Canine Transmissible Venereal Tumor Cells Research Articles

In vitro chemosensitivity of a canine tumor venereal transmissible cancer cell line.

The canine transmissible venereal tumor (CTVT) is the most common malignity in dogs. Because there are reports that this tumor is resistant to vincristine sulfate, the chemotherapeutic options are scarce, and the development of new therapeutic approaches is necessary. In this study, we evaluated the cytotoxic activity of vincristine, doxorubicin, temozolomide, panobinostat, toceranib, gemcitabine, cisplatin, fluorouracil, cyclophosphamide, and methotrexate on a CTVT cell line, determining that all drugs decreased the viability in a dose-dependent manner. Furthermore, they inhibit cellular migration in a time- and drug-dependent manner, as evaluated by the wound healing assay. On the other hand, vincristine, panobinostat, gemcitabine, toceranib, cyclophosphamide, and methotrexate increased the percentage of cells in the subG1 phase, and doxorubicin, temozolomide, gemcitabine, toceranib, and methotrexate decreased the percentage of cells in the synthesis phase. To efficientize the use of vincristine, only toceranib increased the cytotoxic effect of vincristine in a synergistic manner. Our results confirm the use of vincristine as the gold standard for CTVT treatment as monotherapy and suggest the use of a combinatorial and sequential treatment with toceranib.

Recurrent horizontal transfer identifies mitochondrial positive selection in a transmissible cancer

Autonomous replication and segregation of mitochondrial DNA (mtDNA) creates the potential for evolutionary conflict driven by emergence of haplotypes under positive selection for ‘selfish’ traits, such as replicative advantage. However, few cases of this phenomenon arising within natural populations have been described. Here, we survey the frequency of mtDNA horizontal transfer within the canine transmissible venereal tumour (CTVT), a contagious cancer clone that occasionally acquires mtDNA from its hosts. Remarkably, one canine mtDNA haplotype, A1d1a, has repeatedly and recently colonised CTVT cells, recurrently replacing incumbent CTVT haplotypes. An A1d1a control region polymorphism predicted to influence transcription is fixed in the products of an A1d1a recombination event and occurs somatically on other CTVT mtDNA backgrounds. We present a model whereby ‘selfish’ positive selection acting on a regulatory variant drives repeated fixation of A1d1a within CTVT cells.

Conventional-Vincristine Sulfate vs. Modified Protocol of Vincristine Sulfate and L-Asparaginase in Canine Transmissible Venereal Tumor.

Background: Vincristine (VCR) is a mono-chemotherapy for canine transmissible venereal tumor (CTVT). L-asparaginase (LAP) is usually used in combination with other drugs. Previously, LAP-VCR protocol was applied for the CTVT-VCR-resistant cases. However, there were a few reports about using this protocol since the first visit.Aims: To firstly investigate the effectiveness of combining chemotherapy (Vincristine and L-asparaginase, VCR-LAP) in normal CTVT case. Secondly, to compare this protocol with the conventional (Vincristine, VCR) protocol before and during treatment in 24 CTVT dogs.Materials and Methods: Clinical signs, tumor relative volume, and histopathological change [amount of CTVT cells, tumor-infiltrating lymphocytes (TILs), TILs/CTVT ratio, collagen area, and Ki-67 proliferative index (PI)] were the treatment evaluation parameters. Moreover, transcriptome analysis of apoptotic (Bcl-2, Bax), drug-resistant genes (ABCB1, ABCG2), and BCL-2 and BAX expression were also included.Results: Both protocols gave the decreased tumor volume, increased TILs/CTVT ratios and collagen area in the mass. Interestingly, the combination protocol decreased treatment time. There were two resistant cases after treatment with VCR. The expression of Bcl-2 and Bax were decreased, and this may indicate the better response after treatment. Moreover, both drug resistant genes did not increase after treatment.Conclusion: The main finding of this study is that the combination protocol did not only decrease treatment duration time but also gave the effectiveness of treatment outcomes in CTVT cases. Therefore, the application of the new protocol could be used by the field practitioners.

