Investigating TrkA Expression in Canine Appendicular Osteosarcoma

Fan, T.M., Barger, A.M., Sprandel, I.T. and Fredrickson, R.L. (2008), Investigating TrkA Expression in Canine Appendicular Osteosarcoma. Journal of Veterinary Internal Medicine, 22: 1181–1188.

                                                                                                             doi: 10.1111/j.1939-1676.2008.0151.x


Background: The tropomyosin-related kinase A (TrkA) proto-oncogene encodes for a receptor that binds with high affinity to the neurotrophin ligand, nerve growth factor (NGF). Intracellular signaling mediated by the TrkA/NGF axis orchestrates neuronal cell differentiation, mitogenesis, and survival. Interestingly, TrkA also is expressed by bone forming cells, and its signaling promotes antiapoptotic effects in actively dividing osteoblasts.

Hypothesis: In canine immortalized cell lines and naturally occurring tumor samples, osteosarcoma (OSA) cells will express TrkA. In canine OSA cell lines, TrkA signaling will promote cell mitogenesis and survival.

Methods: In vitro, TrkA expression in canine OSA cell lines was assessed by reverse transcriptase-polymerase chain reaction, flow cytometry, and immunocytochemistry. In vitro, the involvement of TrkA-mediated signaling for cell mitogenesis and survival were investigated with commercially available assays. In vivo, TrkA expression was evaluated in primary tumors and pulmonary metastases with immunocytochemistry and immunohistochemistry, respectively.

Results: In vitro, canine OSA cells expressed TrkA mRNA and protein. Ligation of TrkA with exogenous NGF did not induce mitogenesis. Blockade of TrkA signaling with either a protein kinase inhibitor or NGF-neutralizing antibody induced apoptosis of canine OSA cell lines. In vivo, the majority (10/15) of canine OSA primary tumors and pulmonary metastases (9/12) expressed TrkA protein.

Conclusions and Clinical Importance: Canine OSA cells express TrkA, and its signaling protects against apoptosis. Most dogs with spontaneously arising OSA express TrkA within their primary tumors and pulmonary metastatic lesions, warranting further investigations with TrkA antagonists as a novel treatment option for canine OSA.

The tropomyosin-related kinase (Trk) proto-oncogenes encode for high-affinity receptor tyrosine kinases, which include TrkA, TrkB, and TrkC.1,2 The cognate ligands for Trk receptors belong to a family of neurotrophin growth factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5).1 High-affinity binding of Trk receptors with neurotrophins is preferential, with TrkA being the preferred receptor for NGF, TrkB for BDNF, and TrkC for NT-3.3 Signaling through Trk receptors is necessary for neuronal cell differentiation, proliferation, and survival, and thereby regulates neuronal development and maintenance of neural networks.4 In addition to their involvement in the nervous system, Trk receptors and neurotrophins appear to participate in homeostatic and reparative bone morphogenesis5,6 as well as play a role in pathologic processes, including tumorigenesis.3,7,8

With respect to bone biology, the transcription of Trk receptors and neurotrophin growth factors has been demonstrated during the exponential growth phase of an immortalized murine osteoblast cell line, MC3T3-E1.9,10 Additionally, TrkA signaling mediated by exogenous NGF induces MC3T3-E1 cells to actively secrete interleukin-6, a proinflammatory cytokine that promotes osteoclastogenesis and bone resorption.11 In murine and racine models of skeletal fracture repair, the expression of Trk receptors and their cognate neurotrophins has been demonstrated in bone-forming cells, including osteoblasts within the healing bone callus,5,6 and provides in vivo evidence for the participation of Trk signaling in reparative bone biology. Collectively, these in vitro and in vivo studies support the role of Trk receptor signaling in osteoblast proliferation and differentiation.

