In Vitro Expression of the Extracellular Matrix Components Aggrecan,Collagen Types I and II by Articular Cartilage‐Derived Chondrocytes

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan‐based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long‐term results of therapeutic procedures including cell‐based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and in vitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage‐derived chondrocytes were cultivated in vitro and observed microscopically over a time period of 35 days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT‐qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and in vitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells.     

Radioresistance of cancer stem‐like cell derived from canine tumours

Cancer stem‐like cells (CSCs)/cancer‐initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye‐based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage‐independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct‐4 and CD133 gene than those of adherent‐cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent‐cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent‐cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells.     

A three‐dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two‐dimensional (2D) and three‐dimensional (3D) conditions. The 3D was performed in six‐well AlgiMatrix? (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50–125 µm for CC and 175–200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases‐1, ‐2, ‐9 and ‐13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E‐cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)‐derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model.     

Porcine hepatocyte–Kupffer cell co‐culture as an in vitro model for testing the efficacy of anti‐inflammatory substances

As Kupffer cells are highly involved in the regulation of hepatic inflammatory response, the main goal of this study was to improve and to characterize a hepatocyte–Kupffer cell co‐culture of pig origin for modelling endotoxin‐induced hepatic inflammation and for testing the efficacy of potential anti‐inflammatory substances. This monolayer co‐culture was prepared from primary isolated swine hepatocytes and Kupffer cells in the ratio of 6:1 and 2:1, mimicking different states of liver inflammation. The prepared cell cultures were characterized by immunohistochemical CD‐68 detection. Lipopolysaccharide (LPS) challenge of both co‐cultures resulted in elevated interleukin‐8 (IL‐8) and that of 6:1 co‐cultures in increased IL‐6 production with a higher extent than on hepatocyte monocultures, justifying the key role of Kupffer cells in pro‐inflammatory cytokine production. LPS‐induced IL‐8 production was successfully attenuated by concomitant application of both sodium butyrate and terpinen‐4‐ol on hepatocyte monocultures, but not on co‐cultures, demonstrating the importance of the presence of Kupffer cells in cell cultures as inflammatory models. Based on these initial data, the applied porcine primary hepatocyte–Kupffer cell co‐culture is suggested to be a proper tool for in vitro investigations on liver physiology and hepatic inflammation in pigs and can be used as a useful model mimicking in vivo conditions in veterinary research.     

Development of a new canine osteosarcoma cell line

Establishing a canine osteosarcoma (OSA) cell line can be useful to develop in vivo and in vitro models of OSA. The goal of this study was to develop, characterize and authenticate a new canine OSA cell line and a clone. A cell line and a clone were developed with standard cell culture techniques from a naturally occurring OSA in a dog. The clonal cell line induced a tumour after injection in RAG 1‐deficient mouse. Histology was consistent with OSA. The original tumour from the dog and the tumour induced in the mouse were both reactive with vimentin and osteonectin (ON). The parent cell line and clonal cell line were reactive with ON, osteocalcin and alkaline phosphatase. Loss of heterozygosity was found in the same three microsatellite markers in the parent and clonal cell lines, and the tumour tissue grown in the mouse.     

Profile of Steroid Receptors and Increased Aromatase Immunoexpression in Canine Inflammatory Mammary Cancer as a Potential Therapeutic Target

Canine inflammatory mammary cancer (IMC) has been proposed as a model for the study of human inflammatory breast cancer (IBC). The aims of this study were to compare the immunohistochemical expression of aromatase (Arom) and several hormone receptors [estrogen receptor α (ERα), estrogen receptor β (ERβ), progesterone receptor (PR) and androgen receptor (AR)], in 21 IMC cases vs 19 non‐IMC; and to study the possible effect of letrozole on canine IMC and human inflammatory breast cancer (IBC) in vitro using IPC‐366 and SUM‐149 cell lines. Significant elevations of the means of Arom Total Score (TS), ERβ TS and PR TS were found in the IMC group (p = 0.025, p = 0.038 and p = 0.037, respectively). Secondary IMC tumours expressed higher levels of Arom than primary IMC (p = 0.029). Non‐IMC PR‐ tumours contained higher levels of Arom than non‐IMC PR+ tumours (p = 0.007). After the addition of letrozole, the number of IMC and IBC cells dropped drastically. The overexpression of Arom found and the results obtained in vitro further support canine IMC as a model for the study of IBC and future approaches to the treatment of dogs with mammary cancer, and especially IMC, using Arom inhibitors.     

