Retrospective Evaluation of Melphalan, Vincristine, and Cytarabine Chemotherapy for the Treatment of Relapsed Canine Lymphoma.

Dogs diagnosed with multicentric lymphoma often relapse following induction therapy within the first year of treatment. The primary aim of this study was to evaluate the tolerability of a novel drug combination using melphalan, vincristine, and cytarabine (MOC) for the treatment of relapsed lymphoma. On day 1, dogs were treated with vincristine (0.5-0.6 mg/m2 IV) and cytarabine (300 mg/m2 IV over 4-6 hr or subcutaneously over 2 days). On day 7, dogs were treated with melphalan (20 mg/m2per os). This 2 wk protocol was repeated for at least three cycles or until treatment failure. Twenty-six dogs were treated with MOC and met the inclusion criteria. Twenty-three dogs had toxicity data, and all experienced adverse events with the majority graded as mild. The overall response rate was 38%, which included 19% of dogs who achieved a complete response. The median progression-free survival was 29 days (range 1-280 days). The overall clinical benefit was 65% for a median of 37 days (range 33-280 days). MOC is a safe treatment option for relapsed lymphoma in dogs.

Exosomal proteomic signatures correlate with drug resistance and carboplatin treatment outcome in a spontaneous model of canine osteosarcoma.

BACKGROUND: Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. METHODS: In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. RESULTS: Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with < 100 days DFI revealed a proteomic signature that could discriminate between the two cohorts with high accuracy. Furthermore, when the patient's exosomes were compared to exosomes isolated from carboplatin resistant cell lines, several putative biomarkers were found to be shared. CONCLUSIONS: The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Wicking microfluidic approach to separate blood plasma from whole blood to facilitate downstream assays.

Separation of blood plasma or serum from blood is essential for accurate analysis. Conventional blood separation requires instrumentation, reagents, and large sample volumes, limiting this process to laboratory environments with trained personnel. Full implementation of effective blood separation and analysis on microliter sample volumes for point of care (POC) diagnostics has proven extremely challenging resulting in a growing market demand, with common challenges such as expensive device fabrication processes or devices being comprised of materials which are not easily disposable. We developed a membrane-based wicking microfluidic device which is made using a simple fabrication process. This device uses a unique 3D flow channel geometry, fabricated in a polycaprolactone-filled glass microfiber membrane, to efficiently separate microliter sample volumes of blood. Colorimetric assay chemistries were integrated to demonstrate utility of these devices in POC diagnostics. The devices are capable of separating both fresh and anticoagulant-treated blood at microscale sample volumes (<15.0 µL). Modifications to the base device are also reported herein which increased sample volume capacity and separation efficiency. Integrated colorimetric assay enabled semi-quantitative detection of conjugated bilirubin in real blood samples (1.0-1.5 mg/dL). These blood separation devices, fabricated on polycaprolactone-filled glass microfiber, enabled effective blood plasma (anticoagulant-treated blood) and serum (fresh blood) separation with microscale sample volumes. Sample volume capacity and separation efficiency are customizable for specific applications and devices can be integrated with downstream assay chemistries to develop complete POC devices that offer blood separation and diagnostics at the same time on a single membrane.

Nanoparticle-Based Platform for Activatable Fluorescence Imaging and Photothermal Ablation of Endometriosis.

Endometriosis is a painful disorder where endometrium-like tissue forms lesions outside of the uterine cavity. Intraoperative identification and removal of these lesions are difficult. This study presents a nanoplatform that concurrently delineates and ablates endometriosis tissues using real-time near-infrared (NIR) fluorescence and photothermal therapy (PTT). The nanoplatform consists of a dye, silicon naphthalocyanine (SiNc), capable of both NIR fluorescence imaging and PTT, and a polymeric nanoparticle as a SiNc carrier to endometriosis tissue following systemic administration. To achieve high contrast during fluorescence imaging of endometriotic lesions, nanoparticles are constructed to be non-fluorescent prior to internalization by endometriosis cells. In vitro studies confirm that these nanoparticles activate the fluorescence signal following internalization in macaque endometrial stromal cells and ablate them by increasing cellular temperature to 53 ° C upon interaction with NIR light. To demonstrate in vivo efficiency of the nanoparticles, biopsies of endometrium and endometriosis from rhesus macaques are transplanted into immunodeficient mice. Imaging with the intraoperative Fluobeam 800 system reveals that 24 h following intravenous injection, nanoparticles efficiently accumulate in, and demarcate, endometriotic grafts with fluorescence. Finally, the nanoparticles increase the temperature of endometriotic grafts up to 47 °C upon exposure to NIR light, completely eradicating them after a single treatment.