In addition to cellular mitogenesis, TrkA signaling exerts antiapoptotic effects in murine and human immortalized bone-forming cells. In the MC3T3-E1 murine osteoblast cell line, activation of TrkA with NGF reduces apoptotic DNA breakdown in osteoblasts secondary to serum starvation or cytotoxic treatments.12 Mechanistically, the prosurvival effects mediated by NGF in murine osteoblasts may be a result of downregulation of bax, a proapoptotic protein.12 In the human osteoblast cell line hFOB, the antiapoptotic effects of TrkA signaling appear to be cell-cycle dependent, because TrkA signal blockade induces apoptosis of only proliferating, not quiescent human osteoblasts.13 Based on these studies, NGF-mediated signaling through the TrkA receptor appears to protect proliferating osteoblasts cells from programmed cell death.

In mesenchymal neoplasms, TrkA expression and NGF-mediated signaling have been investigated in human sarcoma cell lines.14–16 Similar to osteoblast biology, TrkA signaling participates in cellular mitogenesis and survival, because blockade of TrkA signaling results in decreased cell proliferation and increased apoptosis of human rhabdomyosarcoma and leiomyosarcoma cell lines.14–16 Based on these in vitro studies, NGF-mediated signaling through the TrkA receptor definitively supports cellular mitogenesis and protects against apoptosis in malignantly transformed mesenchymal cells.

Canine appendicular osteosarcoma (OSA) is a highly aggressive tumor of mesenchymal origin comprising a rapidly proliferating population of malignant osteoblasts. Most affected dogs will succumb either to the locally destructive nature of their primary bone tumor or to distant metastases involving the pulmonary parenchyma.17,18 Although amputation of the affected limb and adjuvant systemic chemotherapy increases disease-free intervals and overall survival times in dogs diagnosed with OSA, the identification of novel therapeutic targets warrants investigation. Given the documented mitogenic and antiapoptotic activities of TrkA signaling in murine and human immortalized osteoblast cell lines, as well as similar biologic functions of NGF-mediated signaling in malignantly transformed mesenchymal cells, the TrkA signaling pathway may contribute to the proliferation and survival of canine OSA cells in local and metastatic tumor microenvironments. As such, the purposes of this study were to (1) assess TrkA mRNA and protein expression in immortalized canine OSA cell lines, (2) investigate the in vitro mitogenic and antiapoptotic effects of TrkA signaling in canine OSA cell lines, and (3) determine if spontaneous canine OSA samples derived from either primary tumors or pulmonary metastatic lesions express TrkA receptor.

Materials and Methods

Cell Lines

Two canine OSA lines were investigated for TrkA expression and biologic function. The COS31 canine OSA cell line was provided by Ahmed Shoieb, University of Tennessee.19 The Buck canine OSA cell line, which expresses osteoblastic lineage markers, including receptor activator of nuclear factor κ-B ligand (RANKL) and alkaline phosphatase (unpublished data), was derived from a histologically confirmed primary appendicular osteosarcoma and created in the Comparative Oncology Research Laboratory at the University of Illinois. The PC-12 rat pheochromocytoma cell line, reported to express functional TrkA,20 was purchased commercially.a Before the designated experiments, both canine OSA cell lines were cultured in complete mediab supplemented with 2 mM l-glutaminec and 10% fetal calf serum (FCS). Cell cultures were maintained in subconfluent monolayers at 37°C in 5% CO2, and passaged twice weekly.

Primary and Secondary Antibodies

A rabbit polyclonal anti-human TrkA antibodyd was evaluated for cross-reactivity with canine cells. A corresponding rabbit IgG1e was used as an isotype control for flow cytometric analysis. Secondary antibodies used for flow cytometry and immunocytochemistry were a goat anti-rabbit IgG : FITC conjugatef and a goat anti-rabbit antibody,g respectively. For TrkA neutralization experiments, a monoclonal mouse anti-human NGF-β blocking IgG1h (clone 25623.1) and mouse IgG1i isotype control (clone MOPC-21) were utilized.

