Transcriptomic analysis by RNA sequencing characterises malignant progression of canine insulinoma from normal tissue to metastatic disease.

Insulinomas (INS) are the most common human and canine functioning pancreatic neuroendocrine tumours. The long-term prognosis for malignant INS is poor, because micrometastases are frequently missed during surgery. As human and canine malignant INS share clinical and histopathological features, dogs have been proposed as models for INS research. Using RNA-sequencing, we conducted a pilot study to better understand the underlying molecular mechanisms of canine INS. Normal canine pancreas and lymph node control tissues were compared with primary INS and INS-metastatic lymph nodes, revealing more than 3,000 genes differentially expressed in normal pancreas compared to primary INS. Only 164 genes were differentially expressed between primary INS and INS-metastatic lymph nodes. Hierarchical clustering analysis demonstrated similar genetic profiles in normal pancreas and early clinical stage primary INS, whereas late clinical stage primary INS resembled the genetic profile of INS-metastatic lymph nodes. These findings suggest that markers of malignant behaviour could be identified at the primary site of the disease. Finally, using the REACTOME pathways database, we revealed that an active collagen metabolism, extracellular matrix remodelling, beta-cell differentiation and non-beta-cell trans-differentiation might cause disease progression and hyperinsulinism in INS, identifying major pathways worthy of future research in this currently poorly controlled disease.

Inhibition of canine telomerase in vitro and in vivo using RNAi: further development of a natural canine model for telomerase-based cancer therapies.

Despite advances in cancer therapy, cancer related morbidity and mortality among humans and companion animals remains high, and there is a clear need to develop novel targeted therapies. Expression of the enzyme telomerase has emerged as a central unifying mechanism underlying the immortal phenotype of canine cancer cells and has thus become a candidate for targeted molecular therapies. In this study, the value of telomerase inhibition to target telomerase expressing cancer cells was explored using the novel mechanism of RNA interference (RNAi). Using a Lentiviral expression construct, targeting the RNA component of canine telomerase was effective at inhibiting telomerase in vitro and tumour growth in vivo, but possible resistance mechanisms are highlighted. As canine telomerase biology is more closely related to human telomerase biology than the murine system, it is proposed that this study highlights the value of natural canine models to study anti-telomerase therapies for human patients.

Isolation and characterisation of cancer stem cells from canine osteosarcoma.

There is increasing evidence that cancer is a stem cell disease. This study sought to isolate and characterise cancer stem cells from canine osteosarcoma. One human and three canine cell lines were cultured in non-adherent culture conditions using serum-starved, semi-solid media. Primitive sarcosphere colonies from all cell lines were identified under these conditions and were characterised using molecular and cytochemical techniques for embryonic stem cell markers. Expression of the embryonic stem cell-associated genes Nanog, Oct4 and STAT3 indicated a primitive phenotype. Sarcospheres could be reproduced consistently when passaged multiple times and produced adherent cell cultures when returned to normal growth conditions. Similarities between human and canine osteosarcoma cell lines add credence to the potential of the dog as a model for human disease.

Antibiotic prophylaxis in veterinary cancer chemotherapy: A review and recommendations.

Bacterial infection following cancer chemotherapy-induced neutropenia is a serious cause of morbidity and mortality in human and veterinary patients. Antimicrobial prophylaxis is controversial in the human oncology field, as any decreased incidence in bacterial infections is countered by patient adverse effects and increased antimicrobial resistance. Comprehensive guidelines exist to aid human oncologists in prescribing antimicrobial prophylaxis but similar recommendations are not available in veterinary literature. As the veterinarian's role in antimicrobial stewardship is increasingly emphasized, it is vital that veterinary oncologists implement appropriate antimicrobial use. By considering the available human and veterinary literature we present an overview of current clinical practices and are able to suggest recommendations for prophylactic antimicrobial use in veterinary cancer chemotherapy patients.

Notch pathway inhibition targets chemoresistant insulinoma cancer stem cells.