Evaluation of a Canine Transmissible Venereal Tumour Cell Line with Tumour Immunity Capacity but Without Tumorigenic Property.

IntroductionCanine transmissible venereal tumour (CTVT) is a sexually transmitted tumour affecting dogs worldwide, imposing a financial burden on dog owners. A stable culture cell line in continuous passages for >18 months has only been achieved once. The present study investigated a stable CTVT cell line isolated from a bitch and its potential as a vaccine.Material and MethodsA biopsy from a 2-year-old mongrel bitch with CTVT was obtained for histopathological confirmation and isolation of tumour cells. The isolated cells were cultured to passage 55 and characterised by flow cytometry, with karyotyping by GTG-banding and by PCR detection of myc S-2 and LINE AS1. The isolated CTVT cell line was also used as a preventive vaccine in a canine model.ResultsHistopathological analysis of the isolated tumour cells revealed typical CTVT characteristics. Constant proliferation and stable morphological characteristics were observed during culture. Phenotypic analysis determined the expression of HLA-DR+, CD5.1+, CD14+, CD45+, CD83+, CD163+, and Ly-6G-Ly-6C+. GTG-banding revealed a mean of 57 chromosomes in the karyotype with several complex chromosomal rearrangements. LINE-c-myc insertion in the isolated CTVT cell line at 550 bp was not detected. However, a 340-bp band was amplified. Isolated CTVT cell line inoculation at a concentration of 1×108 did not induce tumour growth in bitches, nor did a challenge with primary CTVT cells.ConclusionThe present study successfully identified and isolated a stable CTVT cell line that may be useful in CTVT prevention.

In vitro Effect of Recombinant Feline Interferon-Ω (rFeIFN-Ω) on the Primary CanineTransmissible Venereal Tumor Culture.

Background: Interferons (IFNs), signaling proteins produced by host cells, are secreted in response to pathogen activity as well as to tumor cells, and display antiviral, antiproliferative, and immunomodulatory effects. Recombinant feline interferon omega (rFeIFN-ω) has in vitro growth inhibition activities on various canine and feline tumor cell lines. Canine transmissible venereal tumor (CTVT) is used as an animal model for immunotherapy due to its specific growth phase. Previous studies have usually focused on the interaction between tumor infiltrating lymphocytes (TILs) and CTVT cells. However, the specific effects of rFeIFN-ω on CTVT cells remains poorly defined.Aims: The aims of this study, therefore, were to evaluate the in vitro effect of rFeIFN-ω on primary CTVT cells and to study the mRNA expression of apoptotic genes and drug resistance genes.Materials and Methods: Purified CTVT cells were treated with various concentrations of rFeIFN-ω and the viability of the cultured cells was ascertained at 24, 48, and 72 h post treatment (hpt) and a dose-response curve plotted. The mRNA expression of apoptotic (BAX and BCL-2) and drug resistance (ABCB1 and ABCG2) genes was performed by reverse transcription quantitative real-time PCR at 72 hpt.Results: rFeIFN-ω displayed an effect against CTVT cell viability, which decreasing viability in a dose-dependent manner within 72 hpt. The relative mRNA expression of BCL-2 was upregulated only at a rFeIFN-ω concentration of 104 IU/100 μl. However, higher concentrations of rFeIFN-ω gave a higher level of relative mRNA expression of ABCB1 transporter gene.Conclusion: This study provided the information of in vitro effect of rFeIFN-ω on CTVT cell viability in a dose dependent manner, as well as, the alteration of BCL-2 and ABCB1 gene expression after treatment. These results encourage future in vivo studies to evaluate the potential efficacy of this treatment in CTVT cases.