TrkA mRNA Assessment

Total RNA was collected from the PC-12, COS31, and Buck cell lines with a commercial kitj and 1 μg of total RNA was reverse transcribed to cDNA.k Five microliters of reverse-transcribed product was used as template in a 50 μL polymerase chain reaction containing 100 ng of each oligonucleotide, 2.5 U of AmpliTaqGold,l and forward (5′-TCT GTG CAG GTC AAC GTC TC-3′) and reverse (5′-GAA CTC GAA AGG GTT GTC CA-3′) degenerate primers for the detection of both rat and canine TrkA.m Reactions were performed in a PTC-200 Peltier thermal cycler with the following cycling conditions: 5 minutes at 94°C denaturing step, followed by 34 cycles (60 seconds at 94°C, 90 seconds at 55°C, and 60 seconds at 72°C), and concluded by 72°C for 10 minutes.

Flow Cytometry

The PC-12, COS31, and Buck cell lines were harvested and resuspended (1 × 106 cells/mL) as a stock concentration in cold phosphate-buffered saline (PBS). Cell stock solutions (200 μL) were added to flow tubes, washed with cold PBS, centrifuged at 1,000 ×g for 5 minutes, resuspended in 1,000 μL of cold fixation/permeabilization buffer,n pulse-vortexed, and incubated and protected from direct light at 4°C for 2 hours. After incubation, cells were washed twice with 2,000 μL of cold permeabilization buffer.n Permeabilized cells were resuspended with 200 μL PBS/2% bovine serum albumin (BSA) (w/v) for 15 minutes at room temperature, and then incubated with primary antibody (anti-TrkA [1 : 25] or isotype control [1 : 25]) at 4°C for 30 minutes. After a wash step with the permeabilization buffer, the secondary antibody (anti-rabbit FITC conjugated, 1 : 10) was incubated with cells at 4°C in the dark for 30 minutes. The cells were washed twice, resuspended in 500 μL of PBS, and analyzed on an Epics-XL flow cytometero based on their forward and side scatter properties and FITC fluorescence.

Immunocytochemistry (Cell Lines)

Cytospin preparations of OSA cell lines were incubated in acetone for 10 minutes, allowed to air dry, and loaded on an autostainer.p To minimize nonspecific peroxidase background staining, all preparations were blocked with 10% hydrogen peroxide for 10 minutes. Cytospin preparations were incubated with the polyclonal anti-human TrkA antibody (1 : 100, 30 minutes). Subsequently, samples were incubated with a secondary goat anti-rabbit immunoglobin for 30 minutes, followed by 3, 3′-diaminobenzidine chromagen solution (7 minutes), and then counterstained with Mayer's hematoxylin (5 minutes). The PC-12 rat pheochromocytoma cell line was used as a TrkA-positive control and for each respective cell line, whereas staining only with the secondary antibody was used as a TrkA-negative control. Positive TrkA expression was characterized by membranous staining with or without cytoplasmic staining.

Exogenous NGF-Induced Mitogenesis

The COS31 or Buck canine OSA cells were plated at a density of 2.0 × 104 cells per 250 μL of starvation media (1% FCS supplemented) or starvation media supplemented with increasing concentrations of human recombinant β-NGFq (range 0.001–10 ng/mL) in a 96-well microtiter plate incubated at 37°C and 5% CO2. After 48 hours, differences in cell mitogenesis were assessed with a nonradioactive colorimetric proliferation assay.r