Insulinomas (INS) are the most common neuroendocrine pancreatic tumours in humans and dogs. The long-term prognosis for malignant INS is still poor due to a low success rate of the current treatment modalities, particularly chemotherapy. A better understanding of the molecular processes underlying the development and progression of INS is required to develop novel targeted therapies. Cancer stem cells (CSCs) are thought to be critical for the engraftment and chemoresistance of many tumours, including INS. This study was aimed to characterise and target INS CSCs in order to develop novel targeted therapies. Highly invasive and tumourigenic human and canine INS CSC-like cells were successfully isolated. These cells expressed stem cell markers (OCT4, SOX9, SOX2, CD133 and CD34), exhibited greater resistance to 5-fluorouracil (5-FU) and demonstrated a more invasive and tumourigenic phenotype in vivo compared to bulk INS cells. Here, we demonstrated that Notch-signalling-related genes (NOTCH2 and HES1) were overexpressed in INS CSC-like cells. Protein analysis showed an active NOTCH2-HES1 signalling in INS cell lines, especially in cells resistant to 5-FU. Inhibition of the Notch pathway, using a gamma secretase inhibitor (GSI), enhanced the sensitivity of INS CSC-like cells to 5-FU. When used in combination GSI and 5-FU, the clonogenicity in vitro and the tumourigenicity in vivo of INS CSC-like cells were significantly reduced. These findings suggested that the combined strategy of Notch signalling inhibition and 5-FU synergistically attenuated enriched INS CSC populations, providing a rationale for future therapeutic exploitation.

Development and Evaluation of a Tissue-Engineered Fibrin-based Canine Mitral Valve Three-dimensional Cell Culture System.

Myxomatous mitral valve disease is the most common cardiac disease of the dog, but examination of the associated cellular and molecular events has relied on the use of cadaveric valve tissue, in which functional studies cannot be undertaken. The aim of this study was to develop a three-dimensional (3D) cell co-culture model as an experimental platform to examine disease pathogenesis. Mitral valve interstitial (VIC) and endothelial (VEC) cells were cultured from normal and diseased canine (VIC only) valves. VICs were embedded in a fibrin-based hydrogel matrix and one surface was lined with VECs. The 3D static cultures (constructs) were examined qualitatively and semiquantitatively by light microscopy, immunofluorescence microscopy and protein immunoblotting. Some constructs were manipulated and the endothelium damaged, and the response examined. The construct gross morphology and histology demonstrated native tissue-like features and comparable expression patterns of cellular (α-smooth muscle actin [SMA] and embryonic smooth muscle myosin heavy chain [SMemb]) and extracellular matrix associated markers (matrix metalloproteinase [MMP]-1 and MMP-3), reminiscent of diseased valves. There were no differences between constructs containing normal valve VICs and VECs (type 1) and those containing diseased valve VICs and normal valve VECs (type 2). Mechanical manipulation and endothelial damage (type 3) tended to decrease α-SMA and SMemb expression, suggesting reversal of VIC activation, but with retention of SMemb+ cells adjacent to the wounded endothelium consistent with response to injury. Fibrin-based 3D mitral valve constructs can be produced using primary cell cultures derived from canine mitral valves, and show a phenotype reminiscent of diseased valves. The constructs demonstrate a response to endothelial damage indicating their utility as experimental platforms.

Preliminary studies of particle-mediated gene delivery to the joints of dogs.

This paper describes a preliminary evaluation of particle-mediated bombardment via the Helios gene gun for the delivery of therapeutic genes to synovial cells in culture. A reporter gene, enhanced green fluorescent protein, was delivered to rabbit synovial fibroblasts (HIG-82) using gold particle (1.0 microm) bombardment to evaluate transfection efficiency at helium pressures of 100 and 150 psi. Transfection of cells occurred at these pressures despite some cell death. The in vitro delivery of gold particles to samples of synovial membrane and articular cartilage from a freshly euthanased dog was also studied to examine depth of penetration of gold particles (1.0 microm) at helium pressures of 250 and 500 psi. Light microscopical examination of histological sections of the synovial membrane showed that particles of gold had penetrated the lining cells of the synovium. However, no gold particles had penetrated the articular cartilage even at 500 psi.

Exploring new biomarkers in the tumour microenvironment of canine inflammatory mammary tumours.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (CIMC) are the most aggressive forms of mammary cancer. Current research aims to identify new therapeutic targets. Here, we investigated gene expression levels of biomarkers associated with the inflammatory microenvironment. A total of 32 formalin-fixed paraffin-embedded samples of canine mammary carcinoma (CIMC = 26; non-CIMC = 6) were used and their cDNA subjected to quantitative polymerase chain reaction (qPCR) to establish gene expression levels for mediators commonly implicated in linking carcinogenesis with inflammation. Gene expression differences between CIMC and non-CIMC types were obtained for cyclooxygenase 2 (COX-2) (P = 0.004), synuclein gamma (SNCG) (P = 0.006), tribbles 1 (P = 0.025), vascular endothelial growth factor (VEGF) (P = 0.017) and CSF1R (P = 0.045). Among these biomarkers correlations were found, particularly between SNCG and tribbles 1 (r = 0.512, P = 0.001). The efficient metastasis of CIMC is intimately linked to components in the tumour microenvironment. This study suggests that upregulation and correlation of SNCG and tribbles 1 deserves to be further explored.