1,25-Dihydroxyvitamin D3, recombinant human transforming growth factor-beta 1, and recombinant human bone morphogenetic protein-2 induce in vitro differentiation of canine osteosarcoma cells.

Effects of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), recombinant human transforming growth factor (rhTGF)-beta 1 and recombinant human bone morphogenetic protein (rhBMP)-2 on differentiation in four different canine osteosarcoma cell lines (POS53B, 53C, 53D and 14A) were examined using markers specifically expressed by phenotypic osteoblasts. 1,25(OH)2D3 increased alkaline phosphatase (ALP) activity in one cell line, osteocalcin production in two lines and type I collagen production in three lines. RhTGF-beta 1 increased ALP activity in one clonal cell, osteocalcin production in one clonal cell and type I collagen production in two clonal cells. RhBMP-2 increased ALP activity in all clonal cells, osteocalcin production in two clonal cells and type I collagen production in three clonal cells. Thus, these agents induced differentiation in osteosarcoma cells at different efficacies. Electron microscopic study revealed that these agents increased cellular activity in all cell lines with no evidence of degeneration of cell organelle by drug cytotoxicity. In some cultures treated with either 1,25(OH)2D3 or rhBMP-2, apoptotic cells were observed. Based on the change in markers, rhBMP-2 and 1,25(OH)2D3 seemed to be more effective than rhTGF-beta 1. These agents are potential inducers of apoptosis.     

Effects of three-dimensional spheroid culture on equine mesenchymal stem cell plasticity

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy fields. We optimize culture conditions of equine adipose tissue-derived MSCs (eAD-MSCs) for treatment of horse fractures. To investigate enhancing properties of three-dimensional (3D) culture system in eAD-MSCs, we performed various sized spheroid formation and determined changes in gene expression levels to obtain different sized spheroid for cell therapy. eAD-MSCs were successfully isolated from horse tailhead. Using hanging drop method, spheroid formation was generated for three days. Quantitative real-time PCR was performed to analyze gene expression. As results, expression levels of pluripotent markers were increased depending on spheroid size and the production of PGE₂ was increased in spheroid formation compared to that in monolayer. Ki-67 showed a remarkable increase in the spheroid formed with 2.0?×?10⁵ cells/drop as compared to that in the monolayer. Expression levels of angiogenesis-inducing factors such as VEGF, IL-6, IL-8, and IL-18 were significantly increased in spheroid formation compared to those in the monolayer. Expression levels of bone morphogenesis-inducing factors such as Cox-2 and TGF-β1 were also significantly increased in spheroid formation compared to those in the monolayer. Expression levels of osteocyte-specific markers such as RUNX2, osteocalcin, and differentiation potential were also significantly increased in spheroid formation compared to those in the monolayer. Therefore, spheroid formation of eAD-MSCs through the hanging drop method can increases the expression of angiogenesis-inducing and bone morphogenesis-inducing factors under optimal culture conditions.     

Prognostic significance of hypermethylation of death‐associated protein kinase (DAPK) gene CpG island in dogs with high‐grade B‐cell lymphoma

Death‐associated protein kinase (DAPK) is a serine/threonine kinase and a tumour suppressor gene. Diffuse large B‐cell lymphomas with inactivated DAPK through hypermethylation of a CpG island is known to result in a biologically aggressive phenotype in humans. This retrospective study was carried out to analyse the prognostic significance of DAPK CpG island hypermethylation in canine lymphoma. We hypothesized that DAPK CpG island hypermethylation can be a negative prognostic indicator in dogs with nodal high‐grade B‐cell lymphoma. Forty‐seven dogs with high‐grade B‐cell lymphoma, according to the updated Kiel classification, were evaluated after being treated with a CHOP (vincristine, cyclophosphamide, doxorubicin and prednisolone)‐based chemotherapy protocol. The methylation status of the DAPK CpG island was examined by methylation‐specific PCR. Progression‐free survival (PFS) and overall survival (OS) were compared using the Kaplan‐Meier analysis and log‐rank test. The cox proportional hazard regression model was used to evaluate the effect of multiple variables. Hypermethylation of the DAPK CpG island was detected in 21 of the 47 dogs. The PFS and OS in dogs with the hypermethylation (median: 220 and 266 days, respectively) were significantly shorter than those of dogs without hypermethylation (median: 301 and 412 days, respectively) (PFS, P = .036; OS, P = .007). In the multivariate analysis, hypermethylation of the DAPK CpG island remained an independent prognostic factor in predicting shortened PFS (P = .047) and OS (P = .021) as well as clinical substage b. Overall, hypermethylation of the DAPK CpG island was a negative prognostic factor in canine high‐grade B‐cell lymphoma.     