Cellular Apoptosis Mediated by TrkA Signaling Blockade

For some TrkA signaling blockade experiments, research grade K-252a, a known small molecule inhibitor of TrkA signaling,21,22 was purchased.s A stock concentration K-252a (100 μg/mL) was prepared with sterile dimethyl sulfoxide (DMSO). The COS31 and Buck canine OSA cells were seeded at 3 × 105 cells per 25 cm2 tissue culture flask with starvation media alone, DMSO vehicle control, or varying concentrations of K-252a (0.01, 0.1, 0.3, and 1.0 nM). Canine OSA cells then were incubated in a 5% CO2 environment for 48 hours at 37°C. After incubation, cells were trypsinized, washed, and assessed for cell viability with a commercially available apoptosis detection kit that uses Annexin-V-FITC.t In some experiments, the apoptotic consequence of TrkA signaling attenuation was investigated by the addition of a NGF-neutralizing antibody. In these studies, COS31 canine OSA cells were seeded at 3 × 105 cells per 25 cm2 tissue culture flask with starvation media alone (control) or with varying concentrations of a monoclonal mouse anti-human NGF-β blocking IgG1 or mouse IgG1 isotype control (0.1, 0.3, 1.0, and 3.0 μg/mL). After incubation for 48 hours, COS31 OSA cells were trypsinized, washed, and assessed for cell viability with a commercially available apoptosis detection kit that uses Annexin-V-FITC.t

Immunocytochemistry (Primary Tumor Samples)

In patients with confirmed appendicular OSA as determined either by histopathology or cytology and alkaline phosphatase positivity, additional fine needle aspirates of the primary tumor were collected for TrkA analysis. Unstained slides were prepared by the same protocol described for immunocytochemical staining of canine OSA cell lines. Cytospin preparations of the PC-12 cell line were used as a TrkA-positive control, and patient samples incubated with secondary antibody only were used as an internal TrkA-negative control. Sample staining for TrkA was evaluated by 1 pathologist (A.M.B.). Cytology samples were dichotomously categorized as positive (> 75% positive staining cells) or negative (≤ 10% positive staining cells).

Immunohistochemistry (Pulmonary Metastatic Lesions)

Formalin-fixed, paraffin-embedded tissue blocks of canine metastatic pulmonary OSA lesions unrelated to the primary tumors assessed for TrkA via immunocytochemistry (as previously described) were retrieved from the Veterinary Diagnostic Laboratory, University of Illinois at Urbana-Champaign for immunohistochemical studies. Using 5 μm tissue sections, slides were deparaffinized in xylene and rehydrated in alcohol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 15 minutes, and slides were rinsed twice with wash buffer for 5 minutes. Nonspecific staining was minimized by blocking slides for 10 minutes with Power Block,u and then blocking for 15 minutes with avidin and biotin block.v Blocked slides were incubated with TrkA antibody (1 : 50) for 60 minutes at room temperature, washed, and then incubated with a biotinylated secondaryw for 20 minutes at room temperature. Slides were washed in buffer for 20 minutes before incubation with a streptavidin-biotinylated horseradish peroxidase complex,x and then the reaction was developed with DAB substrate for 5 minutes and counterstained with hematoxylin for 1 minute. The positive control for TrkA staining was a formalin-fixed, paraffin-embedded PC-12 pheochromocytoma cell pellet, and omission of the primary TrkA antibody served as a negative control. The approximate percentage of TrkA-positive OSA cells identified in 5 high-power fields was assessed by 1 observer (R.L.F.).

Statistical Analysis

Differences among groups for NGF-induced mitogenesis and TrkA blockade-associated apoptosis were evaluated by a 1-way ANOVA with posthoc comparisons made with a Tukey-Kramer multiple comparisons test. All values were reported as mean ± standard deviation. Statistical analysis was performed by commercial computer software.y Significance was defined as P < .05.


TrkA Expression in OSA Cell Lines

Transcription of the TrkA gene was identified for the PC-12 cell line as well as for both canine OSA cell lines evaluated (Fig 1A–1C). Translation of TrkA messenger ribonucleic acid (mRNA) into protein was demonstrated by immunocytochemical staining (Fig 1D–1F) and flow cytometry (Fig 1G–1I). For both immunocytochemical staining and flow cytometry, the majority (≥ 80%) of OSA cells expressed TrkA protein.

