l

1. In Vivo and In Vitro Efficacy of Zoledronate for Treating Oral
Squamous Cell Carcinoma in Cats
J.M. Wypij, T.M. Fan, R.L. Fredrickson, A.M. Barger, L.P. de Lorimier, and S.C. Charney
Background: Feline oral squamous cell carcinoma (OSCC) may cause painful bone destruction. Given the local invasiveness
and rapid clinical progression of OSCC, conventional therapies are often palliative. In human cancer patients, zoledronate
exerts anticancer effects by inhibiting tumor-induced angiogenesis and malignant osteolysis.
Hypothesis: Zoledronate will exert in vitro and in vivo anti-angiogenic and antiresorptive effects in feline OSCC.
Animals: Eight cats with OSCC were prospectively treated with zoledronate and conventional treatment modalities.
Methods: In vitro, zoledronate’s effects in modulating soluble vascular endothelial growth factor (VEGF) secretion and
receptor activator of nuclear factor KB (NF-kB) ligand (RANKL) expression were investigated in a feline OSCC cell line
(SCCF1). In vivo, basal serum C-telopeptide (CTx) concentrations were compared among normal and OSCC-bearing cats, and
the biologic effects of zoledronate administration in cats with naturally occurring OSCC were quantified by serially assessing
circulating serum VEGF and CTx concentrations.
Results: In vitro, zoledronate concentrations greater than 3 mM reduce soluble VEGF secretion in the SCCF1 cell line. The
expression of RANKL in the SCCF1 cell line was also modulated by zoledronate, with low concentrations (3 mM) decreasing
but higher concentrations (30 mM) increasing RANKL expression in comparison with untreated cells. In vivo, cats with bone-
invasive OSCC had greater serum CTx concentrations in comparison with geriatric, healthy controls. Treatment with zoled-
ronate rapidly decreased circulating serum VEGF and CTx concentrations in cats with spontaneously occurring OSCC.
Conclusions and Clinical Importance: Zoledronate exerts in vitro and in vivo effects that may favor the slowing of tumor
growth and pathologic bone turnover associated with OSCC.
Key words: Aminobisphosphonate; Cancer; Focal malignant osteolysis; Serum C-telopeptide; Soluble vascular endothelial
growth factor.
Oral squamous cell carcinoma (OSCC) accounts for
approximately 75% of malignancies involving the
oral cavity of cats. The tumor is invasive, resulting in
osteolysis and cancer-associated pain.1–3
Given the local
invasiveness of OSCC and the morbidity associated with
radical oral surgery in cats,4,5
curative intent resection is
usually not feasible. Treatment options for inoperable
OSCC remain palliative and include systemic chemother-
apy, coarse-fractionated radiation therapy, or a
combination of each. Responses to systemic chemother-
apy or palliative radiation therapy alone have been
disappointing, although radiation therapy combined
with either radiosensitizing agents or hyperthermia may
be more effective.6–12
The long-term prognosis for cats
with OSCC is poor, and for cats with incurable disease,
novel adjuvant therapies that slow down tumor growth
(anti-angiogenic therapies) or minimize cancer-induced
pain (antiresorptive therapies) warrant additional inves-
tigation.