Immunotherapy for the treatment of canine transmissible venereal tumor based in dendritic cells pulsed with tumoral exosomes

Context: Exosomes secreted by tumor cells are a good source of cellular components that stimulate the immune response, such as alarmins (mRNA, tetraspanins (CD9, CD63, CD81), heat-shock proteins, major histocompatibility complex class I molecules) and tumor-associated antigens. These properties permit to pulsed dendritic cells in the immunotherapy for many cancers types. The aim of this study was to demonstrate the use of exosomes derived from canine transmissible venereal tumor (CTVT) as an antigen to pulsed dendritic cells and its administration in dogs with CTVT as treatment against this disease.Material and methods: From primary culture of CTVT cells the exosomes were isolated and characterized by scanning electron microscopy assay, dot blot and protein quantification. The monocytes of each patient were differentiated to dendritic cells (DC) and pulsed with CTVT exosomes (CTVTE). Phagocytosis, tumor size, populations of lymphocytes and IFN-c levels were evaluated.Results: The CTVTE showed a size around 90 nm. CD81, CD63, CD9 and Hsp70 were expressed. Monocytes showed an expression of 85.71% for CD14+, 12.3% for CD80+, 0.1% for CD83+ and 0.8% for DLA-II. In DC 5.1% for CD14+, 86.7% for CD80+, 90.1% for CD83+ and 92.6% for DLA-II and a phagocytosis of 63% was obtained by FITC Dextran test. No side effects were observed in the experimental groups with our therapy. Tumor regression was of 100% at the seventh week, as well as an increase in the level of IFN-γ (142 pg/ml), and CD4+ (28%) and CD8+ (34%) cell percentage.Discusion and conclusion: These results have shown that DC pulsed with tumor exosomes induce regression of the TVT in dogs.

Computerized Cytomorphometric and Cytomorphological Analysis of Canine Transmissible Venereal Tumours

Canine transmissible venereal tumour (CTVT) has three cytomorphological types: plasmacytic, lymphocytic and mixed type. Cytomorphometry, a method of computerized image analysis, has been used recently in veterinary medicine. This study compared the nuclear and cellular morphometric parameters (i.e. radius, diameter, perimeter and area) in different types of CTVT with other canine round cell tumours including lymphoma, mast cell tumour (MCT) and histiocytic tumour (HCT). We also evaluated the relationship between clinical information and the different CTVT cytomorphologic types. CTVT cells from 44 dogs revealed that the measured parameters were significantly different between different round cell tumours and among the CTVT cytomorphological types. CTVT had the largest cells, followed by HCTs, MCTs and lymphomas. The mixed type of CTVT had the largest nuclear and cellular size, followed by the plasmacytic and lymphocytic types. Lymphocytic CTVTs had less aggressive biological behaviour than the other types. Mixed type CTVTs were more likely to show malignant behaviour including metastasis and resistance to chemotherapy; however, there was no significant correlation between cytomorphological type of CTVT and response to chemotherapy.

Cytomorphological, histopathological and immunohistochemical observations on the histiocytic origin of canine transmissible venereal tumour

The cytogenic origin of canine transmissible venereal tumor (CTVT) still remains unknown. Resulting from paucity of information on the histiocytic phenotypic features and behaviours of CTVT, this study was undertaken to show some uncommon cytomorphological features of CTVT and its immunoreactivity with S-100 protein. Nine cases of CTVT were investigated (6 females and 3 males) using nine fine needle aspirates for cytology and four biopsy samples for histopathology and immunohistochemistry. Cytology revealed CTVT cells with pale basophilic, fine granular cytoplasm and distinct intra-nuclear (3/9) and intra-cytoplasmic (9/9) vacuolations. Erythrophagocytosis by a giant binucleated CTVT cell in one dog and nuclear budding in 2 dogs were observed. Histopathology showed loose sheets and cords of uniformly round to ovoid cells with slight indistinct eosinophilic cytoplasm and a tendency towards glandular as well as syncytial formation. Nuclei were large and round with a single centrally placed nucleolus. The mitotic index was high. Immunohistochemistry of the four biopsy samples revealed negative immunoreactivity to pan-cytokeratin, actin and desmin but positive immunoreactivity with vimetin and S-100 proteins. This is the first report in which CTVT showed erythrophagocytosis, nuclear budding and positive immunoreactivity to S-100 protein. Based on these cytomorphological and immunohistochemical features, we conclude that CTVT is of histiocytic/dendritic origin.Keywords: Cytology, histopathology, Histiocytic origin, immunohistochemistry, Transmissible Venereal Tumour

Does the tumour microenvironment alter tumorigenesis and clinical response in transmissible venereal tumour in dogs?

