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Background: Cerebrospinal fluid (CSF) analysis aids in categorizing underlying disease processes in patients with neurologic disease. Convention suggests that CSF should be collected caudal to the lesion. However, little evidence exists to justify this assertion., Hypothesis/objectives: Evaluate the clinicopathologic differences between CSF collected from the cerebellomedullary (CM) and lumbar cisterns in dogs presented for evaluation of neurologic disease., Animals: Fifty-one client-owned dogs undergoing magnetic resonance imaging (MRI) and CSF collection for investigation of neurologic disease., Methods: Cerebrospinal fluid was prospectively collected from the CM and lumbar cisterns in all patients. The total protein (TP) concentration, red blood cell (RBC) count, and total nucleated cell count (TNCC) were analyzed within 30 minutes of collection. Results and cytology findings were interpreted by a single pathologist., Results: Fifty-one paired samples were collected. The TNCC (P < .001), RBC (P < .001), and TP (P < .001) were different between collection sites. When grouped by neurolocalization, TP (intracranial, P < .001; cervical, P < .001; thoracolumbar, P < .001) and RBC (intracranial, P < .001; cervical, P ≤ .002; thoracolumbar, P = .006) counts were significantly different. The TNCC was significantly different in the cervical (P = .04) and thoracolumbar localizations (P = .004) but not for intracranial (P = .30) localizations. The pathologist's interpretation differed between sites in 66.7% of the cases (34/51)., Conclusions: In dogs with lesions that neurolocalized to the brain or cervical spinal cord, there may be clinical benefit in collecting fluid from both the CM and lumbar cisterns. In dogs with thoracolumbar myelopathy, CSF collected from the CM cistern may not be representative of the underlying disease process., (© 2020 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.)

Background: Cathepsin K (CatK) is a lysosomal protease with collagenolytic activity, and its secretion by osteoclasts is responsible for degrading organic bone matrix. People with pathologic bone resorption have higher circulating CatK concentrations., Hypothesis: Canine osteosarcoma (OS) cells will possess CatK, and its secretion will be cytokine inducible. Circulating CatK concentrations will be increased in dogs with OS, and will be a surrogate marker of bone resorption., Animals: Fifty-one dogs with appendicular OS and 18 age- and weight-matched healthy control dogs., Methods: In a prospective study, expressions of CatK mRNA and protein were investigated in OS cells. The inducible secretion and proteolytic activity of CatK from OS cells was assessed in vitro. Serum CatK concentrations were quantified in normal dogs and dogs with OS and its utility as a bone resorption marker was evaluated in dogs with OS treated with palliative radiation and antiresorptive agents., Results: Canine OS cells contain preformed CatK within cytoplasmic vesicles. In OS cells, TGFβ1 induced the secretion of CatK, which degraded bone-derived type I collagen in vitro. CatK concentrations were higher in dogs with OS than healthy dogs (11.3 ± 5.2 pmol/L versus 8.1 ± 5.0 pmol/L, P = .03). In a subset of dogs with OS, pretreatment CatK concentrations gradually decreased after palliative radiation and antiresorptive treatment, from 9.3 ± 3.2 pmol/L to 5.0 ± 3.1 pmol/L, P = .03., Conclusions and Clinical Importance: Canine OS is associated with pathologic bone resorption, and CatK inhibitors might aid in the management of canine OS-related malignant osteolysis., (Copyright © 2011 by the American College of Veterinary Internal Medicine.)

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.

