoplasmic immunoreactivity.
Statistical analyses
For the primary objective of this study, determining
whether Wnt5a expression occurs in canine
osteosarcoma, only descriptive statistics are utilized
given the diversity of tumour locations and
treatments elected by owners. For the study’s
secondary objective, evaluating the relationship
between Wnt5a expression and clinical variables,
cases of appendicular osteosarcoma which had
undergone surgical excision of the primary tumour
and adjuvant chemotherapy were divided into two
sub-groups; those samples staining positive for the
presence of Wnt5a (Wnt5a-positive), and those
samples that were not Wnt5a immunoreactive
(Wnt5a-negative).
Descriptive and comparative statistics were
performed for the overall study population, as well
as the two main sub-groups. Fisher’s Exact Test was
used to compare categorical non-numerical data,
such as gender, between groups. The Wilcoxon
Rank-Sum Test was used to compare continuous
numerical data, such as age and weight, between
groups. Results were considered significant at
P < 0.05.
© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
Two measures of outcome were evaluated.
Progression-free survival (PFS) was defined as the
time (in days) from the date of surgery until the date
of detectable metastatic disease. OS was defined as
the time (in days) from the date of surgery until
the date of death as a result of osteosarcoma or
another cause. Data was considered right-censored
at the time of last veterinary contact if the dogs
remained alive at the conclusion of the study
period, or if they were ultimately lost to follow-
up (n = 2). Kaplan–Meier survival curves were
estimated for both outcomes, along with median
survival times and confidence intervals. PFS and OS
were compared between the study’s two sub-groups
(Wnt5a-positive versus Wnt5a-negative) using the
Log-Rank Test. Results were considered significant
at P < 0.05.
All statistical analyses were performed utilizing
two commercially available software packages:
® ®
Microsoft Excel for Mac 2011 (version 14.2.5)
and R for Mac OS X GUI 2012 (version 2.15.2).
Results
The expression of Wnt5a mRNA in canine osteosar-
coma cell lines was identified in all cell lines tested.
Wnt5a mRNA expression was highest in the Abrams
cell line relative to the other cell lines examined.
Interestingly, the majority of canine osteosar-
coma cell lines had decreased Wnt5a mRNA
expression relative to normal canine osteoblasts
(Fig. 1).
Prior to the performance of Wnt5a IHC on
patient tumour samples, the Wnt5a antibody
was validated for use in canine cells and tissue
samples. Saos-2 cell line lysate, in which Wnt5a
expression has previously been identified, along
with lysates from human (MG-63), murine (K7M2)
and canine (D17, Abrams, UWKOS6, UWKOS7
and UWKOS8) osteosarcoma cell lines and normal
canine osteoblasts were utilized to ensure the
antibody would cross-react with canine tissue and
identify the appropriately-sized protein (∼42 kDa).
In addition, the corresponding tumour tissue used
to generate UWKOS6, UWKOS7 and UWKOS8
was assessed along with canine normal bone. As
shown in the Western blot images, an appropriately-
sized protein product was present in the majority
 Wnt5a expression in canine osteosarcoma 229

Wnt5a Teaching Hospital between May 2002 and Decem-

4.5
ber 2010. The median age of all dogs was 7 years

Relative gene expression

4.0
(range: 1–14). The median weight of all dogs was
3.5
3.0 35 kg (range: 21–76 kg). Of the 30 dogs, there
2.5 were 18 neutered males, 11 spayed females and 1
2.0 intact female. Osteosarcoma samples from 17 dif-
1.5 ferent breeds, purebred or crosses, were included
1.0 in the study, with 8 Labrador retrievers, 2 Golden
0.5 retrievers, 2 Irish Wolfhounds, 2 Saint Bernards,
0.0
2 Great Danes, 2 Boxers, 2 Rottweilers and 1
UW S1