Angiogenesis is considered a fundamental hallmark of
cancer,13
and is necessary for continued primary tumor
growth and successful distant metastases.14
Principally
regulated by vascular endothelial growth factor
(VEGF),15
angiogenesis is characterized by endothelial
cell proliferation, migration, and lumen formation.16
Given that sustained angiogenesis is a prerequisite for
tumor growth, therapeutic strategies that reduce or block
the effects of tumor-associated VEGF are currently being
investigated for the treatment of various cancers.17
Similar to angiogenesis, tissue invasion is another hall-
mark of malignantly transformed cells.13
Focal osteolysis
is a prerequisite for cancer cells to successful invade min-
eralized bone. Tumor-induced bone resorption is
mediated directly by cell surface ligands or indirectly
through the release of soluble factors that promote
osteoclast activity.18
Cancer cells that directly express
surface receptor activator of nuclear factor KB (NF-kB)
ligand (RANKL) are capable of subverting homeostatic
bone turnover mechanisms to cause pathologic bone re-
sorption.19,20
Because malignant osteolysis dramatically
reduces quality-of-life scores in humans with skeletal
neoplasms, antiresorptive therapies are being investigat-
ed for the management of tumor types that preferentially
metastasize to or invade bone.21
Zoledronate, a potent aminobisphosphonate, exerts
several in vitro antineoplastic effects, including the im-
pairment of neoplastic neovascularization, tumor cell
invasion, and migration.22–24
Zoledronate administra-
tion decreases tumor angiogenesis and inhibits
malignant osteolysis in rodent tumor models,22,25–27
and
treatment decreases the number of skeletal-related
events, improves pain scores, decreases serum markers
of bone lysis, and decreases serum VEGF concentrations
From the Comparative Oncology Research Laboratory, Depart-
ment of Veterinary Clinical Medicine (Wypij, Fan, de Lorimier,
Charney) and the Department of Pathology, University of Illinois at
Urbana-Champaign, IL 61802 (Fredrickson, Barger). Findings of
this study were presented in part at the 24th Annual Veterinary Can-
cer Society Conference, Kansas City, MO, 2004; the 25th Annual
Veterinary Cancer Society Conference, Huntington Beach, CA, 2005;
and the 26th Annual Veterinary Cancer Society Conference, Pine
Mountain, GA, 2006.
This study was conducted at the Comparative Oncology Research
Laboratory.
Corresponding author:Timothy M. Fan, DVM, PhD, Department
of Veterinary Clinical Medicine, University of Illinois at Urbana-
Champaign, 1008 West Hazelwood Drive, Urbana, IL 61802; e-mail:
t-fan@uiuc.edu.
Submitted February 24, 2007; Revised May 7, July 7, 2007;
Accepted August 9, 2007.
Copyright r 2008 by the American College of Veterinary Internal
Medicine
10.1111/j.1939-1676.2007.0010.x
J Vet Intern Med 2008;22:158–163

2. in people with metastatic bone tumors.28–31
Zoledronate
is first-line treatment for people with cancers associated
with neovascularization and malignant bone destruction.
Being a standard adjunctive treatment for malignant
osteolytic diseases in human cancer patients, zoledronate
might provide therapeutic benefit for managing cats with
bone-invasive OSCC. Therefore, the first purpose of this
study was to investigate the in vitro effects of zoledronate
on VEGF secretion and surface RANKL expression in
an immortalized feline OSCC cell line (SCCF1). The sec-
ond objective of this study was to characterize serum
CTx concentrations, a bone resorption marker, in
healthy, geriatric cats and in cats with bone-invasive
OSCC. The last purpose of this study was to determine
whether zoledronate administration in cats with bone-
invasive OSCC exerted any potential anticancer effects,
assessed by changes in circulating serum VEGF and
serum CTx concentrations.
Materials and Methods
Cell Lines
A feline OSCC cell line SCCF1 (provided by Dr Thomas J.
Rosol, Ohio State University) was evaluated for soluble VEGF se-
cretion and RANKL expression. The SCCF1 cell line was grown in
Williams E mediaa
supplemented with 2 mM L-glutamine,b
0.05 mg/
mL gentamicin,c
10 ng/mL epidermal growth factor,d
0.01 nM chol-
era toxin,e
and 10% fetal bovine serum (FBS). Cell cultures were
maintained in subconfluent monolayers at 37 1C in 5% CO2 and
passaged twice weekly.