Association of tumour‐infiltrating regulatory T cells with adverse outcomes in dogs with malignant tumours

Regulatory T cells (Tregs) infiltrate into a variety of tumour tissues and associate with poor prognosis in humans. However, data on association of Treg infiltration with prognosis is limited in canine tumours. The purpose of this study was to examine the number of tumour‐infiltrating Tregs and its association with overall survival (OS) in dogs with malignant tumours. The following 168 canine tumours were included: 37 oral malignant melanomas (OMMs); 14 oral squamous cell carcinomas (OSCCs); 16 pulmonary adenocarcinomas (PAs); 37 mammary carcinomas (MCs); 36 mast cell tumours (MCTs) and 28 hepatocellular carcinomas (HCCs). Normal tissues were obtained from 8 healthy dogs as controls. The number of forkhead box P3 (Foxp3)‐positive Tregs in intratumoral and peritumoral areas was investigated by immunohistochemistry. OS was compared between high and low Treg groups. The number of intratumoral and peritumoral Foxp3‐positive Tregs was significantly higher in OMM, OSCC, PA and MC compared with each normal tissue. There were few Foxp3‐positive Tregs in MCT and HCC. With intratumoral Tregs, the OS in the high Treg group was significantly shorter than that in the low Treg group in OMM, OSCC and PA. With peritumoral Tregs, there was no significant difference for OS between the 2 groups in each tumour type. These results suggest that Tregs infiltrate into a variety of canine tumours and the abundance of Tregs are associated with poor prognosis in some solid tumour types.     

Serum vascular endothelial growth factor concentrations and postsurgical outcome in dogs with osteosarcoma

Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor, playing putative roles in both tumour growth and metastasis. The purpose of this study was to correlate pretreatment serum concentrations of VEGF in dogs with osteosarcoma (OSA) with disease‐free interval (DFI) and overall survival (OS). Additionally, the effect of serum from dogs with OSA on ex vivo canine endothelial cell (EC) growth was determined. Pretreatment platelet‐corrected serum VEGF levels correlated significantly with DFI. No other examined variable predicted outcome. The ability of sera from dogs with OSA to stimulate canine EC proliferation did not correlate with VEGF concentration or outcome. These data support a role for VEGF in the development or progression of metastatic disease in dogs with OSA. The VEGF concentration in tested sera was not a major determinant of ex vivo canine EC proliferation in this study.     

Effects of transplantation sites on tumour growth,pulmonary metastasis and ezrin expression of canine osteosarcoma cell lines in nude mice

To determine the influence of the transplantation site of canine osteosarcoma (OS) cell lines on tumour growth and pulmonary metastasis, three OS cell lines were transplanted into nude mice via subcutaneous (SC), intratibial (IT) or intravenous (IV) injection. IT‐xenografts exhibited greater potential for developing primary masses and pulmonary metastasis than SC‐xenografts. In IT and IV xenografts, lung micrometastases along with phosphorylated ezrin–radixin–moesin (p‐ERM) overexpression were found in mice xenografted with HMPOS and OOS cells after 1 week and metastasis was found with decreased p‐ERM expression at later time points. The expression of ezrin and p‐ERM in the primary tumours of IT‐xenografted mice was higher than those in SC‐xenografted mice with HMPOS and OOS cells. The results suggest that the orthotopic transplantation site plays an important role in the spontaneous metastasis of canine OS and that ezrin phosphorylation may be involved in the early metastatic mechanism of canine OS cells.     