Combination toceranib and lomustine shows frequent high grade toxicities when used for treatment of non-resectable or recurrent mast cell tumours in dogs: A European multicentre study.

Mast cell tumours (MCTs) in dogs can present in a variety of forms. Non-resectable, recurrent or metastatic MCTs usually carry a poor prognosis and present a therapeutic challenge. Both toceranib and lomustine have shown single agent activity against MCTs in dogs. In this study, 10 dogs with advanced MCTs were enrolled prospectively and treated with toceranib (median dose 2.7mg/kg orally every other day), lomustine (median dose 60mg/m2 orally every 3 weeks) and prednisolone (1mg/kg orally every other day, alternating with toceranib). Severe adverse events (SAEs), requiring alterations in the protocol, occurred in all dogs. The objective response rate was 50%. Three dogs died or were euthanased due to SAEs and therefore enrolment of new dogs was discontinued prematurely. A long term response (>1year) was observed in two dogs. Modifications of the protocol are required for future prospective studies.

Dual targeting of EGFR and ERBB2 pathways produces a synergistic effect on cancer cell proliferation and migration in vitro.

Members of the epidermal growth factor receptor (EGFR/ERBB) gene family are frequently dysregulated in a range of human cancers, and therapeutics targeting these proteins are in clinical use. We hypothesized that similar pathways are involved in feline and canine tumours and that the same drugs may be of clinical use in veterinary patients. We investigated EGFR and ERBB2 targeting using a panel of feline and canine cell lines. EGFR and ERBB2 were targeted with siRNAs or tyrosine kinase inhibitors (TKIs) and their effect on cellular proliferation, colony formation and migration was investigated in vitro. Here we report that EGFR and ERBB2 combined siRNA targeting produced synergistic effects in feline and canine cell lines similar to that reported in human cell lines. We conclude that dual EGFR and ERBB2 targeting using TKIs should be further evaluated as a potential new therapeutic strategy in feline head and neck and mammary tumours and canine mammary tumours.

The in vitro effects of piroxicam and meloxicam on canine cell lines.

OBJECTIVES: To analyse the direct antiproliferative effects of both piroxicam and meloxicam at a variety of concentrations on a series of canine cancer cell lines and the mechanism of cell death. METHODS: The in vitro effects of piroxicam and meloxicam at various concentrations on canine cell cultures (Madin-Darby canine kidney cells, osteosarcoma, mammary carcinoma, and lymphoma) were assessed with respect to proliferation inhibition and apoptosis induction. Western blot analysis of cyclooxygenase-1 and cyclooxygenase-2 expression was performed on all cell lines. RESULTS: All cell lines used in this study were cyclooxygenase-1 and cyclooxygenase-2 positive apart from Madin-Darby canine kidney cells which were negative for both cyclooxygenase-1 and cyclooxygenase-2. Both meloxicam and piroxicam were able to inhibit proliferation in cell lines in a dose-dependent manner. However, the drug concentration required for a given effect was cell line dependent. CLINICAL SIGNIFICANCE: The results suggest that significant inhibition of proliferation and induction of apoptosis would only occur when drug concentrations were in excess of those that can be achieved in vivo following maximum recommended dose rates. It is possible, however, that local or topical treatment or altered dosing regimens may offer alternative approaches to the use of these drugs as antineoplastic agents.

Cohort study of COX-1 and COX-2 expression in canine rectal and bladder tumours.

OBJECTIVES: To determine the role that cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) play in malignant transformation in canine transitional cell carcinoma and rectal tumours. METHODS: Histological sections of 21 canine rectal adenocarcinomas and 18 canine transitional cell carcinomas were stained for COX-1 and COX-2. Mann-Whitney non-parametric tests were applied to determine if there was any relationship between the percentage of cells expressing COX-1 or COX-2, and between COX-1 and COX-2 staining intensity and age, breed or sex. RESULTS: For rectal adenocarcinomas, 19.0 per cent of the sections were negative for COX-1 and COX-2. A further 38.1 per cent of the sections were negative for COX-2 but positive for COX-1, and 38.1 per cent of the sections had rare or occasional single cells positive for COX-2. No significant differences were found in COX staining when compared with age, breed or sex. For transitional cell carcinomas, all of the sections were positive for COX-1 and COX-2. For COX-2 staining, 16.7 per cent had more than 30 per cent positive cells. For COX-1 staining, 38.9 per cent had more than 30 per cent positive cells. There was a significant increase in the percentage of COX-1 positive cells in small breed dogs (P = 0.0337). CLINICAL SIGNIFICANCE: The variations in COX expression reported in this study may explain the differences in the clinical response of transitional cell carcinomas and rectal adenocarcinomas following treatment with non-steroidal anti-inflammatory drugs.

