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(Background). Canine mammary tumors (CMTs) have emerged as an important model for understanding pathophysiological aspects of human disease. Liquid biopsy (LB), which relies on blood-borne biomarkers and offers minimal invasiveness, holds promise for reflecting the disease status of patients. Small extracellular vesicles (SEVs) and their protein cargo have recently gained attention as potential tools for disease screening and monitoring. (Objectives). This study aimed to isolate SEVs from canine patients and analyze their proteomic profile to assess their diagnostic and prognostic potential. (Methods). Plasma samples were collected from female dogs grouped into CMT (malignant and benign), healthy controls, relapse, and remission groups. SEVs were isolated and characterized using ultracentrifugation (UC), nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Proteomic analysis of circulating SEVs was conducted using liquid chromatography-mass spectrometry (LC-MS). (Results). While no significant differences were observed in the concentration and size of exosomes among the studied groups, proteomic profiling revealed important variations. Mass spectrometry identified exclusive proteins that could serve as potential biomarkers for mammary cancer. These included Inter-alpha-trypsin inhibitor heavy chain (ITIH2 and ITI4), phosphopyruvate hydratase or alpha enolase (ENO1), eukaryotic translation elongation factor 2 (eEF2), actin (ACTB), transthyretin (TTR), beta-2-glycoprotein 1 (APOH) and gelsolin (GSN) found in female dogs with malignant tumors. Additionally, vitamin D-binding protein (VDBP), also known as group-specific component (GC), was identified as a protein present during remission. (Conclusions). The results underscore the potential of proteins found in SEVs as valuable biomarkers in CMTs. Despite the lack of differences in vesicle concentration and size between the groups, the analysis of protein content revealed promising markers with potential applications in CMT diagnosis and monitoring. These findings suggest a novel approach in the development of more precise and effective diagnostic tools for this challenging clinical condition.
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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.
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Neoplasias das Glândulas Suprarrenais , Adrenalectomia , Doenças do Cão , Animais , Cães , Doenças do Cão/cirurgia , Neoplasias das Glândulas Suprarrenais/veterinária , Neoplasias das Glândulas Suprarrenais/cirurgia , Neoplasias das Glândulas Suprarrenais/mortalidade , Neoplasias das Glândulas Suprarrenais/patologia , Estudos Retrospectivos , Masculino , Feminino , Adrenalectomia/veterinária , Análise de Sobrevida , Invasividade Neoplásica , Resultado do TratamentoRESUMO
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.
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Doenças do Cão , Linfoma , Animais , Cães , Melfalan/uso terapêutico , Melfalan/efeitos adversos , Citarabina/uso terapêutico , Vincristina/uso terapêutico , Estudos Retrospectivos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/veterinária , Doenças do Cão/etiologia , Linfoma/tratamento farmacológico , Linfoma/veterinária , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêuticoRESUMO
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., 1â kDa < MW < 30â 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 73â Da of mass through removal of the N-terminal Met (-131â Da), acetylation of the second amino acid (+42â Da), and Met oxidation (+16â 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.
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Neoplasias Ósseas , Osteossarcoma , Animais , Cães , Proteoma/análise , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Peso Molecular , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a DNA/metabolismoRESUMO
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.
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Osteosarcoma prognosis has remained unchanged for the past three decades. In both humans and canines, treatment is limited to excision, radiation, and chemotherapy. Chemoresistance is the primary cause of treatment failure, and the trajectory of tumor evolution while under selective pressure from treatment is thought to be the major contributing factor in both species. We sought to understand the nature of platinum-based chemotherapy resistance by investigating cells that were subjected to repeated treatment and recovery cycles with increased carboplatin concentrations. Three HMPOS-derived cell lines, two resistant and one naïve, underwent single-cell RNA sequencing to examine transcriptomic perturbation and identify pathways leading to resistance and phenotypic changes. We identified the mechanisms of acquired chemoresistance and inferred the induced cellular trajectory that evolved with repeated exposure. The gene expression patterns indicated that acquired chemoresistance was strongly associated with a process similar to epithelial-mesenchymal transition (EMT), a phenomenon associated with the acquisition of migratory and invasive properties associated with metastatic disease. We conclude that the observed trajectory of tumor adaptability is directly correlated with chemoresistance and the phase of the EMT-like phenotype is directly affected by the level of chemoresistance. We infer that the EMT-like phenotype is a critical component of tumor evolution under treatment pressure and is vital to understanding the mechanisms of chemoresistance and to improving osteosarcoma prognosis.