The preliminary study on the effects of growth hormone and insulin‐like growth factor‐I on κ‐casein synthesis in bovine mammary epithelial cells in vitro

The effects of growth hormone (GH) and insulin‐like growth factor‐I (IGF‐I) on protein synthesis and gene expression of κ‐casein in bovine mammary epithelial cell in vitro were studied. The treatments were designed as follows: the growth medium without serum was set as the control group, while the treatments were medium supplemented with GH (100 ng/ml), IGF‐I (100 ng/ml), and GH (100 ng/ml) + IGF‐I (100 ng/ml). The quantity of κ‐casein protein was measured by ELISA, and the κ‐casein gene (CSN3) expression was examined by real‐time quantitative PCR (RT‐qPCR). Compared with the control group, all the experimental groups had greater (p < 0.05) expression of CSN3. The concentration of κ‐casein followed a similar response as CSN3, but the difference between the treatments and the control was not statistically significant (p > 0.05). Furthermore, no synergistic effect of GH and IGF‐I was observed for both the κ‐casein concentration and CSN3 expression. It is therefore concluded that GH or IGF‐I can independently promote the expression of CSN3 in bovine mammary epithelial cells in vitro.     

High COX‐2 expression is associated with increased angiogenesis,proliferation and tumoural inflammatory infiltrate in canine malignant mammary tumours: a multivariate survival study

COX‐2 expression affects mammary tumourigenesis by promoting angiogenesis and cell proliferation, encouraging metastatic spread and tumour‐associated inflammation. Samples of canine mammary tumours (n = 109) were submitted to immunohistochemistry to detect COX‐2, CD31, VEGF, Ki‐67, CD3 and MAC387 expression. Concurrent high expression of COX‐2/CD31, COX‐2/VEGF, COX‐2/Ki‐67, COX‐2/CD3 and COX‐2/MAC was associated with elevated grade of malignancy, presence of intravascular emboli and presence of lymph node metastasis. Tumours with high COX‐2 (P < 0.001) and tumours with concurrent expression of high COX‐2 and high CD31 (P = 0.008); high VEGF (P < 0.001); high Ki‐67 (P < 0.001); high CD3+ T‐lymphocytes (P = 0.002) and elevated MAC387 macrophages (P = 0.024) were associated with shorter overall survival (OS) time. Interestingly the groups with high COX‐2/CD31 and high COX‐2/VEGF retained their significance after multivariate analysis arising as independent predictors of OS. Present data highlight the importance of COX‐2 in canine mammary tumourigenesis.     

Anti‐Muellerian hormone concentration in bitches with histopathologically diagnosed ovarian tumours and cysts

Increased concentrations of Anti‐Muellerian hormone (AMH) can indicate a granulosa cell tumour as shown in women, mares and cows. To investigate AMH to differentiate canine granulosa cell tumour from other ovarian pathologies, we evaluated the ovaries of 63 bitches. Blood serum samples were collected before surgery for AMH analysis. Ovaries were submitted for histopathological examination. Fourteen bitches showed normal ovaries. These bitches had AMH values between 0.12 and 0.99 ng/ml. In 20 bitches ovarian cysts i.e., follicular cysts (n = 8), corpora lutea cysts (n = 7), subsurface cysts (n = 5) were diagnosed. These dogs had AMH values of 0.11–2.09 ng/ml. Bitches with small luteinized follicular cysts had slightly higher AMH values than those without ovarian alteration. In 29 cases ovarian neoplasms i.e., granulosa cell tumour (n = 9), epithelial tumours (n = 16), dysgerminomas (n = 3) and one sarcoma were identified. Anti‐Muellerian hormone values of bitches with an ovarian neoplasm except granulosa cell tumour ranged from 0.18 to 1.18 ng/ml. The AMH values of bitches with granulosa cell tumour ranged from 1.12 to ≤23 ng/ml and were significantly higher (p < .05) than in all of the other bitches. The cut‐off of 0.99 ng/ml gave a sensitivity of 100% and a specificity of 94.44% to diagnose granulosa cell tumour. In conclusion, markedly elevated AMH concentrations in bitches are indicative for a granulosa cell tumour. However, negative testing does not rule out the existence of small one. Differentiation of GCT from luteinized follicular cysts may especially be difficult.     

Tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine‐derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine‐derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor‐alpha‐induced protein 8 (TNFAIP8). CDV replication‐induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV‐induced cancer therapy.