Nucleotide sequence of the porcine p53 cDNA, and the detection of recombinant porcine p53 expressed in vitro with a variety of anti-p53 antibodies.

The cDNA of porcine p53 was cloned and sequenced by a reverse transcriptase polymerase chain reaction (RT - PCR) approach with primers based on regions of homology between all known p53 sequences. The p53 cDNA was found to be 87% conserved to human p53 and 86% conserved to bovine p53 at the nucleotide level. The porcine p53 sequence was inserted into an expression vector and recombinant protein expressed in vitro. An approximately 50 kDa protein was detected by Western blotting using both polyclonal and monoclonal anti-p53 antibodies. The sequence data of porcine p53 and the ability to detect expressed protein with various anti-p53 antibodies will allow the p53 status of the pig population, and the role of p53 in porcine tumours, to be assessed. An understanding of tumour development in the pig may be important if pig cells, tissues or organs are to be used in the treatment of humans.

The canine telomerase catalytic subunit (dogTERT): characterisation of the gene promoter and identification of proximal core sequences necessary for specific transcriptional activity in canine telomerase positive cell lines.

Telomerase biology is complicated by studies that show that telomere expression and telomere biology differs between species, and that existing animal models do not closely resemble the human situation. We have previously reported a description of telomere/telomerase biology in the dog and have suggested this as an alternative model system. To further elucidate telomerase biology in this species we have cloned and characterised the canine reverse transcriptase (dogTERT) promoter. We demonstrate that core promoter activity is contained within a region extending approximately 300 bp upstream of the ATG codon. Transient transfections in telomerase-positive canine cell lines and telomerase negative fibroblasts showed that the promoter is only active in telomerase positive cell lines. Sequence analysis demonstrated that the 5' regulatory region is GC-rich and contains no TATA or CAAT box, similar to the human TERT promoter. Motif searches revealed the presence of multiple transcription factor binding sites common to both the human and canine TERT promoters, including a single E-box, Sp1, AP1, MZF-2 and ER/Sp1 binding sites. These findings suggest that the dogTERT gene shares similar transcriptional control to hTERT. Identification of the core promoter necessary for activity may allow the use of naturally occurring cancers in dogs as a preclinical testing ground for telomerase targeted therapies in human cancer patients.

Gene network and canonical pathway analysis in canine myxomatous mitral valve disease: a microarray study.

Myxomatous mitral valve disease (MMVD) is the single most common acquired heart disease of the dog and is particularly common in small pedigree breed dogs such as the Cavalier King Charles spaniel (CKCS). There are limited data on the mitral valve transcriptome and the aim of this study was to use the microarray technology in conjunction with bioinformatics platforms to analyse transcript changes in MMVD in CKCS compared to normal dogs (non-CKCS). Differentially expressed genes (n = 5397) were identified using cut-off settings of fold change, false discovery rate (FDR) and P <0.05. In total, 4002 genes were annotated to a specific transcript in the Affymetrix canine database, and after further filtering, 591 annotated canine genes were identified: 322 (55%) were up-regulated and 269 (45%) were down-regulated. Canine microRNAs (cfa-miR; n = 59) were also identified. Gene ontology and network analysis platforms identified between six and 10 significantly different biological function clusters from which the following were selected as relevant to MMVD: inflammation, cell movement, cardiovascular development, extracellular matrix organisation and epithelial-to-mesenchymal (EMT) transition. Ingenuity Pathway Analysis identified three canonical pathways relevant to MMVD: caveolar-mediated endocytosis, remodelling of epithelial adherens junctions, and endothelin-1 signalling. Considering the biological relevance to MMVD, the gene families of importance with significant difference between groups included collagens, ADAMTS peptidases, proteoglycans, matrix metalloproteinases (MMPs) and their inhibitors, basement membrane components, cathepsin S, integrins, tight junction cell adhesion proteins, cadherins, other matrix-associated proteins, and members of the serotonin (5-HT)/transforming growth factor -ß signalling pathway.

Tumour-associated macrophages are associated with vascular endothelial growth factor expression in canine mammary tumours.

