Roman Lechowski

What do we know about canine osteosarcoma treatment? – review

Osteosarcoma (OSA) is the most common type of bone tumors in dogs, which has high metastasis ability. 80xa0% of dogs with OSA die due to lung metastasis. As a result its treatment is a challenge for veterinary practitioners. The authors discuss the etiology, pathogenesis and the possible risk factors of OSA. The article focuses on literature review and the study of recent advances in OSA treatment. The authors describe therapies which have significantly prolonged the lives of dogs, as well as those that have proven to be ineffective. Advantages and disadvantages of limb amputation and limb-sparing surgery have been described. Authors present also the results of both single agent’s therapies with the most commonly used drugs as cisplatin, carboplatin and doxorubicin and compare them to the results obtained using combined chemotherapy. The use of nanotechnology as a new approach in OSA treatment in order to avoid multidrug resistance and reduce negative side effects of cytostatic drugs is presented. The main reasons of the therapies failure are also provided in this article.

Enhancing anti-tumor efficacy of Doxorubicin by non-covalent conjugation to gold nanoparticles - in vitro studies on feline fibrosarcoma cell lines.

Background Feline injection-site sarcomas are malignant skin tumors of mesenchymal origin, the treatment of which is a challenge for veterinary practitioners. Methods of treatment include radical surgery, radiotherapy and chemotherapy. The most commonly used cytostatic drugs are cyclophosphamide, doxorubicin and vincristine. However, the use of cytostatics as adjunctive treatment is limited due to their adverse side-effects, low biodistribution after intravenous administration and multidrug resistance. Colloid gold nanoparticles are promising drug delivery systems to overcome multidrug resistance, which is a main cause of ineffective chemotherapy treatment. The use of colloid gold nanoparticles as building blocks for drug delivery systems is preferred due to ease of surface functionalization with various molecules, chemical stability and their low toxicity. Methods Stability and structure of the glutathione-stabilized gold nanoparticles non-covalently modified with doxorubicin (Au-GSH-Dox) was confirmed using XPS, TEM, FT-IR, SAXRD and SAXS analyses. MTT assay, Annexin V and Propidium Iodide Apoptosis assay and Rhodamine 123 and Verapamil assay were performed on 4 feline fibrosarcoma cell lines (FFS1WAW, FFS1, FFS3, FFS5). Statistical analyses were performed using Graph Pad Prism 5.0 (USA). Results A novel approach, glutathione-stabilized gold nanoparticles (4.3 +/- 1.1 nm in diameter) non-covalently modified with doxorubicin (Au-GSH-Dox) was designed and synthesized. A higher cytotoxic effect (p<0.01) of Au-GSH-Dox than that of free doxorubicin has been observed in 3 (FFS1, FFS3, FFS1WAW) out of 4 feline fibrosarcoma cell lines. The effect has been correlated to the activity of glycoprotein P (main efflux pump responsible for multidrug resistance). Conclusions The results indicate that Au-GSH-Dox may be a potent new therapeutic agent to increase the efficacy of the drug by overcoming the resistance to doxorubicin in feline fibrosarcoma cell lines. Moreover, as doxorubicin is non-covalently attached to glutathione coated nanoparticles the synthesized system is potentially suitable to a wealth of different drug molecules.

Derivation of feline vaccine-associated fibrosarcoma cell line and its growth on chick embryo chorioallantoic membrane – a new in vivo model for veterinary oncological studies

Feline vaccine associated fibrosarcomas are the second most common skin tumor in cats. Methods of treatment are: surgery, chemotherapy and radiotherapy. Nevertheless, the usage of cytostatics in feline vaccine associated sarcoma therapy is limited due to their adverse side effects, high toxicity and low biodistribution after i.v. injection. Therefore, much research on new therapeutic drugs is being conducted. In human medicine, the chick embryo chorioallantoic membrane (CAM) model is used as a cheap and easy to perform assay to assess new drug effectiveness in cancer treatment. Various human cell lines have different tumors growth on CAM. In veterinary medicine such model has not been described yet. In the present article derivation of feline vaccine associated fibrosarcoma cell line and its growth on CAM is described. The cell line and the tumor grown were confirmed by histopathological and immunohistochemical examination. As far as we believe, this is the first attempt to create such model, which may be used for further in vivo studies in veterinary oncology.

