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.

Autologous canine immunotherapy: short-time generated dendritic cells loaded with canine transmissible venereal tumor-whole lysate.

OBJECTIVE: The aim of the present study was to evaluate the therapeutic potential of autologous DCs loaded with whole tumor cell lysate of CTVT generated under a simplified and rapid procedure in vitro production process, in a vulvar submucosal model of CTVT in dogs. MATERIALS AND METHODS: We generated a model of intravulvar CTVT in dogs. A CTVT lysate antigen was prepared according to the method of 1-butanol and after administered with complete Freund's adjuvant via subcutaneous in female healthy dogs and challenge with CTVT cells to corroborate the immunogenicity. Short-time generated dendritic cell pulsed with CTVT whole-lysate was performed, and analyzed by FITC-dextran uptake assay and characterized using anti-canine monoclonal antibodies CD14, CD80, CD83, and DLAII by flow cytometry. Dendritic cell therapy was administered in a frequency of three times every 2 weeks when the CTVT had 4 months of growth and 89 ± 5 cm diameter. The CD3+, CD4+ and CD8+ lymphocytes were determined by flow cytometry, and IFN-γ by ELISA assay. RESULTS AND DISCUSSION: The administration of CTVT whole-lysate resulted in tumor prevention. The short-time generated dendritic cell pulsed with CTVT whole-lysate administration resulted in an efficient reduction and elimination of CTVT, probably due to the increase in lymphocyte populations (CD3+, CD4+, and CD8+), IFN-γ production and tumor infiltrating lymphocytes. CONCLUSION: In conclusion, this study demonstrates the efficacy of immunotherapy based in short-time generated dendritic cell pulsed with CTVT whole-lysate for the treatment of CTVT, and offer veterinary oncologists new alternative therapies to treat this and another malignancy.

Immunology of naturally transmissible tumours.

Naturally transmissible tumours can emerge when a tumour cell gains the ability to pass as an infectious allograft between individuals. The ability of these tumours to colonize a new host and to cross histocompatibility barriers contradicts our understanding of the vertebrate immune response to allografts. Two naturally occurring contagious cancers are currently active in the animal kingdom, canine transmissible venereal tumour (CTVT), which spreads among dogs, and devil facial tumour disease (DFTD), among Tasmanian devils. CTVT are generally not fatal as a tumour-specific host immune response controls or clears the tumours after transmission and a period of growth. In contrast, the growth of DFTD tumours is not controlled by the Tasmanian devil's immune system and the disease causes close to 100% mortality, severely impacting the devil population. To avoid the immune response of the host both DFTD and CTVT use a variety of immune escape strategies that have similarities to many single organism tumours, including MHC loss and the expression of immunosuppressive cytokines. However, both tumours appear to have a complex interaction with the immune system of their respective host, which has evolved over the relatively long life of these tumours. The Tasmanian devil is struggling to survive with the burden of this disease and it is only with an understanding of how DFTD passes between individuals that a vaccine might be developed. Further, an understanding of how these tumours achieve natural transmissibility should provide insights into general mechanisms of immune escape that emerge during tumour evolution.

Contagious cancer: lessons from the devil and the dog.

Cancer is generally defined as uncontrollable growth of cells caused by genetic aberrations and/or environmental factors. Yet contagious cancers also occur. The recent emergence of a contagious cancer in Tasmanian devils has reignited interest in transmissible cancers. Two naturally occurring transmissible cancers are known: devil facial tumour disease and canine transmissible venereal tumour. Both cancers evolved once and have then been transmitted from one individual to another as clonal cell lines. The dog cancer is ancient; having evolved more than 6,000 years ago, while the devil disease was first seen in 1996. In this review I will compare and contrast the two diseases focusing on the life histories of the clonal cell lines, their evolutionary trajectories and the mechanisms by which they have achieved immune tolerance. A greater understanding of these contagious cancers will provide unique insights into the role of the immune system in shaping tumour evolution and may uncover novel approaches for treating human cancer.

Extensive conservation of genomic imbalances in canine transmissible venereal tumors (CTVT) detected by microarray-based CGH analysis.

Canine transmissible venereal tumor (CTVT) is an intriguing cancer that is transmitted naturally as an allograft by transplantation of viable tumor cells from affected to susceptible dogs. At least initially, the tumor is able to evade the host's immune response; thus, CTVT has potential to provide novel insights into tumor immunobiology. The nature of CTVT as a "contagious" cancer, originating from a common ancestral source of infection, has been demonstrated previously by a series of studies comparing geographically distinct tumors at the molecular level. While these studies have revealed that apparently unrelated tumors share a striking degree of karyotypic conservation, technological restraints have limited the ability to investigate the chromosome composition of CTVTs in any detail. We present characterization of a strategically selected panel of CTVT cases using microarray-based comparative genomic hybridization analysis at ~one-megabase resolution. These data show for the first time that the tumor presents with an extensive range of non-random chromosome copy number aberrations that are distributed widely throughout the dog genome. The majority of abnormalities detected were imbalances of small subchromosomal regions, often involving centromeric and telomeric sequences. All cases also showed the sex chromosome complement XO. There was remarkable conservation in the cytogenetic profiles of the tumors analyzed, with only minor variation observed between different cases. These data suggest that the CTVT genome demonstrates a vast degree of both structural and numerical reorganization that is maintained during transmission among the domestic dog population.