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Neoplasias Ósseas , Osteossarcoma , Animais , Cães , Humanos , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Transcriptoma/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genéticaRESUMO
Osteosarcoma is the most common type of bone cancer in dogs and humans, with significant numbers of patients experiencing treatment failure and disease progression. In our search for new approaches to treat osteosarcoma, we previously detected multiple chaperone proteins in the surface-exposed proteome of canine osteosarcoma cells. In the present study, we characterized expression of representative chaperones and find evidence for stress adaptation in canine osteosarcoma cells relative to osteogenic progenitors from normal bone. We compared the cytotoxic potential of direct (HA15) and putative (OSU-03012) inhibitors of Grp78 function and found canine POS and HMPOS osteosarcoma cells to be more sensitive to both compounds than normal cells. HA15 and OSU-03012 increased the thermal stability of Grp78 in intact POS cells at low micromolar concentrations, but each induced distinct patterns in Grp78 expression without significant change in Grp94. Both inhibitors were as effective alone as carboplatin and showed little evidence of synergy in combination treatment. However, HMPOS cells with acquired resistance to carboplatin were sensitive to inhibition of Grp78 (by HA15; OSU-03012), Hsp70 (by VER-155008), and Hsp90 (by 17-AAG) function. These results suggest that multiple nodes within the osteosarcoma chaperome may be relevant chemotherapeutic targets against platinum resistance.
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Neoplasias Ósseas , Osteossarcoma , Animais , Neoplasias Ósseas/tratamento farmacológico , Carboplatina , Linhagem Celular Tumoral , Cães , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Osteossarcoma/tratamento farmacológicoRESUMO
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.
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Fasting has been shown to decrease chemotherapy-associated adverse events (AEs), in part through insulin-like growth factor (IGF-1) reduction, and may induce a protective effect on normal cells during chemotherapy treatment in mice and people. The purpose of this study was to evaluate the effect of fasting on constitutional, bone marrow and gastrointestinal (GI) AEs, and serum glucose, IGF-1 and insulin levels in dogs receiving vincristine. The study was a prospective, crossover clinical trial in tumour-bearing dogs. Dogs were randomized to be fasted for 24 to 28 hours prior to and 6 hours following their first or second vincristine treatment, and fed normally for the alternate dose. A significant reduction in nausea, anorexia, lethargy and serum insulin was observed when dogs were fasted; however, no significant differences were found in other GI symptoms, neutrophil count, serum glucose or IGF-1. Fasting prior to vincristine therapy is a safe and effective treatment modality that helped mitigate constitutional and GI AEs in tumour-bearing dogs.
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Antineoplásicos/efeitos adversos , Glicemia/efeitos dos fármacos , Doenças do Cão/induzido quimicamente , Gastroenteropatias/veterinária , Neoplasias/veterinária , Vincristina/efeitos adversos , Ração Animal , Animais , Antineoplásicos/uso terapêutico , Medula Óssea/efeitos dos fármacos , Estudos Cross-Over , Doenças do Cão/tratamento farmacológico , Cães , Privação de Alimentos , Gastroenteropatias/induzido quimicamente , Insulina/sangue , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Neoplasias/tratamento farmacológico , Vincristina/uso terapêuticoRESUMO
BACKGROUND: Lymphoma (LSA) is a common malignancy in dogs. Epigenetic changes are linked to LSA pathogenesis and poor prognosis in humans, and LSA pathogenesis in dogs. Sulforaphane (SFN), an epigenetic-targeting compound, has recently gained interest in relation to cancer prevention and therapy. OBJECTIVE: Examine the impact of oral supplementation with SFN on the lymph node proteome of dogs with multicentric LSA. ANIMALS: Seven client-owned dogs with multicentric LSA. METHODS: Prospective, nonrandomized, noncontrolled study in treatment-naïve dogs with intermediate or large cell multicentric LSA. Lymph node cell aspirates were obtained before and after 7 days of oral supplementation with SFN, and analyzed via label-free mass spectrometry, immunoblots, and Gene Set Enrichment Analysis. RESULTS: There was no clinical response and no adverse events attributed to SFN. For individual dogs, the expression of up to 650 proteins changed by at least 2-fold (range, 2-100) after supplementation with SFN. When all dogs where analyzed together, 14 proteins were significantly downregulated, and 10 proteins were significantly upregulated after supplementation with SFN (P < .05). Proteins and gene sets impacted by SFN were commonly involved in immunity, response to oxidative stress, gene transcription, apoptosis, protein transport, maturation and ubiquitination. CONCLUSIONS AND CLINICAL IMPORTANCE: Sulforaphane is associated with major changes in the proteome of neoplastic lymphocytes in dogs.