Figure 1.  Reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemical staining, and flow cytometric analysis of tropomyosin-related kinase A (TrkA). (Top panel) Amplicons of expected size (∼350 base pairs) generated by RT-PCR for TrkA gene in (A) PC-12, (B) COS31, and (C) Buck. Confirmation of membranous TrkA protein expression via immunocytochemical staining (middle panel) and flow cytometric analysis (bottom panel) in (D/G) PC-12, (E/H) COS31, and (F/I) Buck cell lines stained with 1° rabbit polyclonal anti-human TrkA antibody.

NGF-Induced Mitogenesis

The dose-dependent effect of exogenous human recombinant NGF-β on COS31 and Buck OSA cell mitogenesis was investigated. Increasing concentrations of NGF-β (0.001–10 ng/mL) did not enhance cell proliferation after 48 hours of incubation in comparison with canine OSA cells grown with starvation media (1% FCS supplemented) alone, P > .05 (Fig 2A, B).

Figure 2.  Exogenous recombinant human nerve growth factor (NGF)-induced mitogenesis. Differences in cell proliferation induced by varying concentrations of exogenous NGF in the (A) COS31 or (B) Buck canine OSA cell lines. Proliferation represented as optical densities (OD) and expressed as mean ± standard deviation. Shown data represent findings from 2 independent experiments. Statistical significance represented by * and defined as P < .05.

Cellular Apoptosis Mediated by TrkA Signaling Blockade

The apoptotic effect of attenuating basal TrkA signaling in proliferating COS31 and Buck OSA cells was evaluated with the nonspecific receptor tyrosine kinase inhibitor, K-252a. For the COS31 cell line, the percentage of apoptotic cells after incubation for 48 hours in starvation media (1% FCS supplemented) was 9.1 ±1.7% (Fig 3A). No increase in apoptotic cell death was induced by DMSO vehicle control in comparison with cells incubated with starvation media only (data not shown). The addition of K-252a at concentrations ≥0.1 nM significantly increased the percentage of apoptotic cells (Fig 3A). At the highest concentration of K-252a (1.0 nM), 61.7 ± 1.2% of COS31 OSA cells had undergone programmed cell death after 48 hours of incubation (Fig 3A). Similar to the results observed in the COS31 cell line, Buck OSA cells also were sensitive to TrkA signaling attenuation by K-252a; however, an increase in the percentage of apoptotic cells was observed only at the highest concentration of K-252a (1.0 nM) evaluated in this study (Fig 3B). Incubation of COS31 and Buck cell lines with K-252a for 72 hours produced a similar percentage of apoptotic cells in comparison with cells exposed for 48 hours to K-252a (data not shown).

Figure 3.  Apoptosis induction as a consequence of tropomyosin-related kinase A blockade with K-252a The induction of programmed cell death is caused by a nonspecific protein kinase inhibitor, K-252a, in 2 osteosarcoma cell lines (A) COS31 and (B) Buck. Percentage of apoptotic cells detected by Annexin-V-FITC after 48 hours of incubation expressed as mean ± standard deviation. Shown data represent findings from 2 independent experiments. Statistical difference from control (0 nM) is represented by * and defined as P < .05.

Although K-252a has been shown to block TrkA signaling, its inhibitory activities are not restricted to the TrkA receptor tyrosine kinase. To better elucidate the specific role of TrkA signal attenuation in proliferating canine OSA cells, the COS31 cell line was incubated in starvation media (1% FCS supplemented) with increasing concentrations of a specific NGF-neutralizing antibody or isotype control antibody (Fig 4A and 4B). The percentage of apoptotic COS31 cells after incubation for 48 hours in starvation media (1% FCS supplemented) alone was 13.4 ± 1.2%, whereas coincubation of COS31 cells with NGF-neutralizing antibody at 1.0 and 3.0 μg/mL resulted in a significant increase in apoptosis of 21.2 ± 0.3% (P < .05) and 30.4 ± 5.6% (P < .001), respectively (Fig 4A). Incubation of COS31 cells with isotype control antibody did not change the percentage of apoptotic cells as compared to untreated cells after 48 hours of incubation (Fig 4B).