Long-term outcomes of dogs undergoing surgical resection of mast cell tumors and soft tissue sarcomas: A prospective 2-year-long study.

OBJECTIVE: Report clinical outcomes of dogs with surgically excised mast cell tumors (MCT) and soft tissue sarcomas (STS). STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Fifty-three dogs with 52 MCT (50 low grade, 2 high grade) and 19 STS (12 grade I, 6 grade II, 1 grade III). METHODS: All dogs were examined at 3, 6, 12, 18, and 24 months postoperatively, with cytologic or histopathologic evaluation of suspected local recurrences. Dogs euthanized because of study tumor-related causes underwent necropsy. RESULTS: Median intraoperative margins were 20 mm and 30 mm wide for MCT and STS, respectively, with 1 fascial plane resected en bloc. The narrowest histologic tumor-free margins measured <1 mm in 21 of 52 (40%) MCT and 7 of 19 (37%) STS. All dogs were followed for 24 months. Two of 50 (4%) low-grade MCT were diagnosed, with local recurrence 181 and 265 days postoperatively. Two of 36 (6%) dogs with low-grade MCT developed visceral metastasis 181 and 730 days postoperatively. One of 2 dogs with high-grade MCT developed local recurrence 115 days postoperatively. No local recurrence or metastasis was diagnosed after excision of 19 STS. CONCLUSION: Local recurrence rates among predominantly low- to intermediate-grade MCT and STS were low, despite a high prevalence of histologic tumor-free margins <1 mm. Surgical recommendations for high-grade tumors cannot be extrapolated from this population. CLINICAL SIGNIFICANCE: Surgeons should seek to achieve microscopically complete excision for MCT and STS while minimizing patient morbidity and considering limitations of histopathology in predicting outcomes.

Autocrine production of reproductive axis neuropeptides affects proliferation of canine osteosarcoma in vitro.

BACKGROUND: Osteosarcoma strikes hundreds of people each year, of both advanced and younger ages, and is often terminal. Like many tumor types, these bone tumors will frequently undergo a neuroendocrine transition, utilizing autocrine and/or paracrine hormones as growth factors and/or promoters of angiogenesis to facilitate progression and metastasis. While many of these factors and their actions on tumor growth are characterized, some tumor-derived neuropeptides remain unexplored. METHODS: Using validated canine osteosarcoma cell lines in vitro, as well as cells derived from spontaneous tumors in dogs, we explored the autocrine production of two neuropeptides typically found in the hypothalamus, and most closely associated with reproduction: gonadotropin-releasing hormone (GnRH) and kisspeptin (Kiss-1). We evaluated gene expression and protein secretion of these hormones using quantitative RT-PCR and a sensitive radioimmunoassay, and explored changes in cell proliferation determined by MTS cell viability assays. RESULTS: Our current studies reveal that several canine osteosarcoma cell lines (COS, POS, HMPOS, D17, C4) synthesize and secrete GnRH and express the GnRH receptor, while COS and POS also express kiss1 and its cognate receptor. We have further found that GnRH and kisspeptin, exogenously applied to these tumor cells, exert significant effects on both gene expression and proliferation. Of particular interest, kisspeptin exposure stimulated GnRH secretion from COS, similarly to the functional relationship observed within the neuroendocrine reproductive axis. Additionally, GnRH and kisspeptin treatment both increased COS proliferation, which additionally manifested in increased expression of the bone remodeling ligand rankl within these cells. These effects were blocked by treatment with a specific GnRH receptor inhibitor. Both neuropeptides were found to increase expression of the specific serotonin (5HT) receptor htr2a, the activation of which has previously been associated with cellular proliferation, suggesting that production of these factors by osteosarcoma cells may act to sensitize tumors to circulating 5HT of local and/or enteric origin. CONCLUSIONS: Here we report that kisspeptin and GnRH act as autocrine growth factors in canine osteosarcoma cells in vitro, modulating RANKL and serotonin receptor expression in a manner consistent with pro-proliferative effects. Pharmacological targeting of these hormones may represent new avenues of osteosarcoma treatment.

Transarterial Delivery of a Biodegradable Single-Agent Theranostic Nanoprobe for Liver Tumor Imaging and Combinatorial Phototherapy.

