Sternohyoideus-sternothyroideus muscle flap to reconstruct oronasal fistulas due to maxillary cancer in four dogs.

OBJECTIVE: To describe the repair of oronasal fistulas in dogs treated for maxillary cancer, with a novel sternohyoideus-sternothyroideus muscle flap, and to report the outcome. ANIMALS: Client-owned dogs (n = 4) with oronasal fistulas related to cancer. STUDY DESIGN: Short case series. METHODS: Maxillary defects were caused by tissue destruction by the tumor and tumor response to radiation therapy in two cases and a complication of caudal maxillectomy in two cases, one of which had neoadjuvant radiation therapy. All tumors were >4 cm at the level of the maxilla. Flaps were harvested by transecting the ipsilateral sternothyroideus and sternohyoideus muscles from their origin at the manubrium and costal cartilage. The muscles were rotated around the base of the cranial thyroid artery and tunneled subcutaneously in the neck and through an incision in the caudodorsal aspect of the oral cavity. The muscle flap was sutured to the edges of the oronasal fistula. RESULTS: The flap reached as far rostral as the level of the first premolar without tension. All dogs had clinical signs that improved postoperatively. All dogs had partial dehiscence of the flap. CONCLUSION: This flap was associated with a high rate of complications; however, all flaps were used in challenging cases. Clinical signs related to oronasal fistula were improved in all dogs in this case series.

Evaluation of Myogenin and MyoD1 as Immunohistochemical Markers of Canine Rhabdomyosarcoma.

Canine rhabdomyosarcoma (RMS) presents a diagnostic challenge due to its overlapping histologic features with other soft tissue sarcomas. The diagnosis of RMS currently relies on positive immunohistochemical (IHC) labeling for desmin; however, desmin expression is also observed in non-RMS tumors. Myogenin and MyoD1 are transcription factors reported to be sensitive and specific IHC markers for human RMS, but they are not widely used in veterinary oncology. The goals of this study were to develop an IHC protocol for myogenin and MyoD1, evaluate myogenin and MyoD1 labeling in canine RMS, and report clinical outcomes. Sixteen cases of possible RMS were retrospectively evaluated. A diagnosis of RMS was confirmed in 13 cases based on histological features and immunolabeling for myogenin and MyoD1, with the aid of electron microscopy in 2 cases. Desmin was negative in 3 cases of RMS. Two cases were of the sclerosing variant. The median age of dogs with RMS was 7.2 years. Anatomic tumor locations included previously reported sites such as bladder, larynx, heart, and orbit, as well as other locations typical of soft tissue sarcomas. Survival ranged from 47 to 1480 days for 5 dogs with available data. This study demonstrated that MyoD1 and myogenin should be included with desmin as part of a diagnostic IHC panel for canine RMS. Utilization of these antibodies to improve the accuracy of canine RMS diagnosis will ultimately allow for better characterization of the biological behavior and clinical outcomes of this disease, providing the groundwork for future comparative investigations in canine RMS.

Elucidating the transcriptional program of feline injection-site sarcoma using a cross-species mRNA-sequencing approach.

BACKGROUND: Feline injection-site sarcoma (FISS), an aggressive iatrogenic subcutaneous malignancy, is challenging to manage clinically and little is known about the molecular basis of its pathogenesis. Tumor transcriptome profiling has proved valuable for gaining insights into the molecular basis of cancers and for identifying new therapeutic targets. Here, we report the first study of the FISS transcriptome and the first cross-species comparison of the FISS transcriptome with those of anatomically similar soft-tissue sarcomas in dogs and humans. METHODS: Using high-throughput short-read paired-end sequencing, we comparatively profiled FISS tumors vs. normal tissue samples as well as cultured FISS-derived cell lines vs. skin-derived fibroblasts. We analyzed the mRNA-seq data to compare cancer/normal gene expression level, identify biological processes and molecular pathways that are associated with the pathogenesis of FISS, and identify multimegabase genomic regions of potential somatic copy number alteration (SCNA) in FISS. We additionally conducted cross-species analyses to compare the transcriptome of FISS to those of soft-tissue sarcomas in dogs and humans, at the level of cancer/normal gene expression ratios. RESULTS: We found: (1) substantial differential expression biases in feline orthologs of human oncogenes and tumor suppressor genes suggesting conserved functions in FISS; (2) a genomic region with recurrent SCNA in human sarcomas that is syntenic to a feline genomic region of probable SCNA in FISS; and (3) significant overlap of the pattern of transcriptional alterations in FISS with the patterns of transcriptional alterations in soft-tissue sarcomas in humans and in dogs. We demonstrated that a protein, BarH-like homeobox 1 (BARX1), has increased expression in FISS cells at the protein level. We identified 11 drugs and four target proteins as potential new therapies for FISS, and validated that one of them (GSK-1059615) inhibits growth of FISS-derived cells in vitro. CONCLUSIONS: (1) Window-based analysis of mRNA-seq data can uncover SCNAs. (2) The transcriptome of FISS-derived cells is highly consistent with that of FISS tumors. (3) FISS is highly similar to soft-tissue sarcomas in dogs and humans, at the level of gene expression. This work underscores the potential utility of comparative oncology in improving understanding and treatment of FISS.