Reagents and Antibodies
Zoledronic phosphonic acid monohydratef
was obtained from
Novartis Pharmaceuticals Ltd. Stock solutions (1 mg/mL) were pre-
pared in sterile phosphate-buffered saline (PBS), aliquoted, and
frozen at 20 1C until use. The rabbit polyclonal anti human
RANKL antibodyg
used for flow cytometry has previously been
demonstrated to cross-react with canine and feline neoplastic cells.32
A corresponding rabbit immunoglobulin G (IgG1)h
was used as an
isotype control for flow cytometric analysis. The secondary anti-
body used for flow cytometry was a goat anti rabbit IgG:FITC
conjugate.i
Soluble VEGF Secretion in the SCCF1 Cell Line
SCCF1 cells were plated at a density of 2 104
cells per 250 mL of
complete medium in a 96-well microtiter plate and incubated at
37 1C and 5% CO2. After allowing cells to adhere for 24 hours, the
medium was decanted and replaced with fresh medium containing
various concentrations of zoledronate (0, 1, 3, 10, and 30 mM), and
cells were allowed to grow for an additional 48 hours. Cell culture
supernatants (in quadruplicate) were harvested and soluble VEGF
was determined with a commercially available immunoassayj
previ-
ously demonstrated to be cross-reactive with feline VEGF.33
Differences in soluble VEGF secreted by SCCF1 after zoledronate
exposure were normalized, based on differences in cell proliferation
through the use of a nonradioactive colorimetric proliferation
assayk
in which optical density linearly correlates with viable cell
numbers. Specifically, normalized VEGF concentrations were based
on the average of quadruplicate samples for each experimental
group expressed as the following ratio:
Normalized VEGF ¼ ½Calculated VEGFðpg=mLÞ=optical density:
RANKL Expression in the SCCF1 Cell Line
SCCF1 cells were plated at a density of 5105
cells per T25 tissue
culture flask in complete medium and incubated at 37 1C and 5%
CO2. After allowing the cells to adhere for 24 hours, medium was
decanted and replaced with fresh medium containing various con-
centrations of zoledronate (0, 3, and 30 mM), and cells were allowed
to grow for an additional 48 hours. Adherent SCCF1 cells were col-
lected and washed after trypsinization, and relative RANKL
protein expression by SCCF1 cells was determined by flow cytome-
try by a technique described previously.32
Samples were analyzed
with a Coulter flow cytometer (Beckman Coulter, Fullerton, CA),
and cells were gated based on their forward and side scatter prop-
erties and FITC fluorescence. Relative RANKL protein expressions
were reported as mean fluorescent intensity (MFI).
Basal Serum C-Telopeptide (CTx) Determinations in
Healthy, Geriatric, and OSCC-Bearing Cats
Venous blood samples were collected via jugular venipuncture
from 10 healthy, geriatric cats and 8 cats with histologically con-
firmed, bone-invasive OSCC for the assessment of basal serum CTx
concentrations. The 10 cats used as healthy, geriatric controls were
owned by house officers, and were considered to be in good health
based on history, physical examination, and serum biochemistry
profile. Whole blood samples were centrifuged for 10 minutes at
450 g, and serum was separated and stored at 20 1C in 2 -mL
polypropylene cryovials until analysis. Serum CTx concentrations
were measured by a commercially available immunoassay,l
previ-
ously validated for use in the cat.34
Zoledronate Treatment Study Population
All cats had a diagnosis of OSCC confirmed by histopathology
and palpable, radiographic, or computed tomographic evidence of
bone involvement. All pet owners were informed of available treat-
ment options, and cats were treated in accordance with the animal
care guidelines of the University of Illinois Institutional Animal
Care and Use Committee. All cats were considered eligible to re-
ceive zoledronate by intravenous infusion regardless of prior
treatment or concurrent disease status. As such, the extent of clin-
ical staging was variable. All cats had serum biochemistry profiles
before and after receiving zoledronate. For cats receiving more than
a single dose, all had complete physical examinations and serum
biochemistry profiles before each successive zoledronate treatment
cycle. Zoledronate was administered at 0.2 mg/kg diluted into
25 mL of 0.9% saline, and administered as a 15-minute constant
rate intravenous infusion every 28 days, a regimen derived and
modified from a previous study conducted in healthy dogs.35
In or-
der to assess the immediate biologic effect of single-agent
zoledronate, no other therapies were instituted for the 24-hour pe-
riod after the first zoledronate administration.