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.

In vitro cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity to the transmissible venereal tumor of the dog.

Cell-mediated immunity to the transmissible venereal tumor (TVT) of the dog was studied by use of a 51Cr release cytotoxicity assay. Peripheral blood lymphocytes (PBL) of dogs in which the TVT had regressed were shown to be cytotoxic to the tumor cells in contrast to PBL of normal dogs and animals during progressive tumor growth, which were not cytotoxic. In addition, sera of dogs in which the TVT had regressed could be demonstrated to mediate antibody-dependent cellular cytotoxicity (ADCC) with normal dog lymphocytes or cytotoxic PBL as effector cells. With cytotoxic lymphocytes, the ADCC effect could be observed in addition to the direct cytotoxic effect.

Identification and physicochemical characterization of a tumor-associated antigen from canine transmissible venereal sarcoma.

A tumor antigen associated wtih canine transmissible venereal sarcoma (CTVS) has been identified and partially characterized. The antigen was demonstrated in 3-M KCI and saline extracts of washed CTVS cells. Rabbit anti-CTVS antisera absorbed wih glutaraldehyde cross-linked normal dog serum and pooled homogenates of canine spleen, thymus, lymph node, and liver were used to identify the antigen. The specificity of the rabbit anti-CTVS antiserum for the CTVS antigen was established by use of immunodiffusion, two-dimensional electrophoresis, and the enzyme-linked immunosorbent assay (ELISA). Extracted CTVS antigen was fractionated by Sephadex G-200 chromatography, and the antigen factions were identified with the use of absorbed rabbit anti-CTVS antiserum and the ELISA technique. Antigen activity was detected in samples with estimated molecular weights greater than 70,000 daltons. Antigen fractions reacted with absorbed anit-CTVS antiserum to form a single precipitation band in immunodiffusion studies. Extraction of CTVS antigen with 3 M KCl and saline in the presence of a protease inhibitor did not significantly alter the antigen activity or molecular weight of the CVTS antigen. Cytoplasmic and nucleolar fluorescence were observed in an indirect immunofluorescence test with the use of acetone-fixed CTVS cells and absorbed anti-CTVS antiserum. The CTVs antigen activity was greatly reduced by trypsin digestion, by incubation for 1 hour at 65 degrees C, and by exposure to pH 2.8, 4.0, and 11.0 for 1 hour each.

Lack of beta 2-microglobulin on the surface of canine transmissible venereal tumor cells.

beta 2-Microglobulin (beta 2m) expression on the cell surface of the naturally occurring, allotransplantable canine transmissible venereal tumor (TVT) was investigated by use of indirect membrane immunofluorescence and radioimmunoassay. Two cell populations were identified in animal-derived, collagenase-disaggregated TVT cell suspensions. About 80% of the cells lacked surface beta 2m expression, whereas about 20% of the cells strongly reacted with anti-dog beta 2m serum. With the use of a cell separation technique, beta 2m-negative cells were demonstrated to carry TVT markers on their surface, whereas the beta 2m-positive cells did not express the tumor markers. The beta 2m-positive cells seemed, therefore, to be tumor-infiltrating host cells. These findings were supported by fluorescence staining studies of frozen sections of the TVT. The lack of beta 2m expression on the surface of TVT cells might explain the allotransplantability of this neoplasm, since beta 2m expression on the cell surface appeared to be obligatory for the expression of class I major histocompatibility complex antigens.

Immunoelectron microscopic localization of a tumor-associated antigen (TAA) in the canine transmissible venereal sarcoma by anti-TAA-antiferritin hybrid antibodies.

Immunoferritin and immunoperoxidase reagents were used to localize a tumor-associated antigen (TAA) of the canine transmissible venereal sarcoma (CTVS). Round tumor cells from the CTVS at different stages of growth, i.e., progressive, steady state, and regressing, had TAA diffusely distributed throughout the cytoplasm. In general, TAA was not found on the plasma membrane, within the nucleus, between inner and outer membranes, in cytoplasmic vacuoles, or specifically with any part of the cytocavitary system. Transitional tumor cells, which are intermediate cell types between round cells and spindle-shaped cells and which appear in the tumors at steady state and regressing stages, contained less TAA in their cytoplasm than did the round cell type. The microvilli of tumor cells also contained TAA, suggesting that, in addition to whole cell lysis, shedding of all or parts of these processes may be a mechanism of TAA release as evidenced by the presence of antigenic activity in the extracellular material.