Doxorubicin Conjugated to Glutathione Stabilized Gold Nanoparticles (Au-GSH-Dox) as an Effective Therapeutic Agent for Feline Injection-Site Sarcomas—Chick Embryo Chorioallantoic Membrane Study

Feline injection-site sarcomas are malignant skin tumours with a high local recurrence rate, ranging from 14% to 28%. The treatment of feline injection-site sarcomas includes radical surgery, radiotherapy and/or chemotherapy. In our previous study it has been demonstrated that doxorubicin conjugated to glutathione-stabilized gold nanoparticles (Au-GSH-Dox) has higher cytotoxic effects than free doxorubicin for feline fibrosarcoma cell lines with high glycoprotein P activity (FFS1, FFS3). The aim of the present study was to assess the effectiveness of intratumoural injection of Au-GSH-Dox on the growth of tumours from the FFS1 and FFS3 cell lines on chick embryo chorioallantoic membrane. This model has been utilized both in human and veterinary medicine for preclinical oncological studies. The influence of intratumoural injections of Au-GSH-Dox, glutathione-stabilized gold nanoparticles and doxorubicin alone on the Ki-67 proliferation marker was also checked. We demonstrated that the volume ratio of tumours from the FFS1 and FFS3 cell lines was significantly (p < 0.01) decreased after a single intratumoural injection of Au-GSH-Dox, which confirms the positive results of in vitro studies and indicates that Au-GSH-Dox may be a potent new therapeutic agent for feline injection-site sarcomas.

Prevalence of feline hyperthyroidism in mature cats in urban population in Warsaw

Abstract The purpose of this study was to assess the prevalence of feline hyperthyroidism in a cat population in Warsaw, considering risk factors. The study was conducted between June 2007 and July 2011. Seven-year-old and older cats were examined. Diagnosis of feline hyperthyroidism was based on the results of clinical examination, data from clinical history, and serum concentrations of thyroid hormones. Hyperthyroidism was diagnosed in 20.14% of 417 cats (95% confidence interval (CI): 16.28%-24.01%). Statistically significant risk factors were age (odds ratio (OR) = 1.17; 95% CI: 1.08-1.27), feeding with a commercial wet feed (OR = 6.74, 95% CI: 2.03-22.37), and an indoor lifestyle (OR = 2.25, 95% CI: 1.04-4.84). There were no effects of breed, gender, castration, or the frequency of deworming on the occurrence of hyperthyroidism. Feline hyperthyroidism in Warsaw is a prevalent disease. This probably results from chronic exposure to dietary and environmental factors.

Current knowledge on feline injection-site sarcoma treatment

Feline injection-site sarcomas (FISS) are malignant skin tumours of mesenchymal origin, the treatment of which is a challenge for veterinary surgeons. The role of surgery, radiotherapy and chemotherapy in FISS treatment has been studied, and a correlation between “clean” surgical margins and disease-free survival has been shown. In addition, clean surgical margins are one of the most important factors for achieving a low recurrence rate. The most effective method of FISS treatment includes combining radical surgery with pre- or postoperative radiotherapy. Chemotherapy may be used as a palliative method of treatment or may be considered an adjunctive therapy for surgery and radiotherapy. In cats with FISS without metastasis, the use of immunostimulant treatment with Oncept IL-2, intended as a complementary immunotherapy in association with surgery and brachytherapy, may also be considered to reduce the risk of relapse and increase the time to relapse. Additionally, this review focuses on recent advances in FISS treatment, including the use of novel compounds, such as doxorubicin conjugated to glutathione-stabilized gold nanoparticles, liposomal doxorubicin or tyrosine kinase inhibitors.

3D chick embryo chorioallantoic membrane model as an in vivo model to study morphological and histopathological features of feline fibrosarcomas

BackgroundThe chick embryo chorioallantoic membrane (CAM) model is well described in human medicine as a cost-effective, easy to perform preclinical oncological model for observing pro- and antiangiogenic response, tumor biology and metastasis. The main objective of this article was to present the modification of the CAM assay in order to evaluate tumor growth from two feline fibrosarcoma cell lines (FFS1, FFS3) and describe their morphological and histopathological features.ResultsThe authors described morphological and histopathological features of two feline fibrosarcoma cell lines (FFS1 and FFS3) grown on the CAM. Tumors from the FFS1 cell line showed high malignancy (grade III), while tumors from the FFS3 cell line were grade II. Proliferation markers (Ki-67 and PCNA) were determined and the positive correlation between PCNA and tumor grade (rxa0=xa00.8247; pxa0<xa00.001) was demonstrated, as opposed to Ki-67.ConclusionsThe results obtained indicate that PCNA may be helpful to evaluate the tumor grade, better than Ki-67, for feline fibrosarcomas. However, further investigations of proliferation marker, in bigger number of feline spontaneous fibrosarcomas and feline fibrosarcomas grown on the CAM from different cell lines, are needed to confirm these observations.