Comparison of cross-sectional geometrical properties and bone density of the proximal radius between Saint Bernard and other giant breed dogs.

OBJECTIVE: To compare the geometrical properties and bone mineral density (BMD) of the proximal radius between Saint Bernard and other giant breed dogs. STUDY DESIGN: Observational, cross-sectional, descriptive study. ANIMALS: Thirteen client-owned Saint Bernard dogs and 13 other client-owned giant breed dogs. METHODS: Computed tomography (CT) studies of Saint Bernard and other giant breed dogs were reviewed. Multiplanar reconstruction of the CT images was used to determine cross-sectional variables at the proximal half of the radius, including mean cortical thickness (mCT) and moment of inertia (MOI). Cortical BMD was estimated from Hounsfield unit measurements at each cross-section and averaged per bone. One-way analysis of variance was used to detect differences between groups. RESULTS: Proximal radii of Saint Bernard dogs had a lower cortical/medullary ratio (1.75 vs 2.2, P < .001), mCT (1.96 vs 2.64 mm, P < .001), and MOI in all planes (mediolateral [ML]: 2086.09 vs 2757.69 mm4 , P < .001; craniocaudal [CrCd]: 3736.36 vs 4370.28 mm4 , P = .025; and polar: 5852.45 vs 7127.97 mm4 , P = .002) compared with bones of other breeds. Cross-sectional BMD did not differ between groups of dogs, but the mean BMD of all cross-sections was lower in Saint Bernard dogs (1214.27 vs 1289.80 mg/mm3 , P = .029). CONCLUSION: The proximal radii of Saint Bernard dogs had thinner cortices and lower CrCd, ML, and polar MOI compared with corresponding bones in giant breed dogs. CLINICAL SIGNIFICANCE: The structural properties of the proximal radius of the Saint Bernard differ from those in other giant breeds and could reduce the ability of this region to sustain biomechanical loads. These properties could predispose Saint Bernard dogs to complications after surgical limb-sparing procedures.

Lateral manus translation for limb-sparing surgery in 18 dogs with distal radial osteosarcoma in dogs.

OBJECTIVE: To describe and report outcomes after lateral translation of the manus for limb-sparing management of distal radial osteosarcoma in dogs. STUDY DESIGN: Retrospective case series. STUDY POPULATION: Eighteen client-owned dogs. METHODS: The distal aspect of the affected radius and associated neoplastic tissues were excised. The distal aspect of the ulna was preserved except for its medial cortex, which was removed en bloc with the radial segment. The manus was translated laterally to place the radial carpal bone in contact with the distal aspect of the ulna. A limb-sparing or locking compression plate was placed on the remaining proximal radius and the 3rd metacarpal bone. A 3.5-mm SOP (string of pearls) plate was placed on the lateral aspect of the proximal ulna and the 4th metacarpal bone. Dogs were administered chemotherapy. Data were collected to assess surgical and oncologic outcomes. Limb function was subjectively assessed. RESULTS: The percentage of radius removed ranged from 43% to 94% (median 54%). Complications developed in 12 limbs, with infection in 10, biomechanical complications in 6, and local recurrence in 4. Limb function was subjectively assessed as acceptable. Median disease-free interval was 219 days, and median survival time was 370 days. CONCLUSION: Outcomes after lateral translation of the manus compared favorably to other limb-sparing techniques for dogs with distal radial osteosarcoma, particularly in dogs requiring excision of a large segment of the radius. CLINICAL SIGNIFICANCE: The lateral manus translation provides an alternative limb-sparing technique that does not require an allograft, endoprosthesis, or autograft.