Isolation and characterization of canine venereal tumor-associated inhibitory and blocking factors.

Spontaneous regression of the canine venereal tumor is associated with the production of a serum factor which inhibits in vitro tumor colony-forming units in agar. Logarithmic or persistent tumor growth, on the other hand, is characterized by a serum factor which protects cells against in vitro inhibition (blocking factor). These factors have been characterized by immunochemical methods. Whole regressor and blocking sera were fractionated by Sephadex G-200 filtration and immunoabsorption with rabbit antiserum specific for canine immunoglobulin G2a. Fractions were characterized by immunoelectrophoresis, radial immunodiffusion, and disc gel electrophoresis. In vitro inhibitory and blocking activity of the whole serum was accounted for by the purified immunoglobulin G2a. Blocking activity was also found in protein eluted from logarithmically growing tumors. Preparative polyacrylamide electrophoresis revealed five major fractions with blocking activity only in the immunoglobulin G fraction. Tumor eluates and immunoglobulin G isolated from serially removed tumors demonstrated with the clinical course of the tumor. Using ultrafiltration and sodium dodecyl sulfate electrophoresis of tumor-associated immunoglobulin G at low pH, it was not possible to identify an antigen complexed to the blocking antibody.

Plasmapheresis as immunotherapeutic modality in the treatment of the canine venereal tumor.

Plasmapheresis was evaluated as a treatment modality for the transmissible venereal tumor (TVT) of the dog. The TVT is a unique tumor because of its capability for transplantation as a homograft between untreated randomly bred dogs and is characterized by the presence of dog leukocyte antigen (DLA) determinants on its tumor cells and reactivity in the mixed leukocyte tumor cell culture (MLTC). In the progressing phase, high titers of blocking antibodies measurable in the MLTC were noted. The purpose of this study was to evaluate the influence of plasmapheresis on the growth of established TVT and to correlate tumor response with the removal of blocking factors. Six pairs of DLA-identical dogs were used as experimental and control animals. Plasmapheresis was performed by discontinuous centrifugation using the Hemonetics model 30. An average of 1100 +/- 120 ml of plasma was exchanged on consecutive days during the third week after tumor injection. Consistently lower growth rates were observed in 2 experimental dogs, but there was no early rejection. Sera taken at day 14 of tumor growth contained significant blocking activity in all 12 dogs. Sera obtained post-plasmapheresis showed a decrease of blocking activity in all 6 experimental dogs, P less than 0.001. The TVT appears to be a suitable model for preclinical studies of plasmapheresis alone and in combination with other modalities.

Immunophenotype of the canine transmissible venereal tumour.

The canine transmissible venereal tumour is a naturally occurring contagious round-cell neoplasia which is primarily located in the mucous membrane of the external genitalia in dogs of either sex. In order to specify the controversial cytogenetic origin of this round-cell tumour, 14 cases of canine transmissible venereal tumour, formalin- or Bouin-fixed and paraffin-embedded, were subjected to extensive immunophenotypic analysis using reagents specific to a variety of cytoplasmic or surface antigens: lysozyme, ACM1 antigen, vimentin, neuron-specific enolase, glial fibrillary acidic protein, desmin, alpha smooth muscle actin, CD3, IgG, kappa and lambda light chains, and keratin. Lysozyme immunoreactivity was detected in all cases, ACM1 antigen in 11 of 14, neuron-specific enolase in 11 of 14, vimentin in 10 of 14, glial fibrillary acidic protein in 4 of 14 and desmin in 1 of 14. All the sections were negative to keratins, alpha smooth muscle actin and CD3, whereas in five cases, perivascular tumour cells contained Ig G, kappa and lambda light chains. The immunoreactivity to lysozyme and ACM1 antigen supports the hypothesis of a histiocytic immunophenotype for the canine transmissible venereal tumour.

Lack of MHC expression and retention of ultrastructural characteristics by xenograft transmissible venereal tumor cells in SCID mice.

Canine transmissible venereal tumor (CTVT) is primarily a tumor of adult dogs with a high incidence of spontaneous regression. We recently reported a xenograft model of CTVT (XTVT) in NOD/SCID mice. XTVT cells retain cytological and histological features of CTVT as well as characteristic rearranged LINE/c-MYC junction [Am. J. Vet. Res. 62 (2001) 907]. In this paper, we demonstrate that XTVT cells maintain ultrastructural characteristics of CTVT and do not express MHC classes I and II molecules.