The canine transmissible venereal tumour (CTVT) is a transmissible cancer that is spread naturally between dogs, with the ability to develop and evade the immune system, despite strict immune surveillance of the host. Furthermore, molecular signalling between cells of the immune system and the tumour microenvironment appear to influence the behaviour and development of the tumour. Thus, this study aimed to quantify the expression of genes related to the immune system such as IL-6, IFN-γ, and TGF-β, as well as angiogenic factors (VEGF, CXCR4), in CTVT cells in vivo and in vitro (primary culture), correlating with the clinical response of the animals treated with vincristine. As expected, the most prevalent subtype was plasmacytoid cells, although lymphocytic cells were also found, indicating the possibility of polyclonality. When we compared the gene expressions of IFN-γ and IL-6, we mostly found low expression, concluding that MHC expression was probably not occurring in tumour cells, and no activation of immune cells to eliminate the tumour. The TGF-β gene was normal in the majority of animals but demonstrated decreased expression in vincristine resistant animals, leading to the hypothesis that the concentration of tumour-derived TGF-β was affecting and even suppressing the real TGF-β expression, favouring tumour proliferation and progression in these cases. VEGF expression was extremely high, demonstrating its angiogenic role in tumour growth, while CXCR4 was decreased, possibly because of CTVT's low metastatic potential. Thus, we concluded that the tumour microenvironment, together with the immune system of the host, influences CTVT, presumably altering its tumorigenesis and the animal's clinical response to treatment.

Cell-based polymerase chain reaction for canine transmissible venereal tumor (CTVT) diagnosis.

Canine transmissible venereal tumor (CTVT) is the only naturally contagious tumor that is transmitted during coitus or social behaviors. Based on the tumor’s location, the diagnosis of genital TVT (GTVT) is comparably easier than those in the extragenital area (ETVT) that are more easily incorrectly diagnosed. Fortunately, CTVT cells contain a specific long interspersed nuclear elements (LINE), inserted upstream of the myc gene, allowing a diagnostic polymerase chain reaction (PCR) based detection assay. The objectives of this study were aimed to improve the diagnostic accuracy by applying the diagnostic LINE1-c-myc PCR assay and fine needle aspiration (FNA) collection in direct comparison with standard cytological and histopathological analyses. Seventy-four dogs, comprised of 41 and 31 dogs with tumor masses at their external genitalia and extragenital areas (e.g. skin and nasal cavity), respectively, were included in this study. The signalment of these 65 dogs and clinical history of 20 client-owned dogs were collected. Samples were taken by biopsy for both histopathological examination and FNA for cytological examination and diagnostic PCR. The PCR products from 10 apparently CTVT samples were purified and sequenced. Sixty-one CTVT cases were diagnosed by cytological and histological analyses, but 65 were positive by the PCR assay. Overall, the PCR assay improved the accuracy of diagnostic CTVT results, especially for the more difficult ETVT tumors. Moreover, this PCR-based approach can facilitate the decision as to discontinue chemotherapy by discrimination between residual tumor cell masses and fibrotic tissue.