Bilateral caudal maxillectomy for resection of tumors crossing palatal midline and use of the angularis oris axial pattern flap for primary closure or dehiscence repair in two dogs.

OBJECTIVE: To describe the technique and outcomes for bilateral caudal maxillectomy for resection of large caudal maxillary tumors crossing palatal midline in two dogs. STUDY DESIGN: Clinical case report. ANIMALS: Two client-owned dogs. METHODS: Two client-owned dogs with primary caudal maxillary tumors, a poorly differentiated sarcoma, and a multilobulated osteochondrosarcoma. Bilateral caudal maxillectomies were performed for curative-intent resection of these tumors. The angularis oris axial pattern flap was used for primary closure in one dog and for dehiscence repair in the other. RESULTS: Both tumors were resected with complete histologic margins. The defects were closed with local buccal mucosal flaps, with or without a unilateral angularis oris flap. Esophagostomy tubes were placed at time of surgery to bypass oral feeding. Incisional dehiscence and subsequent oronasal fistula formation occurred as a postoperative complication in both dogs (3 and 10 days, respectively). Both were successfully repaired with a combination of local buccal mucosal flaps and the angularis oris flap. Both dogs had good functional outcome and quality of life after recovery from revision surgery. CONCLUSION: Bilateral caudal maxillectomy allowed resection of caudal maxillary tumors crossing palatal midline, with good function and quality of life after recovery in 2 dogs. CLINICAL SIGNIFICANCE: Good outcomes including complete resections are achievable with bilateral caudal maxillectomy despite complications. Local mucosal and axial pattern flaps can be used for dehiscence repair.

Modification of the metal endoprosthesis limb-salvage procedure for excision of a large distal radial osteosarcoma in a dog: A case report.

OBJECTIVE: To report modification of the endoprosthesis surgical limb-salvage technique to treat a locally extensive osteosarcoma in a dog and associated functional outcome. STUDY DESIGN: Clinical case report. ANIMALS: One client-owned dog. METHODS: A 9-year-old dog was presented for treatment of a locally extensive distal radial osteosarcoma. A limb-salvage surgery was performed with a second-generation Veterinary Orthopedic Implants (VOI) endoprosthesis to reconstruct the radial segmental and carpal osseous defect. The endoprosthesis was stabilized with a 16-mm-wide locking VOI limb-salvage plate. The level of the osteotomy of the radius/ulna was 3 cm proximal to the periosteal reaction seen on radiographs, and the distal osteotomy extended through the proximal metacarpal bones (II-V), 3 cm from the distal extent of the soft tissue component of the tumor. A 3.5-mm SOP (String of Pearls) plate was used as adjunct fixation. RESULTS: The postoperative functional outcome was classified as acceptable. Ambulation was normal, with mild subjective lameness on the treated limb at examinations 20, 43, and 63 days after surgery. The dog was euthanized 92 days after surgery because of progressive metastatic disease. CONCLUSION: The modified endoprosthetic technique allowed complete excision of the carpal joint, which resulted in acceptable functional outcomes in the dog described here. CLINICAL SIGNIFICANCE: Distal ostectomies may include part of the manus during limb salvage surgery of locally extensive distal radial osteosarcoma and using an endoprosthesis implant to reconstruct the defect.

Long-term outcome of dogs treated with ulnar rollover transposition for limb-sparing of distal radial osteosarcoma: 27 limbs in 26 dogs.