UW S2

UW S3

UW S6

UW S7

Ab 8

ts
m
S

as
each of Australian Shepherd, Doberman Pinscher,
KO

KO

KO

KO

KO

KO

ra

bl
eo
UW

German Shepherd, Greyhound, Belgian Turvuren,

st
O
Figure 1. Expression of Wnt5a mRNA in canine
osteosarcoma cell lines relative to a canine normal osteoblast
cell line.
of normal and neoplastic cell line and tissue
lysates suggesting the Wnt5a antibody would be
useful for identifying the Wnt5a protein in canine
osteosarcoma (Fig. 2). In the osteosarcoma samples
having paired cell line and tissue lysates, the
presence of Wnt5a appeared to be increased in
tissue lysates relative to cell lines.
To evaluate Wnt5a expression in canine osteosar-
coma, 48 biopsy samples from 48 dogs were used in
this study. Tumours location were recorded as being
distal radius (n = 16), proximal humerus (n = 10),
distal tibia (n = 6), distal femur (n = 4), car-
pus (n = 2), distal ulna (n = 2), mandible/maxilla
(n = 2), proximal radius (n = 1), proximal femur
(n = 1), proximal tibia (n = 1), scapula (n = 1),
zygomatic arch (n = 1) and rib (n = 1).
Overall, 26 of the 48 (54%) osteosarcoma samples
were positive for Wnt5a expression, whereas 22
of the 48 (46%) were considered negative for
Wnt5a expression (Fig. 3). The Wnt5a staining
was considered cytoplasmic in all 26 cases.
Thirty dogs were included in the second aim
of this study to evaluate the relationship between
Wnt5a expression and various clinical variables
and outcomes in similarly treated dogs. Of the
30 cases, 16 (53%) were considered positive for
Wnt5a expression and 14 (47%) did not have Wnt5a
expression detectable by IHC.
All dogs undergoing surgery and chemotherapy
for treatment of their osteosarcoma presented to
the University of Wisconsin Veterinary Medical
Anatolian Shepherd, Newfoundland, Border Collie,
Leonberger and Dalmatian.
At the time of diagnosis, 21 dogs had a normal
serum Alkaline phosphatase (ALP) concentra-
tion and 9 dogs had an increased serum ALP
concentration. Of the 21 dogs with normal ALP
concentration, 14 were Wnt5a-positive and 7 were
Wnt5a-negative. For the nine dogs with increased
serum ALP concentration, four were Wnt5a-
positive and five were Wnt5a-negative. There was
no correlation between Wnt5a expression and
serum ALP concentration.
Tumour locations for this subset of patients
included the radius in 12 patients, humerus in 7
patients, tibia in 5 patients, femur in 3 patients,
carpus in 2 patients and ulna in 1 patient. Of the
16 Wnt5a-positive tumour locations, 8 were radius
(50%), 3 (19%) humerus, 2 (13%) tibia and 1 in
the ulna (6%), femur (6%) and carpus (6%). For
the 14 Wnt5a-negative tumours, locations included
the radius in 4 (29%), humerus in 4 (29%), tibia
in 3 (21%), femur in 2 (14%) and carpus in 1
(7%). There was no correlation between Wnt5a
expression and tumour location.
Chemotherapy was started within 4 weeks of
amputation for all 30 patients. Initial protocols most
commonly involved platinum drugs (carboplatin
n = 27/30, cisplatin n = 2/30) given intravenously
every 3–4 weeks. Occasionally, carboplatin was
given with additional cytotoxic or palliative agents,
such as either gemcitabine (n = 7) or pamidronate
(n = 7). One patient was treated with an alternating
protocol of cisplatin and doxorubicin. Finally,
one patient was receiving a c-met inhibitor drug
through a clinical trial ongoing at the time. Overall,
the median number of doses of platinum-drug

© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
230 N. Szigetvari et al.

Figure 2. Western blot analyses for the presence of Wnt5a reveals an appropriate sized protein (42 kDa) in murine (K7M2)
and canine osteosarcoma cell lines (D17, Abrams, UWKOS6, and UWKOS8) and tissue (UWKOS6, UWKOS7, UWKOS8)
lysates. Wnt5a protein expression was also detected in canine normal osteoblast cell line and bone tissue. Lysates from
human osteosarcoma cell lines (Saos-2 & MG-63) used as positive controls. The top band represent the loading control,
laminin (250 kDa).