PURPOSE: To assess selective accumulation of biodegradable nanoparticles within hepatic tumors after transarterial delivery for in vivo localization and combinatorial phototherapy. MATERIALS AND METHODS: A VX2 hepatic tumor model was used in New Zealand white rabbits. Transarterial delivery of silicon naphthalocyanine biodegradable nanoparticles was performed using a microcatheter via the proper hepatic artery. Tumors were exposed via laparotomy, and nanoparticles were observed by near-infrared (NIR) fluorescence imaging. For phototherapy, a handheld NIR laser (785 nm) at 0.6 W/cm2 was used to expose tumor or background liver, and tissue temperatures were assessed with a fiberoptic temperature probe. Intratumoral reactive oxygen species formation was assessed using a fluorophore (2',7'-dichlorodihydrofluorescein diacetate). RESULTS: Nanoparticles selectively accumulated within viable tumor by NIR fluorescence. Necrotic portions of tumor did not accumulate nanoparticles, consistent with a vascular distribution. NIR-dependent heat generation was observed with nanoparticle-containing tumors, but not in background liver. No heat was generated in the absence of NIR laser light. Reactive oxygen species were formed in nanoparticle-containing tumors exposed to NIR laser light, but not in background liver treated with NIR laser or in tumors in the absence of NIR light. CONCLUSIONS: Biodegradable nanoparticle delivery to liver tumors from a transarterial approach enabled selective in vivo tumor imaging and combinatorial phototherapy.

Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells.

BACKGROUND: Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells. RESULTS: Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-ß, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree. CONCLUSIONS: Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype.

Reductions in margin length after excision of grade II mast cell tumors and grade I and II soft tissue sarcomas in dogs.

OBJECTIVE: Quantify changes in the circumferential lengths of surgical margins of resected canine mast cell tumors (MCT) and soft tissue sarcomas (STS) between the time of collection and histopathology. STUDY DESIGN: Prospective, hypothesis-driven, clinical study. SAMPLE POPULATION: Two hundred and thirty-seven margins from 69 excised tumors (50 MCT and 19 STS) in 51 client-owned dogs. METHODS: The lengths of surgical margins were recorded (eg, cranial, caudal, dorsal, and ventral) for each tumor at 5 time points: intraoperatively (in vivo), immediately after excision (ex vivo), after formalin fixation (postfixation), once mounted on glass slides (subgross), and as histologically tumor-free margins (HTFMs). RESULTS: Compared to in vivo dimensions, the length of surgical margins at each processing step (ie, ex vivo, postfixation, subgross, and HTFM) was reduced by a median of 3.0, 5.0, 6.0, and 8.8 mm for MCT; 2.5, 2.0, 5.0, and 5.0 mm for STS. All processing steps resulted in significant reductions among MCT samples (P < .0001), except between postfixation vs subgross, and for STS samples (P < .0001), except between ex vivo vs postfixation and subgross vs HTFM. The maximum reduction in the total length of margins (from in vivo to HTFM) was 29.6 and 24.2 mm for MCT and STS, respectively. CONCLUSION: Surgical margin length reductions occur due to a combination of physical factors (eg, tissue elasticity, myofibril contraction, and histologic processing) and biological factors (eg, microscopic tumor infiltration into the grossly normal surgical margin). CLINICAL SIGNIFICANCE: These data provide information relevant to evidence-based surgical planning and may influence patient morbidity in the most commonly encountered cutaneous malignancies of dogs.

Cross-species analysis of the canine and human bladder cancer transcriptome and exome.

We investigated the correspondence between transcriptome and exome alterations in canine bladder cancer and the correspondence between these alterations and cancer-driving genes and transcriptional alterations in human bladder cancer. We profiled canine bladder tumors using mRNA-seq and exome-seq in order to investigate the similarity of transcriptional alterations in bladder cancer, in humans and canines, at the levels of gene functions, pathways, and cytogenetic regions. We found that the transcriptomes of canine and human bladder cancer are remarkably similar at the functional and pathway levels. We demonstrated that canine bladder cancer involves coordinated differential expression of genes within cytogenetic bands, and that these patterns are consistent with those seen in human bladder cancer. We found that genes that are mutated in canine bladder cancer are more likely to be transcriptionally downregulated than non-mutated genes, in the tumor. Finally we report three novel mutations (FAM133B, RAB3GAP2, and ANKRD52) for canine bladder cancer.