Figure 4.  Apoptosis induction with nerve growth factor (NGF)-neutralizing antibody. The induction of apoptosis in COS31 canine OSA cells following specific tropomyosin-related kinase A signaling attenuation by the addition of (A) NGF neutralizing antibody or (B) isotype control antibody. Percentage of apoptotic cells detected by Annexin-V-FITC after 48 hours of incubation expressed as mean ± standard deviation. Shown data represent findings from 2 independent experiments. Statistical difference from control (0 μg/mL) is represented by * and defined as P < .05.

TrkA Expression in Spontaneously Arising Canine Appendicular OSA Samples

The expression of TrkA by OSA cells derived from confirmed primary appendicular OSA lesions (n = 15) was assessed by immunocytochemistry. Ten patient samples demonstrated membranous staining for TrkA in the majority of OSA cells (> 75%) (Fig 5A), whereas 5 patient samples were considered negative because <10% of OSA cells demonstrated TrkA membranous staining (Fig 5B). In addition to immunocytochemical analysis in primary OSA bone lesions, TrkA expression in 12 confirmed OSA pulmonary metastatic lesions also was analyzed by immunohistochemistry. Controls for TrkA immunohistochemistry were PC-12 cell pellets stained with either primary TrkA antibody (positive control, Fig 5C) or buffer only (negative control, Fig 5D). Similar to TrkA staining of primary bone lesions, 75% (9/12) of pulmonary OSA metastatic lesions demonstrated the majority of OSA cells (∼60–70%) to stain positively for TrkA (Fig 5E). Despite the high percentage of positively staining OSA cells comprising pulmonary metastatic lesions, the staining intensity for TrkA was not homogenous and some OSA cells stained much more intensely than neighboring neoplastic cells.

Figure 5.  Tropomyosin-related kinase A (TrkA) expression in primary tumor and pulmonary metastatic lesions from dogs with osteosarcoma. Immunocytochemical results for positive (A) and negative (B) TrkA staining in primary OSA tumor cytologic samples. Negative staining OSA cells demonstrated by “halo” effect surrounding central nucleus. Immunohistochemical results for (C) PC-12 with 1° rabbit polyclonal anti-human TrkA (positive control), (D) PC-12 with buffer only (negative control), and (E) canine OSA cells comprising a pulmonary metastatic OSA lesion from a dog, with * indicating area of osteoid deposition.


In this study, 2 canine OSA cell lines expressed mRNA and protein for the TrkA receptor tyrosine kinase. Based on flow cytometry and immunocytochemistry, TrkA protein expression was membranous, with the majority of cells (> 90%) from each OSA line staining positively. Additionally, TrkA receptors identified on canine OSA cells appeared to be functional and provided a survival signal, because TrkA signaling blockade with either a small molecule inhibitor or NGF-neutralizing antibody induced apoptosis in canine OSA cells cultured in starvation media. Importantly, the membranous expression of TrkA receptors was identified in the majority of spontaneously arising OSA primary tumors and pulmonary metastatic foci as determined by immunocytochemistry and immunohistochemistry, respectively.

With the in vitro experimental system used in this investigation, the exogenous addition of human recombinant NGF (hrNGF) did not appear to promote cell proliferation in the COS31 and Buck canine OSA cell lines. This negative finding was somewhat unexpected, because previous studies with rhabdomyosarcoma and leiomyosarcoma cell lines derived from humans have identified a mitogenic effect mediated by TrkA signaling.16 One potential explanation for why canine OSA cells did not proliferate in response to exogenous hrNGF could be species-specific differences in NGF protein homology. Specifically, canine TrkA receptors simply may not bind hrNGF, and therefore any intracellular signaling mediated by TrkA would not be translated into biologic events such as cell proliferation. However, based on the recently published dog genome, it appears that the canine TrkA receptor shares 92% homology with the human TrkA receptor. Despite the close protein homology between dog and human TrkA receptors, to definitely determine if functional signaling occurred in our experimental system, additional experiments in canine OSA cells that characterize changes in the phosphorylation status of TrkA receptors after exposure to hrNGF would be required.