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Doenças do Cão , Linfoma , Animais , Suplementos Nutricionais , Doenças do Cão/tratamento farmacológico , Cães , Isotiocianatos , Linfonodos , Linfoma/tratamento farmacológico , Linfoma/veterinária , Estudos Prospectivos , Proteoma , SulfóxidosRESUMO
CASE SUMMARY: A case of nasal adenocarcinoma as a suspected secondary malignant neoplasm following definitive radiation therapy and multiagent chemotherapy for nasal lymphoma is described. An 11-year-old spayed female domestic shorthair cat was presented for a 3-week history of progressive facial swelling located over the nasal planum and extending to the medial canthus of the right eye. The cat was previously diagnosed with nasal lymphoma and treated with chemotherapy and definitive radiation 2.5 years prior. Although a definitive diagnosis could not be obtained via cytology, recurrent lymphoma was suspected based on the cat's history and recurrent clinical signs. A lymphoma-directed chemotherapy protocol was attempted, but no clinical response was achieved. The cat was euthanased owing to progressive clinical signs and a diagnosis of nasal adenocarcinoma was made on necropsy examination. Both the original diagnosis of nasal lymphoma and the secondary diagnosis of nasal adenocarcinoma were confirmed with immunohistochemistry. RELEVANCE AND NOVEL INFORMATION: Secondary malignant neoplasm following radiation therapy is infrequently reported in the veterinary literature. In the few reports that exist, most have described sarcoma development in the dog following radiation therapy. In the present report, we describe a cat with a suspected radiation-induced nasal adenocarcinoma that developed 2.5 years after definitive radiation treatment for nasal lymphoma.
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Osteosarcoma (OS) is the most common bone cancer in children and young adults. Solid tumors are characterized by intratumoral hypoxia, and hypoxic cells are associated with the transformation to aggressive phenotype and metastasis. The proteome needed to support an aggressive osteosarcoma cell phenotype remains largely undefined. To link metastatic propensity to a hypoxia-induced proteotype, we compared the protein profiles of two isogenic canine OS cell lines, POS (low metastatic) and HMPOS (highly metastatic), under normoxia and hypoxia. Label-free shotgun proteomics was applied to comprehensively characterize the hypoxia-responsive proteome profiles in the OS cell phenotypes. Hypothesis-driven parallel reaction monitoring was used to validate the differential proteins observed in the shotgun data and to monitor proteins of which we expected to exhibit hypoxia responsiveness, but which were absent in the label-free shotgun data. We established a "distance" score (|zHMPOS - zPOS|), and "sensitivity" score (|zHypoxia - zNormoxia) to quantitatively evaluate the proteome shifts exhibited by OS cells in response to hypoxia. Evaluation of the sensitivity scores for the proteome shifts observed and principal component analysis of the hypoxia-responsive proteins indicated that both cell types acquire a proteome that supports a Warburg phenotype with enhanced cell migration and proliferation characteristics. Cell migration and glucose uptake assays combined with protein function inhibitor studies provided further support that hypoxia-driven adaption of pathways associated with glycolytic metabolism, collagen biosynthesis and remodeling, redox regulation and immunomodulatory proteins typify a proteotype associated with an aggressive cancer cell phenotype. Our findings further suggest that proteins involved in collagen remodeling and immune editing may warrant further evaluation as potential targets for anti-metastatic treatment strategies in osteosarcoma.
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Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Hipóxia/metabolismo , Hipóxia/patologia , Metástase Neoplásica/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Proteoma/metabolismo , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/imunologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Colágeno/metabolismo , Cães , Hipóxia/genética , Metástase Neoplásica/imunologia , Osteossarcoma/genética , Osteossarcoma/imunologia , ProteômicaRESUMO
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.
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Doenças do Cão/cirurgia , Mastocitoma/veterinária , Recidiva Local de Neoplasia/veterinária , Sarcoma/veterinária , Neoplasias de Tecidos Moles/veterinária , Animais , Intervalo Livre de Doença , Doenças do Cão/mortalidade , Cães , Feminino , Estudos Longitudinais , Masculino , Margens de Excisão , Mastocitoma/mortalidade , Mastocitoma/cirurgia , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/cirurgia , Estudos Prospectivos , Sarcoma/mortalidade , Sarcoma/cirurgia , Neoplasias de Tecidos Moles/mortalidade , Neoplasias de Tecidos Moles/cirurgia , Cirurgia VeterináriaRESUMO
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.