Shaved margin histopathology and imprint cytology for assessment of excision in canine mast cell tumors and soft tissue sarcomas.

OBJECTIVE: To determine the feasibility and agreement of margin assessment by imprint cytology, shaved margin histopathology, and radial section histopathology in canine cutaneous and subcutaneous mast cell tumors (MCT) and soft tissue sarcomas (STS). STUDY DESIGN: Prospective clinical study. SAMPLE POPULATION: Three hundred and forty margins from 72 excised tumors (52 MCT and 20 STS) in 54 client-owned dogs. METHODS: Imprint cytology samples were acquired by pressing glass slides to the cut surgical margin of the freshly excised surgical specimen. Shaved margin samples were obtained from the patient wound bed using a scalpel immediately prior to closure. Radial section histopathology was performed as part of routine histopathologic processing. All margins were assessed as either positive or negative for presence of tumor cells at the surgical margin. Agreement among methods was calculated using Fleiss Kappa coefficients and an association of method, margin direction, and tumor type with positive margin status was evaluated using a general linear mixed model. RESULTS: Positive margin detection rates differed for MCT (imprint cytology 21%, radial section histopathology 9%, and shaved margin histopathology 3%; P < .0001) but not for STS. Intermethod agreement was poor (Fleiss Kappa = 0.051 and 0.176 for MCT and STS, respectively). Margin direction did not influence margin status for either tumor type. CONCLUSION: Imprint cytology and shaved margin histopathology are feasible, but their results are frequently disparate from routine radial section histopathology. Future studies are needed to evaluate the correlation of each method with local recurrence rates.

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.

Antiproliferative effects of masitinib and imatinib against canine oral fibrosarcoma in vitro.

BACKGROUND: Canine oral fibrosarcoma (COF) is one of the most common oral tumors in dogs and carries a guarded prognosis due to a lack of effective systemic therapeutic options. Mastinib and imatinib are two commonly used tyrosine kinase inhibitors (TKIs) in veterinary oncology but their potential efficacy against COF is uncharacterized. To begin investigating the rationale for use of these TKIs against COF, the present study tested for the presence TKI targets PDGFR-α, PDGFR-ß, Kit, and VEGFR-2 and examined the in vitro effects on cell viability after TKI treatment alone or with doxorubicin. Immunohistochemistry for PDGFR-α, PDGFR-ß, Kit, and VEGFR-2 was performed in 6 COF tumor biopsies. Presence of these same receptors within 2 COF cell lines was probed by reverse transcription-polymerase chain reaction and, for those with mRNA detected, confirmed via western blot. Effects on cell viability were assessed using an MTS assay after masitinib or imatinib treatment alone (0-100 µM), or in combination with doxorubicin (0-3000 nM doxorubicin). Anti-PDGFRB siRNA knockdown was performed and the effect on cell viability quantified. RESULTS: Expression of the TKI targets evaluated was similar between the 2 COF cell lines and the 6 COF tumor biopsies: PDGFR-α and PDGFR-ß were detected in neoplastic cells from most COF tumor biopsies (5/6 and 6/6, respectively) and were present in both COF cell lines; KIT and KDR were not detected in any sample. Masitinib and imatinib IC50 values ranged from 7.9-33.4 µM, depending on the specific TKI and cell line tested. The addition of doxorubicin resulted in synergistic cytotoxicity with both TKIs. Anti-PDGFRB siRNA transfection reduced PDGFR-ß protein expression by 77% and 67% and reduced cell viability by 24% (p < 0.0001) and 28% (0 = 0.0003) in the two cell lines, respectively. CONCLUSIONS: These results provide rationale for further investigation into the use of TKIs, possibly in combination with doxorubicin, as treatment options for COF.

A multiplex biomarker approach for the diagnosis of transitional cell carcinoma from canine urine.

Transitional cell carcinoma (TCC), the most common cancer of the urinary bladder in dogs, is usually diagnosed at an advanced disease stage with limited response to chemotherapy. Commercial screening tests lack specificity and current diagnostic procedures are invasive. A proof of concept pilot project for analyzing the canine urinary proteome as a noninvasive diagnostic tool for TCC identification was conducted. Urine was collected from 12 dogs in three cohorts (healthy, urinary tract infection, TCC) and analyzed using liquid chromatography tandem mass spectrometry. The presence of four proteins (macrophage capping protein, peroxiredoxin 5, heterogeneous nuclear ribonucleoproteins A2/B, and apolipoprotein A1) was confirmed via immunoblot. Of the total 379 proteins identified, 96 were unique to the TCC group. A statistical model, designed to evaluate the accuracy of this multiplex biomarker approach for diagnosis of TCC, predicted the presence of disease with 90% accuracy.