Serum VEGF and CTx in Zoledronate-Treated
OSCC-Bearing Cats
Venous blood samples were collected via jugular venipuncture
for the assessment of serum soluble VEGF and CTx concentrations.
To ensure that changes in serum VEGF and CTx concentrations
were indeed an effect of zoledronate and not other conventional
therapies, tumor-bearing cats were treated only with zoledronate on
Day 1, and, if applicable, additional conventional therapies, includ-
ing radiation therapy, chemotherapy, or NSAIDs, were instituted
on Day 2 after collection of serum samples. Whole blood samples
were centrifuged for 10 minutes at 450 g, and serum was separated
and stored at 20 1C in 2-mL polypropylene cryovials until analysis.
Serum VEGF and CTx concentrations were measured by
159
Zoledronate and OSCC

3. commercially available immunoassays,j,l
respectively, both of which
have been previously validated for use in the cat.33,34
Statistical Analysis
To assess the dose-dependent, biologic activity of zoledronate in
the SCCF1 cell line, reductions in soluble VEGF secretion and
RANKL MFI in comparison with untreated cells were evaluated
with a repeated measure ANOVA, and post hoc comparisons were
made with a Tukey-Kramer multiple comparisons test. Differences
in basal serum CTx concentrations between healthy, geriatric, and
OSCC-bearing cats were analyzed by means of a Wilcoxon rank-
sum test. The immediate (o24 hours) effects of zoledronate admin-
istration in OSCC-bearing cats on serum VEGF and CTx
concentrations were analyzed by means of a Student’s t-test and a
Wilcoxon signed-rank test, respectively. Normal distributed data
sets were expressed as mean standard deviation, and nonnormal
distributed data sets were expressed as median and range. All sta-
tistical analysis was performed by commercial computer software.m
Significance was defined as P o.05.
Results
In Vitro Studies
The basal secretion of soluble VEGF by untreated
SCCF1 cells was 632 108 pg/mL after normalization
for differences in cell densities caused by zoledronate ex-
posure. When incubated with zoledronate concentrations
of 3, 10, and 30 mM, SCCF1 secretion of soluble VEGF
concentrations was significantly reduced in comparison
with untreated cells to 449 80, 312 74, and 342
35 pg/mL, respectively (P o.05 for all comparisons). In
addition, zoledronate also influenced the expression of
surface RANKL. Untreated SCCF1 cells expressed
RANKL with a mean fluorescent intensity (MFI) of 24.1
1.7 units. Zoledronate at a concentration of 3 mM qual-
itatively decreased RANKL expression in comparison to
baseline (19.6 1.1 units, P 4.05), whereas zoledronate
at 30 mM significantly increased RANKL expression
above baseline (35.3 1.0 units, P o.01) (Fig 1).
In Vivo Studies
The median basal serum CTx concentration in cats
with bone-invasive OSCC was 601 pg/mL (range 298–
2,260), which was significantly higher than in healthy,
geriatric cats, 336 pg/mL (range 231–642), P 5 .02.
Eight cats with naturally occurring OSCC with con-
firmed bone involvement were treated with zoledronate
(Table 1). The median number of zoledronate treatments
administered to each cat was 1 (range 1–4). For cats re-
ceiving more than a single dose, the median
intertreatment interval was 28 days (range 21–30). In all
8 cats treated with zoledronate, the basal serum VEGF
and CTx concentrations were significantly reduced 24
hours after zoledronate administration. Serum VEGF
concentrations were 124 46.8 pg/mL before treatment
and 74.7 28.6 pg/mL after zoledronate treatment
(P 5 .007) (Fig 2). The average reduction in serum
VEGF concentrations 24 hours after zoledronate treat-
ment was 49.1 37.2 pg/mL. Serum CTx concentrations
were 600.9 pg/mL (range 298–2,260) before and 404 pg/
mL (range 153–1,980) after zoledronate administration
(P 5 .008) (Fig 3). The median reduction in serum CTx
concentrations 24 hours after zoledronate treatment was
170 pg/mL (range 61.3–472).