Tumour-associated macrophages (TAMs) have been implicated in carcinogenesis including an important role in angiogenesis. In this study, we describe the relationship between TAMs and angiogenesis in canine mammary tumours (CMT). Formalin-fixed paraffin-embedded CMT samples [(n = 128: malignant (n = 97) and benign (n = 31)] were submitted to immunohistochemical staining to detect MAC387, vascular endothelial growth factor VEGF and CD31 expression. A statistical analysis was carried out to assess possible associations with clinicopathological variables and biological markers of tumour angiogenesis. TAMs, detected by MAC387 expression, were significantly associated with malignant CMT (P < 0.001) and VEGF positive tumours (P = 0.002) and also associated with VEGF expression within malignant CMT (P = 0.043). Associations with clinicopathological variables were found between TAMs and the presence of infiltrative growth (P = 0.031), low tubule formation (P = 0.040) and lymph node metastasis (P = 0.016). The results support the hypothesis that TAMs influence angiogenesis in CMT suggesting TAMs may represent a therapeutic target in this disease.

Inflammation and cancer: till death tears them apart.

Advances in biotechnology have enabled the collection of an immeasurable amount of information from genomic, transcriptomic, metabolomic and proteomic studies of tumours within their microenvironments. The dissection of cytokine and chemokine networks has provided new clues to the interactions between cancer cells and their surrounding inflammatory landscape. To bridge the gap between chronic inflammation and cancer, dynamic participants in the tumour microenvironment have been identified, including tumour-associated macrophages (TAMs) and regulatory T cells (Tregs). Both of these cell types are notable for their ability to cause immunosuppressive conditions and support the evasion of tumour immune surveillance. It is clear now that the tumour-promoting inflammatory environment has to be included as one of the major cancer hallmarks. This review explores the recent advances in the understanding of cancer-related inflammation and how this is being applied to comparative oncology studies in humans and domestic species, such as the dog.

Culture and characterisation of canine mitral valve interstitial and endothelial cells.

Valve interstitial cells (VICs) have an important role in the aetiopathogenesis of myxomatous mitral valve disease (MMVD) in the dog. Furthermore, there is evidence that valve endothelial cells (VECs) also contribute to disease development. In addition to examining native valve tissue to understand MMVD, another strategy is to separately examine VIC and VEC biology under in vitro culture conditions. The aim of this study was to isolate and characterise canine mitral VICs and VECs from normal dog valves using a combination of morphology, immunohistochemistry and reverse transcription PCR (RT-PCR). Canine mitral VECs and VICs were isolated and cultured in vitro. The two cell populations exhibited different morphologies and growth patterns. VECs, but not VICs, expressed the endothelial markers, platelet endothelial cell adhesion molecule (PECAM-1 or CD31) and acetylated low density lipoprotein (Dil-Ac-LDL). Both VECs and VICs expressed vimentin and embryonic non-smooth muscle myosin heavy chain (SMemb), an activated mesenchymal cell marker. The myofibroblast marker, alpha smooth muscle actin (α-SMA), was detected at the mRNA level in both VEC and VIC cultures, but only at the protein level in VIC cultures. The morphological heterogeneity and expression of non-endothelial phenotypic markers in VEC cultures suggested that a mixture of cell types was present, which might be due to cell contamination and/or endothelial-mesenchymal transition (EndoMT). The use of a specific endothelial culture medium for primary VEC cultures enhanced the endothelial properties of the cells and reduced α-SMA and SMemb expression.

Telomere lengths and telomerase activity in dog tissues: a potential model system to study human telomere and telomerase biology.

Studies on telomere and telomerase biology are fundamental to the understanding of aging and age-related diseases such as cancer. However, human studies have been hindered by differences in telomere biology between humans and the classical murine animal model system. In this paper, we describe basic studies of telomere length and telomerase activity in canine normal and neoplastic tissues and propose the dog as an alternative model system. Briefly, telomere lengths were measured in normal canine peripheral blood mononuclear cells (PBMCs), a range of normal canine tissues, and in a panel of naturally occurring soft tissue tumours by terminal restriction fragment (TRF) analysis. Further, telomerase activity was measured in canine cell lines and multiple canine tissues using a combined polymerase chain reaction/enzyme-linked immunosorbent assay method. TRF analysis in canine PBMCs and tissues demonstrated mean TRF lengths to range between 12 and 23 kbp with heterogeneity in telomere lengths being observed in a range of normal somatic tissues. In soft tissue sarcomas, two subgroups were identified with mean TRFs of 22.2 and 18.2 kbp. Telomerase activity in canine tissue was present in tumour tissue and testis with little or no activity in normal somatic tissues. These results suggest that the dog telomere biology is similar to that in humans and may represent an alternative model system for studying telomere biology and telomerase-targeted anticancer therapies.