Blockade of TrkA signaling with either K-252a or NGF-neutralizing antibody increased apoptosis in canine OSA cell lines cultured in starvation media. Both COS31 and Buck cells appeared sensitive to the antiapoptotic signals mediated by TrkA signaling. However, in the COS31 cell line, attenuation of TrkA signaling with K-252a had a greater apoptotic effect than that achieved with a NGF-neutralizing antibody. The percentage of apoptotic cells after treatment with the highest concentration of K-252a (1.0 nM) was 61.7%, whereas the highest concentration of NGF-neutralizing antibody (3 μg/mL) induced only an apoptotic percentage of 30.4%. One explanation for the discrepancy in the percentage of apoptotic cells induced by K-252a or NGF-neutralizing antibody could be the extent of TrkA signaling attenuation achieved by these 2 different blockade strategies. The greater cell death associated with K-252a treatment could simply reflect a more complete TrkA signal blockade than that achieved with NGF-neutralizing antibody. Furthermore, the higher rate of apoptosis induced by K-252a could also be a function of its multikinase inhibitory properties, because K-252a blocks multiple intracellular signaling pathways mediated not only by TrkA but also protein kinase C and cAMP-dependent protein kinases.21,22 Regardless of the different apoptotic rates induced by either K-252a or NGF-neutralizing antibody, the collective findings derived from this study support the hypothesis that TrkA signaling provides a survival signal for proliferating canine OSA cells.

Cytologic samples derived from the primary tumor of OSA-bearing dogs (n = 15) demonstrated distinct differences in TrkA staining. The majority of malignant osteoblasts collected from primary tumor samples (10/15), strongly and uniformly expressed TrkA, with >75% of all cells demonstrating membranous staining. In the minority of samples evaluated (5/15), staining for TrkA was considered negative because <10% of all malignant osteoblasts stained positive. In accordance with the results derived from primary bone lesions, immunohistochemical evaluation of metastatic pulmonary OSA samples (n = 12) also demonstrated that the majority of metastatic foci (9/12) contain large populations of OSA cells (60–70% of malignant osteoblasts) that stained positively for TrkA. In 25% (3/12) of pulmonary metastatic lesions, few OSA cells stained positively and the lesions were considered negative for TrkA expression. Based on the naturally occurring OSA samples evaluated in this study, the majority of OSA cells derived from either primary tumors or metastatic lesions seem to express the TrkA receptor.

The potential clinical utility of TrkA signaling blockade is being actively investigated for the treatment of various human tumors types,23 with specific emphasis on prostate carcinoma.24–26 In addition to its direct anticancer effects, the inhibition of TrkA signaling also is being evaluated for the treatment of inflammatory and neuropathic pain associated with cancer-induced malignant osteolysis.27–29 Dogs diagnosed with appendicular OSA often either succumb to distant pulmonary metastases or are euthanized for unacceptable pain secondary to focal malignant osteolysis. Based on these common clinical presentations of canine OSA, novel therapies with the potential to induce cancer cell apoptosis in metastatic and primary tumor lesions, as well as to alleviate pain associated with localized bone destruction, could greatly improve management of canine OSA. Given the relatively high and uniform expression of TrkA in canine OSA lesions, as well as the induction of apoptosis in canine OSA cell lines after signaling blockade, TrkA potentially may serve as a novel therapeutic target in dogs diagnosed with OSA.