Discussion
In the current study, we demonstrate that when
SCCF1 cells were exposed to various concentrations of
zoledronate (3–30 mM), the basal secretion of soluble
VEGF was reduced by 30–50%. This finding supports
the anti-angiogenic potential of zoledronate for slowing
the growth of naturally occurring OSCC in cats. Zoled-
ronate’s capacity to attenuate SCCF1’s soluble VEGF
secretion observed in this study is consistent with a pre-
vious report showing a novel nonaminobisphosphonate
Fig 1. Flow cytometric analysis of SCCF1 receptor activator of
nuclear factor KB (NF KB) ligand (RANKL) protein expression.
Modulatory effect of zoledronate on RANKL protein expression in
the SCCF1 cell line. Negative control, isotype staining (thin, dotted
line), basal RANKL expression (thin solid line), zoledronate 3 mM
effect on RANKL expression (cross-hatch), and zoledronate 30 mM
effect on RANKL expression (thick solid line).
Table 1. Study population characteristics: OSCC and healthy,
geriatric cats.
OSCC
(n 5 8)
Healthy, geriatric
(n 5 10)
Age (years)
Median 15 12
Range 8–18 7–15
Weight (kg)
Median 3.3 5.0
Range 2.2–8.8 3.6–7.3
Sex
Female spayed 5 2
Male neutered 3 8
Breed
Domestic shorthair 3 9
Domestic longhair 4 1
Mixed 1 0
Tumor location
Maxilla 3
Mandible 3
Intermandible 1
Lingual base 1
OSCC, oral squamous cell carcinoma.
160 Wypij et al

4. to suppress soluble VEGF secretion from a human
squamous carcinoma cell line.36
The in vitro molecular
mechanism for reduced soluble VEGF secretion after
zoledronate exposure is currently undetermined, but it is
possible that cellular sequestration or isoform shifts may
account for the reduction in soluble VEGF secretion ob-
served in this investigation.
Although the principal antiresorptive mechanism ex-
erted by zoledronate is the induction of osteoclast
apoptosis through inhibition of the mevalonate pathway,
the ability to down-regulate tumor-associated RANKL
expression would also favor bone protection. In this
study, zoledronate at a concentration of 3 mM qualita-
tively reduced the MFI of RANKL in SCCF1, indicating
decreased RANKL protein expression in comparison to
baseline, which would theoretically decrease bone re-
sorption. However, zoledronate at a concentration of
30 mM resulted in an apparent rebound effect, with an
actual increase in MFI of RANKL, which could possibly
enhance bone resorption. These in vitro findings suggest
that a narrow therapeutic window exists for zoledronate
to regulate SCCF1 cell RANKL expression that favors
bone protection. Although it was unexpected that higher
concentrations of zoledronate (30 mM) would induce a
rebound effect for RANKL expression based on flow
cytometric analysis, zoledronate’s potent and direct
osteoclast apoptotic effects are likely to mitigate any en-
hanced bone resorptive consequences associated with
increased tumor cell RANKL expression.