IMMUNE CHARACTERIZATION OF PERIPHERAL BLOOD MONONUCLEAR CELLS OF THE DOGS RESTORED FROM INOCULATION OF CANINE TRANSMISSIBLE VENEREAL TUMOR CELLS

Canine transmissible venereal tumor (CTVT) is a tumor which can be transmitted naturally through mucosa contact between dogs. When CTVT cells are experimentally inoculated on dogs, they will grow rapidly (Progressive/P phase) and then regress (Regressive/R phase) spontaneously. Therefore, it is a good model to investigate the interactions between tumor cells and host immune system. Previous studies have shown that CTVT cells cannot grow in the dogs restored from CTVT inoculation. To investigate the possible mechanism, this study characterized the CTVT-specific immune response of the peripheral blood mononuclear cells (PBMCs) which isolated from the blood of "naïve" or "CTVT-restored" dogs. The phenotypes (CD3, CD4, CD8, or CD21) of PBMCs were examined by flowcytometry. In response to CTVT stimulation, proliferation, IFN-γ secretion, and cytotoxicity of PBMCs were analyzed. Expression level of proinflammatory cytokines (TNF-α, IL-1β, IL-6, TGF-β), Th1 (IL-2, IFN-γ), and Th2 cytokines (IL-4, IL-10) and cytotoxic proteins (Granzyme B, Perforin) in PBMCs was also evaluated by real-time RT-PCR. The results indicated that there is no significant difference between two groups on lymphocyte phenotypes. Proliferation, IFN-γ secretion, and cytotoxicity of PBMCs between two groups showed no significant difference, except naïve PBMCs present higher proliferation after Con-A stimulation. Production of IL-1β and IL-6 in naïve PBMCs was higher than that in CTVT-restored PBMCs (p < 0.05). The production difference of IL-1β and IL-6 between two groups might be the reason why CTVT cannot be reinoculated on CTVT-restored dog. However, further investigations are necessary to explore the exact role of these cytokines in CTVT growth.

Overexpression of chemokine ligand 7 is associated with the progression of canine transmissible venereal tumor

BackgroundChemokines play multiple roles in the development and progression in a variety of tumors. Chemokine (C-X-C motif) ligand 7 (CXCL7) has been found associated with pro-inflammatory responses, but its role in cancer growth remains unclear. Our previous study showed that R phase tumor infiltrating lymphocytes (TILs) produced large amounts of interleukin (IL)-6 which antagonized transforming growth factor (TGF)-β derived from CTVT to diminish the immune-suppressive microenvironment. Now we intend to determine the expression pattern of CXCL7 and the role of IL-6/TGF-β in CXCL7 induction during spontaneous progressive (P) and regressive (R) phases in canine transmissible venereal tumor (CTVT).ResultsWe have demonstrated that CXCL7 expressed at high level in P phase and down-regulated in R phase by western blot and real-time PCR. This suggested that CXCL7 expression was negatively correlated with the tumor growth. Co-culturing TILs with CTVT cells was found to reduce CXCL7 expression, while adding IL-6 blocking antibody reversed it. Moreover, in P phase CTVT, while IL-1β and TGF-β had no obvious effect on CXCL7 expression, IL-6 was found significantly to reduce CXCL7 expression in a dose-dependent manner. The mRNA expression results of CXCL7 receptor, CXCR2, further confirmed the effects of IL-6 concentration on the CXCL7 expression.ConclusionCXCL7 overexpression might be associated with the progressive growth of CTVT. The results shown here also suggest the role of CXCL7 in cancer development and the potential as the anti-cancer therapeutic target.

Clinicopathological study of canine transmissible venereal tumour in leishmaniotic dogs

Canine transmissible venereal tumour is occasionally observed in leishmaniotic dogs, and Leishmania amastigotes can be harboured in canine transmissible venereal tumour cells. The aim of this paper was to investigate the clinicopathological significance of the association of both diseases. Nineteen dogs affected by canine transmissible venereal tumour and canine leishmaniasis were studied retrospectively. In these dogs, the tumour manifested a large size and often aggressive behaviour (42%) and no predictive sign of spontaneous regression was observed. Sporadic Leishmania amastigotes were found within the canine transmissible venereal tumour in three cases, probably transported by infected macrophages often infiltrating the tumour. A high Leishmania parasitisation of canine transmissible venereal tumour was observed in two other cases and verified by immunohistochemistry. Canine transmissible venereal tumour is a tumour of the dog able to harbour a large number of Leishmania parasites. Alternatively, the systemic disease (canine leishmaniasis) may lower the immune defence against malignancy (canine transmissible venereal tumour).