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Meios de Contraste/administração & dosagem , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/terapia , Nanopartículas , Imagem Óptica/métodos , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Silanos/administração & dosagem , Nanomedicina Teranóstica/métodos , Animais , Linhagem Celular Tumoral , Feminino , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Projetos Piloto , Valor Preditivo dos Testes , Coelhos , Espécies Reativas de Oxigênio/metabolismoRESUMO
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.
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Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Hormônio Liberador de Gonadotropina/metabolismo , Kisspeptinas/genética , Kisspeptinas/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Animais , Comunicação Autócrina , Remodelação Óssea/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cães , Retroalimentação Fisiológica/fisiologia , Expressão Gênica , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/farmacologia , Técnicas In Vitro , Kisspeptinas/farmacologia , Terapia de Alvo Molecular , Ligante RANK/genética , Receptores de Kisspeptina-1/genética , Receptores de Kisspeptina-1/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo , Receptores de Serotonina/genética , Reprodução/fisiologia , Serotonina/metabolismo , Serotonina/farmacologia , Agonistas do Receptor de Serotonina/metabolismo , Agonistas do Receptor de Serotonina/farmacologiaRESUMO
The role of epigenetic alterations during cancer has gained increasing attention and has resulted in a paradigm shift in our understanding of mechanisms leading to cancer susceptibility. Sulforaphane (SFN) is a naturally occurring isothiocyanate derived from the precursor glucosinolate, glucoraphanin (GFN), which is found in cruciferous vegetables such as broccoli. Sulforaphane has been shown to suppress tumour growth by several mechanisms including inhibiting histone deacetylases. The objective of this study was to provide a detailed analysis of sulforaphane absorption following a single oral dose of a broccoli sprout supplement in normal dogs. A single dose of broccoli sprout supplement (with active myrosinase) was orally administered to 10 healthy adult dogs. Blood and urine samples were collected prior to dosing, and at various time points post-dosing. Plasma total SFN metabolite levels peaked at 4 h post-consumption and were cleared by 24 h post-consumption. Urinary SFN metabolites peaked at 4 h post-consumption, and remained detectable at 24 and 48 h post-supplement consumption. A trend for decrease in histone deacetylase activity was observed at 1 h post-consumption and a significant decrease was observed at 24 h post-consumption. The data presented herein indicate that oral SFN is absorbed in dogs, SFN metabolites are detectable in plasma and urine post-dosing, and SFN and its metabolites have some effect on histone deacetylase activity following a single dose.
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Brassica/química , Cães , Histona Desacetilases/metabolismo , Isotiocianatos/farmacocinética , Extratos Vegetais/farmacologia , Animais , Cães/sangue , Cães/urina , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacocinética , Inibidores de Histona Desacetilases/farmacologia , Isotiocianatos/metabolismo , Isotiocianatos/farmacologia , SulfóxidosRESUMO
OBJECTIVE To evaluate the usefulness of injection of indocyanine green (ICG) solution with near-infrared (NIR) fluorescence imaging for transcutaneous detection of sentinel lymph nodes (SLNs) and their associated lymphatic vessels in the oral mucosa of healthy dogs. ANIMALS 6 adult purpose-bred research hounds. PROCEDURES Each dog was sedated, and 1 mL of ICG solution was injected into the gingival mucosa dorsal to the right maxillary canine tooth. Subsequently, NIR fluorescence imaging was used to transcutaneously detect the lymphatic vessels and SLNs. The distance between the injection site and each SLN was measured. Time to first evidence of node fluorescence was recorded, and velocity of ICG movement was calculated. A slide preparation of a fine-needle aspiration sample of the fluorescing structure underwent cytologic examination (to confirm presence of lymphatic tissue) and NIR fluorescence imaging (to confirm presence of ICG). RESULTS The ipsilateral mandibular lymphocentrum was the SLN in all dogs. The time to visually detectable fluorescence ranged from 4 to 15 minutes (mean ± SD, 8.8 ± 3.76 minutes). The mean velocity was 1.94 ± 0.93 cm/min. Fluorescence was not observed in the contralateral lymph nodes. Each fluorescing structure was confirmed to be lymphatic tissue, and NIR fluorescence imaging revealed that ICG was present in the sampled SLN. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that injection of ICG solution with NIR fluorescence imaging can be used to transcutaneously identify SLNs along with associated lymphatic vessels in the oral mucosa of healthy dogs. Time from injection to identification of fluorescence was rapid with prolonged retention of material within the SLN, indicating that this procedure could be performed during surgery.