A B C

Figure 3. Cytoplasmic expression of Wnt5a in canine appendicular osteosarcoma. (A) A typical neoplastic mesenchymal
population with deposition of neoplastic osteoid (asterisk). Subsets exhibit (B) strong cytoplasmic immunoreactivity or (C)
negative cytoplasmic immunoreactivity for Wnt5a. ×60. HE.

chemotherapy received by each patient was 4
(1–4). The median number of doses of platinum-
drug chemotherapy (4) was the same between
patients with Wnt5a-positive and Wnt5a-negative
tumours.
Metastatic disease developed in 22 of the 30
patients (73%), based on thoracic radiographs,
aspirate or biopsy of palpable lesions, and/or
necropsy. Of the remaining eight patients, six
patients had no information on development
of metastatic disease prior to date of death,
one patient was euthanized for non-osteosarcoma
reasons without evidence of metastatic disease,
one patient remains alive without evidence of
metastatic disease. The median PFS for all 30
patients was 108 days (35–1784 days) (Fig. 4A).
The sites of metastatic disease included the
lung (n = 16), other bone sites (n = 6), subcutis,
lymph node, kidney, liver, spleen, diaphragm,
brain and myocardium. Metastatic disease was
documented in 12 of the 16 (75%) of the Wnt5a-
positive cases and in 10 of the 14 (71%) of the
Wnt5a-negative cases. The median PFS for Wnt5a-
positive samples was 112 days (69–549 days)
and 108 days (35–1784 days) for Wnt5a-negative
samples (Fig. 4B). There was no difference in PFS
of patients with Wnt5a-positive tumours compared
to patients with Wnt5a-negative tumours (P = 0.4).
No other individual factors were identified as being
associated with PFS. A total of five patients were
known to have received additional chemotherapy
following the development of metastatic disease.
Three of the five patients were in the Wnt5a-positive
population and they were treated with c-met study
drug (2) or a combination of toceranib phosphate
and cyclophosphamide (1). The two patients in the
Wnt5a-negative group were treated with the c-met
study drug or doxorubicin.
The median OS time for all 30 dogs was 234 days
(69–1784 days) (Fig. 5A). Two cases were censored;

© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
 Wnt5a expression in canine osteosarcoma 231

A B

1.0

1.0
Proportion progression free
0.8
Wnt5a Neg

0.8
Wnt5a Pos

0.6

0.6
0.4

0.4
0.2
0.2

0.0
0.0

0 500 1000 1500 0 500 1000 1500

Time (days)

Figure 4. (A) The median progression free survival (PFS) for all dogs undergoing amputation and adjuvant chemotherapy
(n = 30) was 108 days. The lower and upper-dashed lines represent 95% lower and upper-confidence levels, respectively. (B)
The median PFS according to Wnt5a status of tumour tissue in dogs treated with at least surgery. There was no difference in
PFS of Wnt5a(+) versus Wnt5(−) patients (112 versus 108 days, respectively; P = 0.4).

A B

1.0
1.0

Wnt5a Neg
0.8
0.8

Wnt5a Pos
Proportion surviving
Proportion surviving

0.6
0.6

0.4
0.4

0.2
0.2

0.0
0.0

0 500 1000 1500 0 500 1000 1500

Time (days) Time (days)

Figure 5. (A) The median overall survival time (OS) for all dogs undergoing amputation and adjuvant chemotherapy
(n = 30) was 234 days. The lower and upper-dashed lines represent 95% lower and upper-confidence levels, respectively. (B)
The median OS in similarly-treated dogs with osteosarcoma according to Wnt5a status of tumour tissue in dogs treated with
at least surgery. Dogs with Wnt5a-positive osteosarcoma samples trended towards shorter OS compared to patients with
Wnt5a-negative samples (212 versus 292 days, P = 0.1).