In cats with OSCC, a subpopulation has cancer pain as
a consequence of focal malignant osteolysis. Cancer-
induced bone resorption may increase circulating con-
centrations of collagen type I breakdown products,
which can be assessed in urine and blood, and have
proved to be valuable in monitoring response to anti-
resorptive therapies for human cancer patients.37
Few
studies in companion animals have investigated bone re-
sorption markers and cancer; however, there are
increases in urine N-telopeptide in dogs with append-
icular osteosarcoma.38
Cats in the current study with
histologically confirmed, bone-invasive OSCC had sig-
nificantly higher serum concentrations of CTx than
healthy, geriatric, control cats. One possible explanation
for the increased serum CTx concentration in cats with
OSCC could be the ongoing focal bone destruction with-
in the oral cavity, as has been previously demonstrated in
a small subset of cats that bone-invasive OSCC express
RANKL, a principal mediator for osteoclastogenesis.32
In human patients suffering from skeletal metastases
of breast carcinoma, zoledronate treatment decreases cir-
culating serum VEGF and bone turnover marker
concentrations, changes that correlate with improved
performance status. In the current study, zoledronate
was administered to cats with bone-invasive OSCC, and
attempts were made to verify whether the dose used ex-
erted biologic activity as determined by reductions in
serum VEGF and CTx concentrations within 24 hours
after zoledronate administration. For all cats treated (n
5 8), significant decreases in both serum VEGF and CTx
concentrations were identified after the first dose of
zoledronate, supporting the notion that zoledronate ad-
ministered at a dosage of 0.2 mg/kg IV exerts biologic
activity in OSCC-bearing cats. Interestingly, the magni-
tude of reduction for either serum VEGF or CTx after
zoledronate treatment varied in the 8 cats evaluated, and
could possibly reflect individual differences in biologic
and therapeutic responsiveness to zoledronate treatment.
The reduction in serum CTx concentrations after
zoledronate administration observed in this study was
expected, and most likely attributable to the potent anti-
resorptive effects of zoledronate on both homeostatic
and pathologic bone turnover. Unlike the straightfor-
ward explanation for reduced serum CTx con-
centrations, the potential mechanisms for reduced serum
VEGF concentrations after zoledronate administra-
tion are theoretical and multiple, and could include a
Fig 3. In vivo effects of zoledronate on serum C-telopeptide (CTx)
concentrations. Changes in serum CTx concentrations in cats with
bone-invasive oral squamous cell carcinoma (n 5 8) immediately
before (preZOL) and within 24 hours after (postZOL) treatment
with zoledronate (0.2 mg/kg) administered intravenously. Reduc-
tions in serum CTx concentrations after zoledronate treatment were
statistically significant, P o.05.
Fig 2. In vivo effects of zoledronate on serum vascular endothelial
growth factor (VEGF) concentrations. Changes in serum VEGF
concentrations in cats with bone-invasive oral squamous cell carci-
noma (n 5 8) immediately before (preZOL) and within 24 hours
after (postZOL) treatment with zoledronate (0.2 mg/kg) adminis-
tered intravenously. Reductions in serum VEGF concentrations
after zoledronate treatment were statistically significant, P o.05.
161
Zoledronate and OSCC

5. combination of the following: (1) reduced malignant
osteolysis with subsequent diminished release of bone-
derived TGF-b, a potent promoter of the VEGF gene; (2)
direct attenuation of soluble VEGF release by OSCC
cells as similarly demonstrated in vitro with the SCCF1
cell line; and (3) direct cytotoxicity to OSCC cells, there-
by decreasing the absolute number of tumor cells capable
of releasing soluble VEGF.
Although the findings of this investigation are novel
and important, several limitations should be addressed.