OBJECTIVE: To determine outcomes in dogs with distal radial osteosarcoma treated with ulnar rollover transposition (URT) limb-sparing surgery including: viability of the ulnar graft, complications, subjective limb function, disease-free interval (DFI), and survival time (ST). STUDY DESIGN: Retrospective case series. ANIMALS: Twenty-six client-owned dogs with distal radial osteosarcoma and no involvement of the ulna. METHODS: Data of dogs treated with URT were collected at the time of surgery and retrospectively from medical records and by contacting owners and referring veterinarians. RESULTS: URT technique was performed on 27 limbs in 26 dogs. The ulnar graft was determined to be viable in 17 limbs, nonviable in 3, and unknown in 7. Complications occurred in 20 limbs. Infection was diagnosed in 12 limbs. Biomechanical complications occurred in 15 and local recurrence in 2 limbs. Limb function graded by veterinarians or owners was poor in 2 limbs, fair in 4, good in 14, excellent in 3, and unknown in 4. Median DFI was 245 days and median ST was 277 days. CONCLUSION: The URT technique maintained the viability of the ulnar graft. The complication rate was high but limb function appeared acceptable. Although sufficient length of the distal aspect of the ulna must be preserved to perform this technique, local recurrence was not increased compared to other limb-sparing techniques when cases were appropriately selected.

Osteoprotegerin activates osteosarcoma cells that co-express RANK and RANKL.

BACKGROUND: Osteosarcoma (OS) is an aggressive and often fatal cancer that afflicts over 1000 humans and 10,000 dogs per year in the United States. Recent evidence suggests deregulation in the signaling triad, receptor activator of nuclear factor kappa B (RANK), its activating ligand (RANKL), and the RANKL inhibitor, osteoprotegerin (OPG) plays a key role in the pathogenesis of OS. This study investigated the expression of RANK and RANKL in osteosarcoma tumors and cell lines and describes an activating effect of OPG on OS cells in vitro. RESULTS: Canine OS tumors and cell lines co-express mRNA for both RANK and RANKL. Expression of these proteins in OS cell lines was confirmed by Western blot and immunofluorescence microscopy. Expression of the soluble form of RANKL was not detected in media from OS cells. OPG-Fc incubation increased the phosphorylation status of ERK, AKT and the p65 subunit of nuclear factor kappa B (NFκB) and induced NFκB translocation from the cytoplasm to the nucleus in canine OS cells. OPG increased proliferation in both canine and human derived OS cell lines. CONCLUSION: RANKL is produced by OS tumors and cell lines that also express RANK. This data provides preliminary evidence for a potential autocrine and or paracrine activation pathway in canine OS. An activating effect of exogenous OPG on signal transduction proteins, NFκB and proliferation in OS is described. These data provide new information concerning aberrant signaling in OS and could be important to those considering OPG as a therapeutic agent for osteosarcoma.

Bilateral Superior Labial Mucosal Transposition Flaps to Correct Stenosis of the Nares Following Bilateral Rostral Maxillectomy Combined with Nasal Planum Resection in a Dog.

OBJECTIVE: To describe a technique using labial mucosal flaps to correct stenosis of the nares subsequent to bilateral rostral maxillectomy and nasal planum resection. STUDY DESIGN: Case report ANIMALS: Client-owned dog. METHODS: A 10-year-old, neutered male Golden Retriever developed repeated stenosis of the nares, at first after bilateral rostral maxillectomy and nasal planum resection, and again after revision surgery. Bilateral, superior labial mucosal transposition flaps were created and interpolated between the nasal mucosa and skin after debridement of scar tissue. RESULTS: The stenosis did not recur after mucosal flap transposition and the dog returned to normal quality of life (last follow-up 25 months postoperative). CONCLUSION: Single-stage, superior labial mucosal transposition flaps can be used to correct nares stenosis subsequent to previous surgery.

Effect of Metoclopramide on the Incidence of Early Postoperative Aspiration Pneumonia in Dogs with Acquired Idiopathic Laryngeal Paralysis.

