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Keywords:

  • Aminobisphosphonate;
  • Cancer;
  • Focal malignant osteolysis;
  • Serum C-telopeptide;
  • Soluble vascular endothelial growth factor

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

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-κB) 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 μM 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 μM) decreasing but higher concentrations (30 μM) 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 zoledronate 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.

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 chemotherapy, coarse-fractionated radiation therapy, or a combination of each. Responses to systemic chemotherapy 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 investigation.

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 hallmark of malignantly transformed cells.13 Focal osteolysis is a prerequisite for cancer cells to successful invade mineralized 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-κB) ligand (RANKL) are capable of subverting homeostatic bone turnover mechanisms to cause pathologic bone resorption.19,20 Because malignant osteolysis dramatically reduces quality-of-life scores in humans with skeletal neoplasms, antiresorptive therapies are being investigated 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 impairment of neoplastic neovascularization, tumor cell invasion, and migration.22–24 Zoledronate administration 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 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 second 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

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

Cell Lines

A feline OSCC cell line SCCF1 (provided by Dr Thomas J. Rosol, Ohio State University) was evaluated for soluble VEGF secretion 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 cholera toxin,e and 10% fetal bovine serum (FBS). Cell cultures were maintained in subconfluent monolayers at 37 °C 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 prepared in sterile phosphate-buffered saline (PBS), aliquoted, and frozen at −20 °C 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 antibody 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 μL of complete medium in a 96-well microtiter plate and incubated at 37 °C 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 μM), 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 previously 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:

  • image

RANKL Expression in the SCCF1 Cell Line

SCCF1 cells were plated at a density of 5 × 105 cells per T25 tissue culture flask in complete medium and incubated at 37 °C and 5% CO2. After allowing the cells to adhere for 24 hours, medium was decanted and replaced with fresh medium containing various concentrations of zoledronate (0, 3, and 30 μM), and cells were allowed to grow for an additional 48 hours. Adherent SCCF1 cells were collected and washed after trypsinization, and relative RANKL protein expression by SCCF1 cells was determined by flow cytometry 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 properties 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 confirmed, 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 °C in 2 -mL polypropylene cryovials until analysis. Serum CTx concentrations were measured by a commercially available immunoassay,l previously 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 treatment 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 receive zoledronate by intravenous infusion regardless of prior treatment or concurrent disease status. As such, the extent of clinical 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 order to assess the immediate biologic effect of single-agent zoledronate, no other therapies were instituted for the 24-hour period 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, including 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 °C in 2-mL polypropylene cryovials until analysis. Serum VEGF and CTx concentrations were measured by 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 (<24 hours) effects of zoledronate administration 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 statistical analysis was performed by commercial computer software.m Significance was defined as P <.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

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 exposure. When incubated with zoledronate concentrations of 3, 10, and 30 μM, 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 <.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 μM qualitatively decreased RANKL expression in comparison to baseline (19.6 ± 1.1 units, P >.05), whereas zoledronate at 30 μM significantly increased RANKL expression above baseline (35.3 ± 1.0 units, P <.01) (Fig 1).

image

Figure 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 μM effect on RANKL expression (cross-hatch), and zoledronate 30 μM effect on RANKL expression (thick solid line).

Download figure to PowerPoint

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 =.02. Eight cats with naturally occurring OSCC with confirmed 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 receiving 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 =.007) (Fig 2). The average reduction in serum VEGF concentrations 24 hours after zoledronate treatment 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 =.008) (Fig 3). The median reduction in serum CTx concentrations 24 hours after zoledronate treatment was 170 pg/mL (range 61.3–472).

Table 1.   Study population characteristics: OSCC and healthy, geriatric cats.
 OSCC (n = 8)Healthy, geriatric (n = 10)
  1. OSCC, oral squamous cell carcinoma.