First, the beneficial in vitro effects of zoledronate in re-
ducing soluble VEGF secretion and modulating
RANKL protein expression in the SCCF1 cell line might
not be applicable to natural disease states, because it is
unknown what concentrations of zoledronate are
achieved within the osteolytic tumor micro environment
associated with naturally occurring OSCC. Second, the
maximal attenuation of soluble VEGF by zoledronate
both in vitro (50%) and in vivo (45%) was incom-
plete, and given the large family of angiogenic peptides
that exert redundant activities, partial reductions in only
VEGF might not translate into a meaningful decrease in
cancer cell-induced angiogenesis. Similarly, anti-an-
giogenic effects of novel therapies could require
significant time before measurable responses are ob-
served,39
and given the rapid invasiveness and clinical
morbidity associated with OSCC in cats, any beneficial
anti-angiogenic effects exerted by zoledronate might be
too delayed to alter the natural course of disease. As
such, the evaluation of zoledronate in an inducible xeno-
graft murine tumor model would have provided more
information in determining the biologic relevance of
VEGF attenuation and RANKL modulation in the
SCCF1 cell line. Third, although basal serum CTx con-
centrations were significantly higher in cats with bone-
invasive OSCC when compared with healthy, geriatric
cats, it was not possible to determine whether increased
CTx concentrations could be solely attributed to focal
malignant osteolysis caused by local disease progression
in the oral cavity. Other possibilities that may have ac-
counted for the difference in basal CTx concentrations
between healthy, geriatric, and bone-invasive OSCC-
bearing cats could have been occult endocrine or meta-
bolic disease states associated with increases in global
skeletal resorption, such as hyperparathyroidism, hype-
radrenocorticism, chronic renal insufficiency, and
idiopathic hypercalcemia. Fourth, the number of cats
with bone-invasive OSCC treated with zoledronate was
very small (n 5 8); therefore, strong conclusions regard-
ing the clinical effectiveness of zoledronate cannot be
stated in this study. However, it was not a study objective
to determine whether zoledronate could exert measur-
able clinical effects on naturally occurring OSCC, but
rather the intent was to assess whether single-agent
zoledronate demonstrated theoretical anticancer activi-
ties (anti-angiogenic and antiresorptive), as was
supported by significant reductions in both serum VEGF
and CTx concentrations. Although both serum VEGF
and CTx concentrations were reduced within 24 hours
after zoledronate infusion, we did not evaluate the dy-
namic changes in either serum VEGF or CTx
concentrations as a function of time, and therefore the
maximal duration and magnitude of suppression of these
two surrogate markers could not be determined in this
study. Last, zoledronate has been incriminated in the
rare development of jaw osteonecrosis in human cancer
patients,40
which could mean that its institution in cats
with preexisting mandibular or maxillary bone lesions
might be contraindicated. However, it should be stated
that the exact etiology for bisphosphonate-induced
osteonecrosis remains to be elucidated, but appears to
preferentially develop in human patients treated with
long-term (436 months) antiresorptive therapies. Given
the poor prognosis of bone-invasive OSCC in cats, it is
unlikely that many cats would survive long enough to be
treated with chronic aminobisphosphonate treatment;
thus the potential for developing jaw osteonecrosis
would appear remote.
Despite these limitations, this report provides new in-
formation regarding the bone resorptive characteristics
of naturally occurring OSCC, and is the first description
of serum CTx concentrations in companion animals with
bone-invasive neoplasms. Furthermore, findings from
this study provide in vitro and in vivo evidence to sup-
port future clinical investigations for evaluating
zoledronate in cats diagnosed with bone-invasive OSCC.
Additional prospective studies will be required to define
the clinical effectiveness and long-term tolerability of
zoledronate in both dogs and cats suffering from skeletal
malignancies, and it is hoped that the findings of this
study will provide a conceptual platform for exploring
the use of surrogate markers of bone resorption and ami-
nobisphosphonate treatment for monitoring and treating
painful neoplastic osteolytic processes in companion an-
imals, respectively.
Footnotes
a
Biosource, Rockville, MD
b
Sigma-Aldrich, St Louis, MO
c
Sigma, St Louis, MO
d
Pepro Tech, Rocky Hill, NJ
e
Calbiochem, La Jolla, CA
f
Zoledronate, Basel, Switzerland
g
Axxora Platform, San Diego, CA
h
SCB, Santa Cruz, CA
i
Serotec, Raleigh, NC
j
Quantikine, RD Systems, Minneapolis, MN
k
CellTiter96, Promega, Madison, WI
l
Serum Crosslaps, Nordic Biosciences, Herlev, Denmark
m
GraphPad, Instat3, San Diego, CA
Acknowledgments
The authors would like to thank Jane Chladny and
Lisa Shipp of the Veterinary Diagnostic Laboratory and
Ian Sprandel of the Comparative Oncology Research
Laboratory for their technical assistance.
162 Wypij et al

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