OBJECTIVE: Evaluate the effect of a perioperative intravenous continuous rate infusion (CRI) of metoclopramide on the incidence of aspiration pneumonia in the short term postoperative period in dogs undergoing unilateral arytenoid lateralization. STUDY DESIGN: Prospective, randomized, multi-center clinical trial. ANIMALS: 61 client-owned dogs with idiopathic laryngeal paralysis and normal preoperative thoracic radiographs. METHODS: All dogs underwent unilateral arytenoid lateralization with a uniform anesthetic, analgesic, and management protocol. Dogs in the treatment group received an intravenous CRI of metoclopramide for 24 hours perioperative. All dogs were assessed for clinical signs of aspiration pneumonia based on the results of physical examination and owner interview up to the point of suture removal (10-14 days postoperative). Any dog with suspected aspiration pneumonia had thoracic radiographs performed. RESULTS: Six dogs developed aspiration pneumonia in the short term postoperative period (2/28 control dogs and 4/33 treated dogs), accounting for an overall frequency of 10% with no significant difference between control and treated dogs. No variables measured in the study were significantly different between control and treated dogs. CONCLUSIONS: Perioperative metoclopramide, at the doses used in this study, did not affect the incidence of aspiration pneumonia in the short term postoperative period in dogs with idiopathic laryngeal paralysis undergoing unilateral arytenoid lateralization.

Attributing physical and biological impacts to anthropogenic climate change.

Significant changes in physical and biological systems are occurring on all continents and in most oceans, with a concentration of available data in Europe and North America. Most of these changes are in the direction expected with warming temperature. Here we show that these changes in natural systems since at least 1970 are occurring in regions of observed temperature increases, and that these temperature increases at continental scales cannot be explained by natural climate variations alone. Given the conclusions from the Intergovernmental Panel on Climate Change (IPCC) Fourth Assessment Report that most of the observed increase in global average temperatures since the mid-twentieth century is very likely to be due to the observed increase in anthropogenic greenhouse gas concentrations, and furthermore that it is likely that there has been significant anthropogenic warming over the past 50 years averaged over each continent except Antarctica, we conclude that anthropogenic climate change is having a significant impact on physical and biological systems globally and in some continents.

Updates in Surgical Oncology.

New knowledge and data can influence the treatment options of dogs and cats affected by neoplasms. Partial limb amputation with the use of a prosthesis is possible in dogs. Newer studies attempt to define better and understand the complications and limb function associated with this approach. Limb sparing is an alternative to amputation, and three-dimensional printing allows the manufacturing of personalized endoprostheses. Finally, the recommended approach for the excision of cutaneous mast cell tumors (MCTs) is with proportional margins. In dogs, grade shifting might have occurred when removing a recurrent MCT or soft tissue sarcoma.

Patient-specific finite element modeling for fracture risk prediction in a canine model of osteosarcoma.

Background: In cancer patients with bone tumors, pathological fractures are a major concern. Making treatment decision for these patients requires an evaluation of fracture risk, which is currently based on semi-qualitative criteria that lack patient-specificity. Because of this, there exists a need for quantitative fracture risk prediction tailored to the patient's individual bone geometry. To address this need, this study aims to develop and validate a finite element (FE) technique that can be used to create patient-specific models and more accurately identify fracture risk. Model validation was performed using canine radii. Methods: Radii were harvested from eight canines euthanized for reasons unrelated to the study. A semicircular osteotomy was made in the distal portion of each bone to simulate tumor lysis. Samples underwent computed tomography (CT) scanning and were randomly assigned to loading groups for destructive mechanical testing. Three samples were tested in torsion, three in cantilever bending, and two in compression. FE models were created for each bone from the corresponding CT scan to replicate patient-specific geometry. Material properties were based on equations relating scan properties to elastic modulus. Boundary conditions and loads were added to the models based on the sample's treatment group. Stiffness and strain data were collected from both the mechanical testing and FE simulation, and yield load predictions were made based on maximum principal strain. Experimental and computational results were compared using a linear regression. Results: The FE models were most accurate in predicting stiffness, followed by strain, with yield load having the lowest accuracy. Linear regressions resulted in R2 values of 0.9335 for bending and compression and 0.8798 for torsion. Conclusions: The proposed FE technique is a valid method for predicting fracture in a canine model of osteosarcoma. This method could provide patient-specific, quantitative data to aid clinicians in decisions regarding surgical intervention for patients with bone tumors.

Comparative Transcriptomes of Canine and Human Prostate Cancers Identify Mediators of Castration Resistance.