one case was euthanized for non-osteosarcoma
related reasons and one case remains alive at the
time of writing. The median survival times based on
Wnt5a expression were 212 days (69–549 days) for
patients with Wnt5a-positive tumours and 292 days
(94–1784 days) for patients with Wnt5a-negative
tumours (Fig. 5B). The decreased survival time in
patients with Wnt5a-positive tumours compared to
the patients with Wnt5a-negative tumours did not
reach significance (P = 0.1)
Discussion
Osteosarcoma is a highly aggressive disease with
median OS times plateaued at approximately
8–12 months with standard of care therapy.1 While

© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
232 N. Szigetvari et al.
amputation or limb-sparing procedures provide
the benefit of relieving pain due the primary
tumour, they offer minimal improvement in
slowing metastatic disease for the roughly 80% of
cases considered to have micrometastasis at the time
of diagnosis. The use of adjuvant chemotherapy
is recommended and associated with improved
survival times, however, minimal improvements
have been reported over the past 20 years.1
Alternatives, or adjuvants, to standard cytotoxic
chemotherapy appear necessary, and optimism
has formed around molecular pathway inhibitors,
or targeted therapies.12 – 14 The use of targeted
therapies in osteosarcoma should be based on a
thorough understanding of the signalling pathways
contributing to the development and progression
of the disease. Signalling pathways such as the PI-
3 kinase/mTOR, receptor tyrosine kinase pathways
and the canonical and non-canonical Wnt pathways
are being studied in the context of osteosarcoma for
their potential to serve as therapeutic targets.12 – 16
The non-canonical Wnt signalling pathway
encompasses Wnt signalling independent of ß-
catenin (i.e. canonical signalling) and includes the
Wnt-5a/calcium pathway and Wnt-5a/c-JNK path-
ways. Wnt5a, a non-canonical Wnt ligand, has
been associated with multiple cancer types includ-
ing osteosarcoma.17 – 21 Nakano et al.19 initially
reported on Wnt5a being one of seven genes with
increased expression in a more highly metastatic
subline of the HuO9 osteosarcoma cell line. Using
the human osteosarcoma cell lines, Saos-2 and
U2OS, Enmoto et al. demonstrated Wnt5a was
associated with invasion and migration of these cell
lines.2 Similarly, reduction of Wnt5a expression
was recently associated with reduced tumourige-
nesis in vitro and in vivo of murine and human
osteosarcoma cell lines.21 The precise mechanisms
by which Wnt5a signalling results in enhanced
migration, invasion and subsequent tumour metas-
tasis are yet to be elucidated, though the abil-
ity to enhance invadopodia and increase matrix
metalloproteinase production may contribute to
these characteristics.22,23 These studies highlight
the biological relevance of Wnt5a in osteosarcoma
development and progression. The identification of
Wnt5a expression in canine osteosarcoma cell lines
© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
(D17, Abrams, UWKOS6 and UWKOS8) and tis-
sue lysate (UWKOS6, UWKOS7 and UWKOS8)
suggests canine osteosarcoma will continue to
play an invaluable role in contributing to the
broader knowledge regarding the pathogenesis of
osteosarcoma.13,24 However, our results suggest
further evaluation of the cell autonomous effects of
Wnt5a in canine osteosarcoma would be best stud-
ied in the Abrams cell line, as this cell line appears
to over-express Wnt5a relative to non-neoplastic
canine osteoblasts. In addition, it is interesting to
note the diminished expression of Wnt5a protein in
the canine osteosarcoma cell lines relative to their
paired tumour tissue. This could be the result of
selecting a clonal cell population that does not over-
express Wnt5a. Alternatively, this may indicate the
Wnt5a ligand is being produced in the tumour
micro-environment that would be included in the
tumour tissue lysate but not the cell line lysate. If
Wnt5a ligand is being produced by the surrounding
tumour microenvironment, it may act in be acting
in a non-cell autonomous fashion on osteosarcoma
cells. This has been reported in squamous cell car-
cinoma, where Wnt5a expression is localized to
neoplastic cells and tumour-associated fibroblasts
at the leading edge of the cancer.25 Further studies
are necessary to explain these findings and their
significance.
The clinical implication of Wnt5a in human
osteosarcoma was recently assessed in a study by
Lu et al.24 that found Wnt5a was expressed in
81% of patient tumour samples. This is greater
than the 54% of canine osteosarcomas assessed
here, but is consistent with Wnt5a expression
being a frequent finding in osteosarcoma. The
apparent disparity between Wn5a expression in
human and canine OSA may be due to differences
in IHC interpretation, as the Lu et al. study
considered tumour samples with as few as 6%
of neoplastic cells staining positive for Wnt5a to
be considered a Wnt5a-positive tumour. For our
study, a much more conservative estimation of
positivity was employed, requiring over 90% of
tumour cell positivity, thereby reducing the number
of samples we considered positive for Wnt5a. It is
also possible the decalcification process adversely
effects the ability to detect Wnt5a in the tissue,