Detection of the tumour suppressor gene TP53 and expression of p53, Bcl‐2 and p63 proteins in canine transmissible venereal tumour

Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.

Trans-rectal Ultrasound–coupled Spectral Optical Tomography of Total Hemoglobin Concentration Enhances Assessment of the Laterality and Progression of a Transmissible Venereal Tumor in Canine Prostate

To evaluate whether trans-rectal spectral optical tomography of total hemoglobin concentration (HbT) can image longitudinal and lateral developments of a canine transmissible venereal tumor (TVT) in a canine prostate. A near-infrared (NIR) applicator was integrated with a trans-rectal ultrasound (TRUS) transducer to perform ultrasound (US)-coupled optical tomography of the canine prostate. Spectral detection at 785 and 830 nm enabled quantitation of HbT. Canine TVT cells were injected into the right lobe of a dog's prostate gland. Longitudinal imaging assessment of the post-injection prostate was performed by coupled US/NIR imaging over a 45-day duration. By day 7, NIR indicated TVT infiltration in the noninjected left prostatic lobe with the gray-scale US indistinct. By day 31, both NIR and gray-scale US revealed more widespread TVT involvement in the left than in the right lobe, as well as an extensive TVT mass in the caudal aspect of the gland, of which the peak HbT increased 3-fold and the mass volume grew exponentially over the 45-day duration. Increased blood supply to the mass was also observed on Doppler US. TRUS-coupled spectral optical tomography enhances assessment of the laterality and progression of prostate tumor compared with using gray-scale and Doppler TRUS.

Deterioration and Metastasis of a Canine Transmissible Venereal Tumor in a Dog Associated with a CPV-2

Virus infection has been documented to be associated with tumor progression. A 2- year-old female beagle was inoculated in the dorsal flank with viable canine transmissible venereal tumor (CTVT) cells for routine laboratory tumor passage. Tumor masses were palpable 2 weeks after inoculation, then grew progressively, and the tumor size remained stable. Parvovirus (CPV-2) infection was diagnosed by RT-PCR at week 11 after tumor inoculation and, 2 weeks later, the primary tumors began to grow again. Besides, palpable tumor masses were found scattered around the primary tumor sites. Because of the deteriorating condition of the animal and the dramatic increase in tumor size and number, the dog was euthanized and venous blood collected for a complete blood count and serum chemistry analysis. Postmortem examination revealed a disseminated CTVT involving the subcutaneous tissue, spleen, liver, lung, and multiple lymph nodes, which was confirmed by histopathological examination and the presence of LINE gene insertion in the 5 end of c-myc gene, a characteristic of CTVT cells, shown using RT-PCR. Lymphoid cell depletion in the spleen and lymph nodes, and focal micro-necrosis of the cryptic epithelium of the small intestine were also seen. Blood analysis showed decreased levels of albumin and total blood protein and an extremely low lymphocyte count. Here, we report a rare case in which a stable CTVT became a devastating metastatic CTVT after a CPV-2 infection. The immunosuppressive effects of CPV-2 might account for the breakdown in the symbiotic balance between the CTVT and host that caused the stable tumor to develop into a metastatic cancer. The correlation between parvovirus infection and aggravated CTVT progression is worthy of further investigation.