Background: Chemokine receptors (CXCRs) are transmembrane proteins classically studied for their participation in leukocyte homing. By their binding of cognate ligands, CXCRs orchestrate key cellular processes, including directional migration. Several different CXCRs are expressed on cancer cells and dictate tissue-specific metastases. In pediatric osteosarcoma (OSA), CXCR4 expression by tumor cells may participate in metastasis to tissues containing CXCL12, the partnering ligand for CXCR4. Canine and pediatric OSA share many biological similarities, including preferential metastasis to lung, bone, and lymph node., Hypothesis: In canine immortalized cell lines and naturally occurring tumor samples, OSA cells will express CXCR4. In canine OSA cell lines, CXCR4 will participate in directional cell migration., Methods: In vitro, CXCR4 expression in canine OSA cell lines was assessed by reverse-transcriptase polymerase chain reaction, Western blot analysis, flow cytometry, and immunocytochemistry. In vitro, involvement of CXCR4-mediated signaling for directional migration was investigated with a commercial assay. In vivo, CXCR4 expressions were evaluated in primary tumors and pulmonary metastases with immunocytochemistry and immunohistochemistry, respectively., Results: In vitro, canine OSA cells express CXCR4 mRNA and protein. Ligation of CXCR4 with exogenous CXCL12 results in directional migration of canine OSA cell lines. In vivo, majority (8/11) of the canine OSA primary tumors, but minority (2/8) of the pulmonary metastases express CXCR4 protein., Conclusions and Clinical Importance: Canine OSA cells express CXCR4, and its signaling participates in directional migration. Most dogs with spontaneously arising OSA express CXCR4 within their primary tumors.

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 kappaB (NF-kappaB) 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 microM 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 microM) decreasing but higher concentrations (30 microM) 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.

Background: Cyclooxygenase-2 (COX-2) and its principle enzymatic metabolite, prostaglandin E2 (PGE2), are implicated in cancer progression. Based upon immunohistochemical (IHC) evidence that several tumor types in animals overexpress COX-2 protein, COX-2 inhibitors are used as anticancer agents in dogs and cats., Hypothesis: IHC is inaccurate for assessing tumor-associated COX-2 protein and enzymatic activity., Methods: Five mammalian cell lines were assessed for COX-2 protein expression by IHC and Western blot analysis (WB), and functional COX-2 activity was based upon PGE2 production., Results: Detection of COX-2 protein by IHC and WB were in agreement in 4 of 5 cell lines. In 1 cell line that lacked COX-2 gene transcription because of promoter hypermethylation (HCT-116), IHC produced false-positive staining for COX-2 protein expression. Functional COX-2 enzymatic activity was dissociated from relative IHC-based COX-2 protein expression in 2 cell lines (RPMI 2650 and SCCF1). The RPMI 2650 cell line demonstrated strong COX-2 protein expression but minimal PGE2 production., Conclusions and Clinical Importance: Western blot is more accurate than IHC for the detection of COX-2 protein in the cell lines studied. Furthermore, the semiquantitative identification of COX-2 protein by IHC or WB does not necessarily correlate with enzymatic activity. Based upon the potential inaccuracy of IHC and dissociation of COX-2 protein expression from enzymatic activity, the practice of instituting treatment of tumors with COX-2 inhibitors based solely on IHC results should be reconsidered.

Background: Receptor activator of nuclear factor kappa-B (RANK), RANK-ligand (RANKL), and the soluble decoy receptor osteoprotegerin (OPG) form a key axis modulating osteoclastogenesis. In health, RANKL-expressing bone stromal cells and osteoblasts activate osteoclasts through RANK ligation, resulting in homeostatic bone resorption. Skeletal tumors of dogs and cats, whether primary or metastatic, may express RANKL and directly induce malignant osteolysis., Hypothesis: Bone malignancies of dogs and cats may express RANKL, thereby contributing to pathologic bone resorption and pain. Furthermore, relative RANKL expression in bone tumors may correlate with radiographic characteristics of bone pathology., Animals: Forty-two dogs and 6 cats with spontaneously-occurring tumors involving bones or soft tissues were evaluated., Methods: A polyclonal anti-human RANKL antibody was validated for use in canine and feline cells by flow cytometry and immunocytochemistry. Fifty cytologic specimens were collected from bone and soft tissue tumors of 48 tumor-bearing animals and assessed for RANKL expression. In 15 canine osteosarcoma (OSA) samples, relative RANKL expression was correlated with radiographic characteristics of bone pathology., Results: Expression of RANKL by neoplastic cells was identified in 32/44 canine and 5/6 feline tumor samples. In 15 dogs with OSA, relative RANKL expression did not correlate with either radiographic osteolysis or bone mineral density as assessed by dual energy x-ray absorptiometry., Conclusions and Clinical Importance: In dogs and cats, tumors classically involving bone and causing pain, often may express RANKL. Confirming RANKL expression in tumors is a necessary step toward the rational institution of novel therapies targeting malignant osteolysis via RANKL antagonism.