Age (years)
 Median1512
 Range8–187–15
Weight (kg)
 Median3.35.0
 Range2.2–8.83.6–7.3
Sex
 Female spayed52
 Male neutered38
Breed
 Domestic shorthair39
 Domestic longhair41
 Mixed10
Tumor location
 Maxilla3 
 Mandible3 
 Intermandible1 
 Lingual base1 
image

Figure 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 carcinoma (n=8) immediately before (preZOL) and within 24 hours after (postZOL) treatment with zoledronate (0.2 mg/kg) administered intravenously. Reductions in serum VEGF concentrations after zoledronate treatment were statistically significant, P <.05.

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image

Figure 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=8) immediately before (preZOL) and within 24 hours after (postZOL) treatment with zoledronate (0.2 mg/kg) administered intravenously. Reductions in serum CTx concentrations after zoledronate treatment were statistically significant, P <.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

In the current study, we demonstrate that when SCCF1 cells were exposed to various concentrations of zoledronate (3–30 μM), 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. Zoledronate's capacity to attenuate SCCF1's soluble VEGF secretion observed in this study is consistent with a previous report showing a novel nonaminobisphosphonate 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 observed in this investigation.

Although the principal antiresorptive mechanism exerted 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 μM qualitatively reduced the MFI of RANKL in SCCF1, indicating decreased RANKL protein expression in comparison to baseline, which would theoretically decrease bone resorption. However, zoledronate at a concentration of 30 μM 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 μM) 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 enhanced 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 concentrations of collagen type I breakdown products, which can be assessed in urine and blood, and have proved to be valuable in monitoring response to antiresorptive therapies for human cancer patients.37 Few studies in companion animals have investigated bone resorption markers and cancer; however, there are increases in urine N-telopeptide in dogs with appendicular osteosarcoma.38 Cats in the current study with histologically confirmed, bone-invasive OSCC had significantly 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 within 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 circulating 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 exerted biologic activity as determined by reductions in serum VEGF and CTx concentrations within 24 hours after zoledronate administration. For all cats treated (n = 8), significant decreases in both serum VEGF and CTx concentrations were identified after the first dose of zoledronate, supporting the notion that zoledronate administered at a dosage of 0.2 mg/kg IV exerts biologic activity in OSCC-bearing cats. Interestingly, the magnitude 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 antiresorptive effects of zoledronate on both homeostatic and pathologic bone turnover. Unlike the straightforward explanation for reduced serum CTx concentrations, the potential mechanisms for reduced serum VEGF concentrations after zoledronate administration are theoretical and multiple, and could include a combination of the following: (1) reduced malignant osteolysis with subsequent diminished release of bone-derived TGF-β, 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, thereby 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 reducing 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 incomplete, 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-angiogenic effects of novel therapies could require significant time before measurable responses are observed,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 xenograft 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 concentrations 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 accounted for the difference in basal CTx concentrations between healthy, geriatric, and bone-invasive OSCC-bearing cats could have been occult endocrine or metabolic disease states associated with increases in global skeletal resorption, such as hyperparathyroidism, hyperadrenocorticism, chronic renal insufficiency, and idiopathic hypercalcemia. Fourth, the number of cats with bone-invasive OSCC treated with zoledronate was very small (n = 8); therefore, strong conclusions regarding the clinical effectiveness of zoledronate cannot be stated in this study. However, it was not a study objective to determine whether zoledronate could exert measurable clinical effects on naturally occurring OSCC, but rather the intent was to assess whether single-agent zoledronate demonstrated theoretical anticancer activities (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 dynamic 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 (>36 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 information 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 support 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 aminobisphosphonate treatment for monitoring and treating painful neoplastic osteolytic processes in companion animals, respectively.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

aBiosource, Rockville, MD

bSigma-Aldrich, St Louis, MO

cSigma, St Louis, MO

dPepro Tech, Rocky Hill, NJ

eCalbiochem, La Jolla, CA

fZoledronate, Basel, Switzerland

gAxxora Platform, San Diego, CA

hSCB, Santa Cruz, CA

iSerotec, Raleigh, NC

jQuantikine, R&D Systems, Minneapolis, MN

kCellTiter96, Promega, Madison, WI

lSerum Crosslaps, Nordic Biosciences, Herlev, Denmark

mGraphPad, Instat3, San Diego, CA

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References

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.

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  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. Acknowledgments
  8. References
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