Abstract B58: Gene expression study on a highly aggressive canine transmissible venereal tumor of a NOD/SCID mice model

Abstract We have set up a xenograft NOD/SCID mice model bearing canine transmissible venereal tumor (CTVT) (designated as MCTVT). In addition to the similarity of the tumor cells between CTVT and MCTVT, the insertion of the specific gene fragment in the 5′ end of c-myc characteristic for a CTVT cell was also confirmed in MCTVT. However, as inoculating back to dogs, the MCTVT showed a much more aggressive growth and much higher metastatic frequency than a common CTVT. The goal of this research was to explore the gene expression differences between CTVT and MCTVT, and tried to delineate the mechanisms of their growth pattern differences. The differential gene expressions between the 2 tumors were studied using Affymetrix. Studied by hierarchical analysis and filtering the differential expressed genes by 2-fold method, we have found 105 genes significantly up-regulated and 37 down-regulated in MCTVT comparing to CTVT. Many of these genes were previously published to be involved in the areas such as malignant candidate genes, important biological processes, tumor development and immune modulation. We then used Real-Time RT-PCR to study the top 30 up-regulated genes. Three genes (APOC1, MMP1 and KMO) were further confirmed that were significantly up-regulated in MCTVT. The three genes were then tested in 22 spontaneous canine mammary gland tumors (CMGT) using Real-Time RT-PCR, the results indicated that two out of the three up-regulated genes discriminated malignant from benign CMGT and their gene expression was significantly higher in grades 4 and 5 malignant CMGT than those from lower grade tumors. We are currently studying the activities of KMO in association with the tumor cell growth. This study presented the novel candidate gene (KMO) that might associate with the malignant growth of tumor cells and has potential to be used as a biological marker to differentiate degrees of malignancy. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B58.

Electroporation‐mediated IL‐12 gene therapy in a transplantable canine cancer model

Interleukin-12 (IL-12) is effective in treating many types of rodent tumors, but has been unsuccessful in most human clinical trials, suggesting that animal models of more clinical relevance are required for evaluating human cancer immunotherapy. Herein, we report on the effectiveness of gene therapy with plasmid encoding human IL-12 (pIL-12) through in vivo electroporation in the treatment of beagles with a canine tumor, the canine transmissible venereal tumor (CTVT). The optimal electroporation conditions for gene transfer into CTVTs were tested by luciferase activity and determined to be a voltage of 200 V and duration of 50 msec, with the number of shocks set at 10 pulses, and the use of an electrode with 2 needles. Under these conditions, intratumoral administration of as little as 0.1 mg pIL-12 followed by electroporation significantly inhibited the growth of well-established tumors and eventually led to complete tumor regression. Furthermore, local pIL-12 treatment also induced a strong systemic effect that prevented new tumor growth and cured established tumors at distant locations. Intratumoral administration of pIL-12 greatly elevated the IL-12 level in the tumor masses, but produced only a trace amount in the serum. A high level of IFN-gamma mRNA was also detected in the treated tumor masses. pIL-12 gene therapy attracted significantly more lymphocytes infiltrating the tumors, including CD4(+) and CD8(+) T cells, and the surface expression of MHC I and MHC II molecules on CTVT cells was greatly increased after pIL-12 therapy. This treatment also induced apoptosis of the tumor cells as detected by Annexin V. More importantly, delivery of pIL-12 with intratumoral electroporation did not result in any detectable toxicity in the dogs. We conclude that intratumoral electroporation of the pIL-12 gene could cause profound immunologic host responses and efficiently treat CTVT in beagle dogs. The results also indicate that CTVT is an excellent large animal cancer model for testing immunogene therapies mediated by electroporation.

Effects of immunotherapy of IL-6 and IL-15 plasmids on transmissible venereal tumor in beagles