Experimental tumor growth of canine osteosarcoma cell line on chick embryo chorioallantoic membrane (in vivo studies)

The chick embryo chorioallantoic membrane (CAM) model is extensively used in human medicine in preclinical oncological studies. The CAM model has several advantages: low cost, simple experimental approach, time saving and following “3R principles”. Research has shown that the human osteosarcoma cell lines U2OS, MMNG-HOS, and SAOS can form tumors on the CAM. In veterinary medicine, this has been described only for feline fibrosarcomas, feline mammary carcinomas and canine osteosarcomas. However, in case of canine osteosarcomas, it has been shown that only non-adherent osteosarcoma stem cells isolated from KTOSA5 and CSKOS cell lines have the ability to form microtumors on the CAM after an incubation period of 5xa0days, in contrast to adherent KTOSA5 and CSKOS cells. In the presented study, we have proven that the commercial adherent canine osteosarcoma cell line (D-17) can form vascularized tumors on the CAM after the incubation period of 10xa0days.

The Use of Liposomes and Nanoparticles as Drug Delivery Systems to Improve Cancer Treatment in Dogs and Cats

Background: Cancer remains a leading cause of death in companion animals. In human medicine, liposomes and nanoparticles have been extensively investigated as drug delivery systems (DDS) for anticancer agents due to their ability to target cancerous cells and reduce the negative side effects of free cytostatic drugs. In this review, the authors discuss the results of clinical trials using liposomes and polymer-based nanoparticles as DDS to improve cancer treatment in dogs and cats, indicating which ones seem worth further evaluation. The authors then overview ongoing animal cancer clinical trials, evaluating nano-DDS registered on the American Veterinary Medical Association Animal Health Studies Database. Finally, the authors indicate the nano-drugs that require further in vivo evaluation based on the encouraging results obtained from in vitro studies. Conclusions: Liposomes have been the most investigated nano-DDS in veterinary medicine. The lack of cardiotoxicity of the commercially available liposomal doxorubicin (Doxil/Caelyx) suggests it should be used in dogs with cardiac disorders, rather than using free doxorubicin. Cisplatin-incorporated hyaluronic acid nanoparticles, nanocrystals of cisplatin, and paclitaxel are the most promising nano-drugs for potent applications in treating various canine cancers (e.g. oral melanoma, oral sarcoma, and anal gland adenocarcinoma) and their translation into the treatment of human diseases.

Stellate cells in livers of dogs with portal vein hypoperfusion

Hepatic stellate cells play a crucial role in the development of liver fibrosis. In a damaged liver, stellate cells undergo activation, which is manifested as a change of their phenotype: differentiation of stellate cells to myofibroblast-type cells, expression of alpha-Smooth Muscle Actin, their proliferation and a reduction in the size of cytoplasmic lipid droplets. The aim of this study was to determine the number and morphology of stellate cells in the canine liver affected by congenital portosystemic shunt (PSS) and portal vein hypoplasia – hepatic microvascular dysplasia (PVH-HMD). The material for investigation were archived paraffin blocks with liver samples collected supravitally from six dogs with PSS, six dogs with PVH-HMD and six healthy dogs. On the HE-stained sections, the number of stellate cells per 100 hepatocytes was counted (Sztark method) and the diameter of veins in the hepatic triads was measured (light microscope Olympus BX 43, SC30 camera, CellSens Entry 2011 Olympus). In addition, the diameter of lipid droplets in stellate cells was measured (computed image analysis system LUCIA 4.21). The results were analysed statistically (the Kruskal-Wallis test followed by Dunn’s post-hoc procedure; significance level (α) at 0.05; Statistica 12 StatSoft Inc.). The degree of liver fibrosis was determined (Masson’s method of slide stain; Scheuer scale). The liver samples from the dogs with PSS and PVH-HMD were stained immunohistochemically with Monoclonal Mouse Anti-Human Smooth Muscle Actin (α-SMA), clone 1A4, antibodies (DAKO). Portosystemic shunt and primary portal vein hypoplasia in the dog results in a reduction in the diameter of portal vein branches and in insufficient portal blood flow through the liver. In the material investigated, this was particularly evident in the animals affected by PSS: such dogs had a significantly smaller diameter of the veins than did the healthy dogs (p < 0.001) or the dogs with PVH-HMD (p = 0.023). Fibrosis and the expression of α-SMA were stronger in the dogs with PSS than in those with PVH-HMD. Moreover, the dogs with PSS had a significantly higher average number of stellate cells than the healthy animals (p = 0.007) did. However, the examination of the material revealed an enlargement of cytoplasmic lipid droplets: the dogs with PSS had a significantly larger diameter of lipid vacuoles in the cytoplasm of stellate cells than did the healthy animals (p < 0.001) or the dogs with PVH-HMD (p = 0.043); the dogs with PVH-HMD had lipid droplets with a significantly larger diameter than the healthy animals (p < 0.001) did. Hypoperfusion of the liver and the accompanying regressive lesions in hepatocytes result mainly in an increased number of stellate cells and stronger expression of α-SMA, while cytoplasmic lipid droplets in the stellate cells are not reduced in size. The present study indicates the need for detailed analyses of clinical cases and warrants further comprehensive studies of comparative hepatopathology because it demonstrates differences between humans and dogs in the morphological indicators of hepatic stellate cell activation in chronic liver damage.