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Mucosa Bucal/patologia , Boca/diagnóstico por imagem , Linfonodo Sentinela/diagnóstico por imagem , Espectroscopia de Luz Próxima ao Infravermelho , Animais , Cães , Estudos de Viabilidade , Feminino , Fluorescência , Verde de Indocianina , Linfonodos , Metástase Linfática , Masculino , Anormalidades da Boca/diagnóstico por imagemRESUMO
Circulating cancer exosomes are microvesicles which originate from malignant cells and other organs influenced by the disease and can be found in blood. The exosomal proteomic cargo can often be traced to the cells from which they originated, reflecting the physiological status of these cells. The similarities between cancer exosomes and the tumor cells they originate from exhibit the potential of these vesicles as an invaluable target for liquid biopsies. Exosomes were isolated from the serum of eight osteosarcoma-bearing dogs, five healthy dogs, and five dogs with traumatic fractures. We also characterized exosomes which were collected longitudinally from patients with osteosarcoma prior and 2 weeks after amputation, and eventually upon detection of lung metastasis. Exosomal proteins fraction were analyzed by label-free mass spectrometry proteomics and were validated with immunoblots of selected proteins. Ten exosomal proteins were found that collectively discriminate serum of osteosarcoma patients from serum healthy or fractured dogs with an accuracy of 85%. Additionally, serum from different disease stages could be distinguished with an accuracy of 77% based on exosomal proteomic composition. The most discriminating protein changes for both sample group comparisons were related to complement regulation, suggesting an immune evasion mechanism in early stages of osteosarcoma as well as in advanced disease.
RESUMO
Fluorescence image-guided surgery combined with intraoperative therapeutic modalities has great potential for intraoperative detection of oncologic targets and eradication of unresectable cancer residues. Therefore, we have developed an activatable theranostic nanoplatform that can be used concurrently for two purposes: (1) tumor delineation with real-time near infrared (NIR) fluorescence signal during surgery, and (2) intraoperative targeted treatment to further eliminate unresected disease sites by non-toxic phototherapy. Methods: The developed nanoplatform is based on a single agent, silicon naphthalocyanine (SiNc), encapsulated in biodegradable PEG-PCL (poly (ethylene glycol)-b-poly(É-caprolactone)) nanoparticles. It is engineered to be non-fluorescent initially via dense SiNc packing within the nanoparticle's hydrophobic core, with NIR fluorescence activation after accumulation at the tumor site. The activatable nanoplatform was evaluated in vitro and in two different murine cancer models, including an ovarian intraperitoneal metastasis-mimicking model. Furthermore, fluorescence image-guided surgery mediated by this nanoplatform was performed on the employed animal models using a Fluobeam® 800 imaging system. Finally, the phototherapeutic efficacy of the developed nanoplatform was demonstrated in vivo. Results: Our in vitro data suggest that the intracellular environment of cancer cells is capable of compromising the integrity of self-assembled nanoparticles and thus causes disruption of the tight dye packing inside the hydrophobic cores and activation of the NIR fluorescence. Animal studies demonstrated accumulation of activatable nanoparticles at the tumor site following systemic administration, as well as release and fluorescence recovery of SiNc from the polymeric carrier. It was also validated that the developed nanoparticles are compatible with the intraoperative imaging system Fluobeam® 800, and nanoparticle-mediated image-guided surgery provides successful resection of cancer tumors. Finally, in vivo studies revealed that combinatorial phototherapy mediated by the nanoparticles could efficiently eradicate chemoresistant ovarian cancer tumors. Conclusion: The revealed properties of the activatable nanoplatform make it highly promising for further application in clinical image-guided surgery and combined phototherapy, facilitating a potential translation to clinical studies.
Assuntos
Neoplasias Experimentais/terapia , Fototerapia/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Cirurgia Assistida por Computador/métodos , Nanomedicina Teranóstica/métodos , Animais , Feminino , Corantes Fluorescentes/farmacocinética , Células HEK293 , Humanos , Lactonas/química , Camundongos , Camundongos Nus , Nanopartículas/química , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/cirurgia , Polietilenoglicóis/química , Porfirinas/farmacocinéticaRESUMO
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.