thereby reducing the overall percentage of positive-
staining samples. This may explain the disparate
IHC-staining of UWKOS8 relative to the results
of the Western blot analyses in the associated cell
line and tissue. Conversely, it is interesting to note
the IHC and Western blot analysis on the tumour
tissue of UWKOS7 was Wnt5a-postive, whereas
UWKOS7 cell line lysate was Wnt5a-negative. This
finding would be supportive of the theory that
Wnt5a noted within tumours could be produced
in a non-cell autonomous fashion by the tumour
microenvironment in addition to an osteosarcoma-
cell autonomous origin.
The study by Lu et al. found the expression
of Wnt5a was positively correlated with ROR2
expression, a receptor tyrosine kinase capable of
binding Wnt5a ligand and initiating the non-
canonical Wnt signalling pathway. Unfortunately,
we have been unable to successfully identify ROR2
expression in formalin-fixed canine tissue using the
currently available antibodies, so the correlation
between the Wnt5a ligand and ROR2 could not
be addressed in this study. In the Lu et al. study,
expression of Wnt5a was associated with increasing
Enneking surgical stage and tumour metastasis,
suggesting the expression of Wnt5a is associated
with more aggressive disease. In this study, Wnt5a
expression was not associated with PFS in a similarly
treated group of dogs. The use of PFS as a measure
of biological/clinical significance is likely to be a
stronger endpoint, relative to OS, due to the option
of humane euthanasia for the veterinary patient
population. However, the two populations were
relatively similar in regard to patients receiving
additional therapy following the onset of metastatic
disease and there was a trend towards reduced OS in
patients with Wnt5a-positive tumours compared to
patients with Wnt5a-negative tumours (212 versus
292 days, respectively; P = 0.1).
A limitation of this study is the relatively small
number of patients (30) that were treated in a
similar fashion that had histopathology samples
available for IHC. Similarly, while most patients
received a platinum-based chemotherapy protocol
following amputation, this was not true for all
patients and may hold some impact on our
analyses.
© 2013 John Wiley & Sons Ltd, Veterinary and Comparative Oncology, 14, 3, 225–235
Wnt5a expression in canine osteosarcoma 233
In summary, Wnt5a is expressed in canine
osteosarcoma cell lines, tissue lysate and paraffin-
embedded sections of canine osteosarcoma. Fifty-
four percent of the canine osteosarcoma samples
evaluated were positive for Wnt5a expression.
When assessing the association of Wnt5a and
clinical variables there was no association with
PFS, however, there was a trend towards shorter
OS for patients with Wnt5a-positive tumours.
Findings of this study continue to draw parallels
previously recognized of osteosarcoma in people
and dogs.8 – 10,26,27 While our findings provide
additional support for the canine model in the
study of spontaneous osteosarcoma, further studies
are necessary to determine whether the presence
of Wnt5a in canine osteosarcoma is indicative
of an activated non-canonical Wnt signalling
pathway; if activated, does Wnt5a function in
a cell autonomous or non-cell autonomous
fashion; and perhaps most importantly, how
this pathway contributes to the pathogenesis of
osteosarcoma.
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