Outcome in dogs with invasive adrenal gland tumors that did not pursue adrenalectomy.

OBJECTIVE: To report the survival times in dogs diagnosed with adrenal tumors with vascular or soft tissue invasion that did not undergo adrenalectomy. ANIMALS: Retrospective case series of 32 client-owned dogs. METHODS: The medical records of a referral veterinary hospital were reviewed to identify dogs that were diagnosed with an invasive adrenal mass and did not undergo adrenalectomy between January 2013 and December 2022. Data collected included signalment, examination findings, and diagnostic results from the initial presentation. Descriptive statistics were calculated to summarize dog signalment information, and Kaplan-Meier survival analysis was performed for calculation of median survival time. RESULTS: Most dogs (n = 28) had vascular invasion, primarily into the caudal vena cava. Surgery was offered but not pursued due to perceived risk of sudden death (n = 5), risk of hemorrhage (4), or concurrent diagnosis of disseminated intravascular coagulation (1). Only 1 dog pursued stereotactic body radiation therapy, and 1 was prescribed toceranib phosphate (Palladia). Of these 32 dogs, 30 (93.8%) died or were euthanized and 2 (6.2%) dogs survived. The median follow-up time was 49 days (range, 0 to 1,910 days). The median survival time was 50 days (95% CI, 4 to 194 days). The most common cause of death or euthanasia was hemoabdomen (n = 8). CLINICAL RELEVANCE: Nonsurgical management of invasive adrenal tumors was associated with short survival times in this case series.

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.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

BACKGROUND: Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. RESULTS: Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. CONCLUSIONS: The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

Top-down mass spectrometry for characterizing the low molecular weight proteome of canine osteosarcoma cell phenotypes.

Osteosarcoma (OS) is the most common primary malignant tumor of bone, which occupying about 20% of all bone cancers. To increase understanding of the biology of OS, we developed and evaluated a top-down mass spectrometry approach to detect, identify and quantify low molecular weight (MW) proteins (i.e., 1 kDa < MW < 30 kDa) in osteosarcoma cells. Top-down proteomic (TDP) data was acquired using reversed phase nano-liquid chromatography in conjunction with high-resolution mass spectrometry and resulted in the assignment of 328 proteins and 820 proteoforms or degradation products with high confidence. Eight post-translational modifications (PTMs) were identified in the present study, including N-terminal acetylation, lysine acetylation, succinylation, malonylation, serine/tyrosine phosphorylation, histidine methylation and N-acetylleucine. We confirmed that a truncated N-terminal proteoform lost 73 Da of mass through removal of the N-terminal Met (-131 Da), acetylation of the second amino acid (+42 Da), and Met oxidation (+16 Da). The results showed that the levels of proteoforms/biodegradable peptides correlated with the metastatic phenotypes of osteosarcoma cell lines. This study demonstrates the benefits of TDP for the characterization and relative quantification of proteoforms with relevance to OS biology and the potential of small molecular weight proteoforms to serve as a still underappreciated source of biomarkers.

Potential Promises and Perils of Human Biological Treatments for Immunotherapy in Veterinary Oncology.

The emergence of immunotherapy for the treatment of human cancers has heralded a new era in oncology, one that is making its way into the veterinary clinic. As the immune system of many animal species commonly seen by veterinarians is similar to humans, there is great hope for the translation of human therapies into veterinary oncology. The simplest approach for veterinarians would be to adopt existing reagents that have been developed for human medicine, due to the potential of reduced cost and the time it takes to develop a new drug. However, this strategy may not always prove to be effective and safe with regard to certain drug platforms. Here, we review current therapeutic strategies that could exploit human reagents in veterinary medicine and also those therapies which may prove detrimental when human-specific biological molecules are used in veterinary oncology. In keeping with a One Health framework, we also discuss the potential use of single-domain antibodies (sdAbs) derived from camelid species (also known as Nanobodies™) for therapies targeting multiple veterinary animal patients without the need for species-specific reformulation. Such reagents would not only benefit the health of our veterinary species but could also guide human medicine by studying the effects of outbred animals that develop spontaneous tumors, a more relevant model of human diseases compared to traditional laboratory rodent models.