Prostate cancer continues to be one of the most lethal cancers in men. While androgen-deprivation therapy is initially effective in treating prostate cancer, most cases of advanced prostate cancer eventually progress to castration-resistant prostate cancer (CRPC), which is incurable. Similarly, the most aggressive form of prostatic carcinoma occurs in dogs that have been castrated. To identify molecular similarities between canine prostate cancer and human CRPC, we performed a comparative analysis of gene expression profiles. Through this transcriptomic analysis, we found that prostatic carcinoma in castrated dogs demonstrate an androgen indifferent phenotype, characterized by low androgen receptor and neuroendocrine associated genes. Notably, we identified two genes, ISG15 and AZGP1 that were consistently up- and downregulated, respectively, in both canine prostatic carcinoma and human CRPC. Additionally, we identified several other genes, including GPX3, S100P, and IFITM1, that exhibited similar expression patterns in both species. Protein-protein interaction network analysis demonstrated that these 5 genes were part of a larger network of interferon-induced genes, suggesting that they may act together in signaling pathways that are disrupted in prostate cancer. Accordingly, our findings suggest that the interferon pathway may play a role in the development and progression of CRPC in both dogs and humans and chart a new therapeutic approach.

Comparative Transcriptomes of Canine and Human Prostate Cancers Identify Mediators of Castration Resistance.

Prostate cancer continues to be one of the most lethal cancers in men. While androgen deprivation therapy is initially effective in treating prostate cancer, most cases of advanced prostate cancer eventually progress to castration-resistant prostate cancer (CRPC), which is incurable. Similarly, the most aggressive form of prostatic carcinoma occurs in dogs that have been castrated. To identify molecular similarities between canine prostate cancer and human CRPC, we performed a comparative analysis of gene expression profiles. Through this transcriptomic analysis, we found that prostatic carcinoma in castrated dogs demonstrates an androgen-indifferent phenotype, characterised by low-androgen receptor and neuroendocrine-associated genes. Notably, we identified two genes, ISG15 and AZGP1, that were consistently up- and down-regulated, respectively, in both canine prostatic carcinoma and human CRPC. Additionally, we identified several other genes, including GPX3, S100P and IFITM1, that exhibited similar expression patterns in both species. Protein-protein interaction network analysis demonstrated that these five genes were part of a larger network of interferon-induced genes, suggesting that they may act together in signalling pathways that are disrupted in prostate cancer. Accordingly, our findings suggest that the interferon pathway may play a role in the development and progression of CRPC in both dogs and humans and chart a new therapeutic approach.

A new approach for prediction of tumor sensitivity to targeted drugs based on functional data.

BACKGROUND: The success of targeted anti-cancer drugs are frequently hindered by the lack of knowledge of the individual pathway of the patient and the extreme data requirements on the estimation of the personalized genetic network of the patient's tumor. The prediction of tumor sensitivity to targeted drugs remains a major challenge in the design of optimal therapeutic strategies. The current sensitivity prediction approaches are primarily based on genetic characterizations of the tumor sample. We propose a novel sensitivity prediction approach based on functional perturbation data that incorporates the drug protein interaction information and sensitivities to a training set of drugs with known targets. RESULTS: We illustrate the high prediction accuracy of our framework on synthetic data generated from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and an experimental dataset of four canine osteosarcoma tumor cultures following application of 60 targeted small-molecule drugs. We achieve a low leave one out cross validation error of <10% for the canine osteosarcoma tumor cultures using a drug screen consisting of 60 targeted drugs. CONCLUSIONS: The proposed framework provides a unique input-output based methodology to model a cancer pathway and predict the effectiveness of targeted anti-cancer drugs. This framework can be developed as a viable approach for personalized cancer therapy.

A case study of personalized therapy for osteosarcoma.

BACKGROUND: Effective targeted therapies are needed in sarcomas, but the biological heterogeneity of these tumors has presented a challenge to clinical integration of small molecule inhibitors in sarcoma treatment. Here we outline a process to personalize therapy for sarcomas through a case study of a canine with spontaneous osteosarcoma. PROCEDURE: Rapid establishment of a primary tumor cell culture is described, followed by efficient functional characterization of the tumor that identified the Src inhibitor dasatinib as the most effective targeted therapy for this individual dog. RESULTS: Adjuvant dasatinib was administered for a total of 26 weeks following treatment with chemotherapy. Pharmacokinetic studies confirm that a therapeutic serum concentration was achieved at a tolerable dose of 0.75 mg/kg/day. The canine patient remains without evidence of recurrent disease 24 months following initial diagnosis. CONCLUSIONS: The approach described through this illustrative case study is broadly applicable and might be used for other solid tumors in canines as well as in humans.