Canine transmissible venereal tumor (CTVT) is a tumor with low MHC antigen expression and is an ideal tumor model for studying the interactions between host immunity and cancer cells. CTVTs produce high concentrations of TGF-β to hamper the host immune responses and facilitate their growth progression. However, during the later stages of tumor progression, tumor-infiltrating lymphocytes secrete IL-6. This cytokine antagonizes TGF-β and restores the IFN-γ activities in promoting MHC antigen expression, and the NK cytotoxicity that has been repressed by TGF-β is also activated. In this study, we applied combinatory treatment of IL-6 plasmid and IL-15 plasmid ( pIL-6/pIL-15) to CTVT-bearing beagles. IL-6 was used as an anti-TGF-β cytokine; IL-15 was used to promote NK- and CTVT-specific cytotoxicity. After intratumoral pIL-6/pIL-15 delivery mediated by electroporation, MHC antigen expression on CTVT cells was dramatically increased from in less than 5.9% to up to 34% of the tumor cells. The proportion of CD8 + T cells infiltrating the tumor was also significantly elevated from 6.96 ± 0.23% to 21.63 ± 5.40%. In addition, the tumor-specific cytotoxicity was enhanced along with a marked increase in tumor-specific IFN-γ-producing cells. These immune responses are believed to be the important forces driving the tumor towards regression. The results indicate that pIL-6/pIL-15 combinatory immunotherapy may facilitate a promising and effective means of treating tumors.

Review Of Canine Transmissible Venereal Tumour (TVT) In Dogs

Transmissible Venereal tumour (TVT) also known as stickers sarcoma (Sticker, 1904) transmissible venereal tumour, venereal granuloma, canine condyloma or infectious sarcoma (Madewell and Theilen, 1987; Nielsen and Kennedy, 1990 and Roger, 1997). Canine transmissible venereal Sarcoma (TVS) (Yang, 1988) is the most notable among the three recognized transmissible tumours in domestic animals. It is a naturally occurring contagious round – cell neoplasia of dogs. It is a well characterized sexually transmitted neoplasm commonly affecting the external genitalia in dogs (Brown et al., 1980; Richardson, 1981 and Cohen, 1985). TVT is primarily located in the mucous membrane of the external genitalia of either sex. This tumour is unique in oncology, because it was the first tumour to be transmitted experimentally. This was achieved by the Russian Veterinarian, Nowinsky in 1876 according to Epstein and Bennett (1974) and Yang, et al (1991). Canine transmissible venereal tumour (CTVT) is virtually the only tumour transmitted by cell transplantation under natural conditions (Yang, 1988). Viable CTVT cells can be transmitted to susceptible animals and transfer, in a given dog, to other mucous membranes (e.g. the oral cavity) spontaneously through injured skin, or mucosa during coitus, licking, fighting/ biting, sniffing or scratching (Stubbs and Furth, 1934). In addition to the genital system (Higgins, 1966), CTVT can occur any where on the body including the skin, oral and nasal cavities, conjunctiva and even the anal mucosa (Neilsen and Kennedy, 1990, Calvert, 1984; Harmir, 1986; Amber and Adeyanju, 1986; Adeyanju et al., 1987 and Betamuzi and Bittegeko, 1991) gluteal region, maxilla gingival, dorsal skin of thorax (Gurel et al., 2002). TVT can be experimentally transmitted to wolves, Jackals, Coyotes and red foxes (Madewell and Theilen, 1987; Neilsen and Kennedy, 1990 and Roger, 1996).Recent research in Australia which revealed the existence of a newly emerged tumour in Tasmanian Devils (Devils are an endangered marsupial species) that appears to be caused by transmissible cancer cells, in this case by biting (Claudio et al., 2006). The tumour is recognized in all dog breeds in various part of the world, especially in tropical and sub tropical zones (Gurel, et al., 2002). Due to the nature of transmission by sexual contact, free roaming, sexually active dogs in region with poorly enforced leash laws are at greatest risk for contrasting this disease. Although, CTVT does not often metastasized, it has been reported in the regional lymph nodes, liver, pancreas, spleen, lung, kidney, ocular cavity and brain (Mcleod and Lewis, 1972; Oduye et al., 1973; Adams and Slaughter, 1970; Prier and Brodey, 1963, Yang, 1987, Perez et al., 1998, Ferreira et al., 2000). CTVT transplantability in animal model showed that tumour cell differentiation and, in turn, spontaneous regression could be induced. (Yang, 1988 and Angell, 2000). Nigerian Veterinary Journal Vol. 28 (1) 2007 pp. 54-70