Although this study offers new information on the expression and function of TrkA in canine OSA cell lines, as well as TrkA expression in spontaneously arising canine OSA tissue samples, several limitations should be addressed. First, previous studies investigating human cancer cell lines have suggested the existence of an autocrine or paracrine survival loop in which neoplastic cells not only express TrkA but also produce endogenous NGF.16,30–32 In this study, we evaluated expression of TrkA in only a limited number of canine OSA cell lines and did not thoroughly evaluate if these same cell lines produced and secreted biologically active NGF. As such, we are not able to confirm or deny the existence of an autocrine or paracrine loop involving TrkA and its cognate ligand, NGF. However, based on previous studies that demonstrated the coexpression of Trk receptors and associated neurotrophins by osteoblasts participating in reparative callus formation, it is expected that malignantly transformed osteoblasts would also have the capacity for simultaneous receptor and ligand expression. Second, although exogenous hrNGF (ng/mL) failed to induce mitogenesis in canine OSA cell lines cultured in starvation media (1% FCS) in this study, we did not ascertain if the addition of a NGF-neutralizing antibody at saturating concentrations to canine OSA cells would diminish their proliferative capacity in culture. Therefore, it remains possible that low levels of NGF (pg/mL) produced endogenously by canine OSA cells may be adequate to achieve a mitogenic plateau; and extremely low concentrations of NGF actually may exert mitogenic activity in canine OSA cells. Third, this study was not designed to characterize the intermediate signaling partners associated with TrkA activation, specifically the intracellular signaling consequences associated with TrkA signaling blockade achieved by either K-252a or NGF-neutralizing antibody. Given that PI3K is a documented downstream signal pathway after TrkA activation,2 the increased apoptosis observed in canine OSA cell lines after TrkA blockade could be a result of decreased Akt phosphorylation. Finally, despite identification of TrkA-positive malignant osteoblasts from the majority of naturally occurring primary tumors (10/15) and metastatic pulmonary lesions (9/12), this study was not designed to investigate the clinical or biologic relevance of these findings. However, given that TrkA expression in several human tumor types currently serves as a target for investigational receptor tyrosine kinase inhibitors, future studies evaluating the clinical relevance of TrkA expression in canine OSA appear justified.

Despite the limitations of this study, it is the first report to describe expression of the neurotrophin receptor TrkA in canine OSA. Given that TrkA signaling appears important for providing antiapoptotic signals to several mesenchymal cancer cell lines, neurotrophin-mediated signaling may contribute to the growth and survival of common sarcomas diagnosed in companion animals. This investigation provides necessary information for designing prospective studies to evaluate TrkA expression in canine OSA patients, which potentially may provide useful information on disease treatment for this common and highly metastatic neoplasm.


aAmerican Type Culture Collection, Manassas, VA

bDulbecco's Modified Eagle's Medium, Gibco, Grand Island, NY

cSigma-Aldrich, St Louis, MO

dAxxora LLC, San Diego, CA

eSanta Cruz Biotechnology, Santa Cruz, CA

fSerotec, Raleigh, NC

gDAKO Corporation, Carpinteria, CA


iBD Biosciences, San Jose, CA

jRNeasy mini kit, Qiagen, Valencia

kSuperScript Double-Stranded cDNA Synthesis Kit, Invitrogen, Carlsbad, CA

lApplied Biosystems, Foster City, CA

mIntegrated DNA Technologies, Coralville, IA

neBiosciences, San Diego, CA

oBeckman Coulter, Hialeah, FL

pDAKO Corporation

qHuman recombinant β-NGF, R&D Systems, Minneapolis, MN

rCellTiter96, Promega, Madison, WI

sAxxora LLC

tBD Biosciences

uPower Block, Biogenex

vAvidin and biotin block, Signet, Dedham, MA

wSupersensitive biotinylated rabbit link, BioGenex

xSupersensitive HRP labeling Streptavidin Peroxidase, BioGenex

yGraphPad InStat, GraphPad Software Inc, San Diego, CA


The authors would like to thank Lisa Shipp and Jane Chladny for their technical assistance in optimizing immunocytochemistry and immunohistochemical staining methodologies. Additionally, the authors thank Drs Lorin Hillman, Jackie Wypij, and Pamela Lucas, and Mrs Nancy George, Jenny Rose, and Rebecca Moss of the Cancer Care Clinic for patient management.


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