The expression and role of serotonin receptor 5HTR2A in canine osteoblasts and an osteosarcoma cell line.

BACKGROUND: The significance of the serotonergic system in bone physiology and, more specifically, the importance of the five hydroxytryptamine receptor 2A (5HTR2A) in normal osteoblast proliferation have been previously described; however the role of serotonin in osteosarcoma remains unclear. Particularly, the expression and function of 5HTR2A in canine osteosarcoma has not yet been studied, thus we sought to determine if this indoleamine modulates cellular proliferation in vitro. Using real time quantitative reverse transcription PCR and immunoblot analyses, we explored receptor expression and signaling differences between non-neoplastic canine osteoblasts (CnOb) and an osteosarcoma cell line (COS). To elucidate specific serotonergic signaling pathways triggered by 5HTR2A, we performed immunoblots for ERK and CREB. Finally, we compared cell viability and the induction of apoptosis in the presence 5HTR2A agonists and antagonists. RESULTS: 5HTR2A was overexpressed in the malignant cell line in comparison to normal cells. In CnOb cells, ERK phosphorylation (ERK-P) decreased in response to both serotonin and a specific 5HTR2A antagonist, ritanserin. In contrast, ERK-P abundance increased in COS cells following either treatment. While endogenous CREB was undetectable in CnOb, CREB was observed constitutively in COS, with expression and exhibited increased CREB phosphorylation following escalating concentrations of ritanserin. To determine the influence of 5HTR2A signaling on cell viability we challenged cells with ritanserin and serotonin. Our findings confirmed that serotonin treatment promoted cell viability in malignant cells but not in normal osteoblasts. Conversely, ritanserin reduced cell viability in both the normal and osteosarcoma cells. Further, ritanserin induced apoptosis in COS at the same concentrations associated with decreased cell viability. CONCLUSIONS: These findings confirm the existence of a functional 5HTR2A in a canine osteosarcoma cell line. Results indicate that intracellular second messenger signal coupling of 5HTR2A is different between normal and malignant cells, warranting further research to investigate its potential as a novel therapeutic target for canine osteosarcoma.

The effects of taurolidine alone and in combination with doxorubicin or carboplatin in canine osteosarcoma in vitro.

BACKGROUND: Osteosarcoma (OS) affects over 8000 dogs/year in the United States. The disease usually arises in the appendicular skeleton and metastasizes to the lung. Dogs with localized appendicular disease benefit from limb amputation and chemotherapy but most die within 6-12 months despite these treatments. Taurolidine, a derivative of taurine, has anti-tumor and anti-angiogenic effects against a variety of cancers. The following in vitro studies tested taurolidine as a candidate for adjuvant therapy for canine OS. Tests for p53 protein status and caspase activity were used to elucidate mechanisms of taurolidine-induced cell death. RESULTS: Taurolidine was cytotoxic to osteosarcoma cells and increased the toxicity of doxorubicin and carboplatin in vitro. Apoptosis was greatly induced in cells exposed to 125 µM taurolidine and less so in cells exposed to 250 µM taurolidine. Taurolidine cytotoxicity appeared caspase-dependent in one cell line; with apparent mutant p53 protein. This cell line was the most sensitive to single agent taurolidine treatment and had a taurolidine-dependent reduction in accumulated p53 protein suggesting taurolidine's effects may depend on the functional status of p53 in canine OS. CONCLUSION: Taurolidine's cytotoxic effect appears dependent on cell specific factors which may be explained, in part, by the functional status of p53. Taurolidine initiates apoptosis in canine OS cells and this occurs to a greater extent at lower concentrations. Mechanisms of cell death induced by higher concentrations were not elucidated here. Taurolidine combined with doxorubicin or carboplatin can increase the toxicity of these chemotherapy drugs and warrants further investigation in dogs with osteosarcoma.