Canine transmissible venereal tumor cell depletion of B lymphocytes: molecule(s) specifically toxic for B cells.

Canine transmissible venereal tumor (CTVT) is an excellent model for investigating the interaction between host immunity and tumor growth. Although CTVT is an allograft, initially the host immune system is unable to destroy the tumor cells, and the tumor grows progressively for about 4-6 months (P phase). After a short stable phase, the tumor undergoes regression (R phase). In this study, CTVT inoculation significantly reduced the proportion of B lymphocytes among all peripheral blood lymphocytes (PBL), but the proportion of B lymphocytes returned to normal after complete removal of CTVT. Following CTVT inoculation, immunoglobulin concentrations decreased gradually, coincident with B lymphocyte decline. Furthermore, CTVT secreted a soluble, heat- and protease K-sensitive cytotoxic molecule(s) that destroyed peripheral blood B lymphocytes (PBBL) but spared other types of immune cells regardless of whether mitogens, such as IL-2 or Con A, were present. The decrease in the proportion and viability of PBBL was caused by a cytotoxic molecule(s) that induced apoptosis. The molecular weight of the CTVT-derived cytotoxic molecule(s) was 30-100kDa. Human, domestic cat, horse and mouse B cells were also sensitive to the substance.

Effect of tumor infiltrating lymphocytes on the expression of MHC molecules in canine transmissible venereal tumor cells.

Canine transmissible venereal tumor (CTVT) can be allo-transplanted across major histocompatibility complex barriers. The expression of MHC molecules is usually low in the progression (P) stage and then greatly increases during tumor regression (R). We investigated the effects of tumor infiltrating lymphocytes (TIL) on the expression of MHC molecules of CTVT cells. Isolated, viable CTVT cells were inoculated at each of 12 sites (1 x 10(8) CTVT cells per site) on the back of six, mixed-breed dogs. Tumor masses were collected every 2-3 weeks and prepared for histopathologic, immunocytochemistry, flow cytometry and immunoblotting studies. The level of MHC expression on tumor cells from different stages of growth was measured. Initially, expression of MHC I and II molecules in P phase CTVT was low. Twelve weeks post-inoculation (PI), expression increased dramatically and it continued to increase during R phase. Tumor growth slowed after 12 weeks PI and tumors entered R phase around 17 weeks PI. We hypothesize that CTVT evades host immunosurveillance and grows progressively for 12 weeks, when it becomes vulnerable and subject to the host's anti-tumor immune responses. We further demonstrated that R phase, but not P phase, TIL were closely associated with the over-expression of MHC I and II molecules by CTVT cells. The number and proportion of TIL were higher in R phase tumors. Supernatants, from R phase co-cultures (CTVT+TIL) and TIL only, promoted MHC I and II expression on P phase CTVT cells. After culturing alone for 1 month, expression of MHC classes I and II molecules in R phase CTVT cells decreased to the level of P phase CTVT cells. However, the above-mentioned supernatants restored their expression of MHC I and II molecules. In contrast, supernatants from P phase TIL or CTVT cells increased expression slightly or had no effect. Therefore, TIL, not CTVT cells, produce the effective substance (s) to promote the expression of MHC molecules by the tumor cells. Heat treated supernatant was unable to promote the expression of MHC I and II molecules by CTVT cells. In conclusion, TIL isolated from R phase CTVT secreted a heat-sensitive, soluble substance(s) that triggered over-expression of MHC I and II after 12 weeks PI. This caused the tumor to enter R phase and helped stop CTVT growth. Our findings will facilitate the understanding and further investigation of the mechanisms that initiate host immune surveillance against tumors.

Immunohistochemical study of the local inflammatory infiltrate in spontaneous canine transmissible venereal tumour at different stages of growth.

In this study, the immunohistochemical distribution of CD3 (T lymphocytes), CD79 (B lymphocytes and plasma cells), IgG, IgM, IgA, IgG subclasses (IgG2, IgG3 and IgG4) L1 (macrophages) and MHC Class II antigen was analysed in the inflammatory infiltrates associated with spontaneous canine transmissible venereal tumours (CTVT) at different stages of growth. With all antibodies used, except IgM and IgA, the number of immunoreactive cells was significantly higher (p < 0.05) in the infiltrate of CTVT undergoing spontaneous regression or with stable growth (14 cases), than in tumours undergoing progressive growth (nine cases). This result suggests that T lymphocytes in addition to B cells, plasma cells expressing IgG, IgG2 and IgG4, and macrophages participate in the effective immune response against CTVT and mediate spontaneous regression of the tumour. MHC Class II antigen was expressed by infiltrating lymphocytes and macrophages, and also by fibroblasts within and around the tumours. Class II was also expressed by a variable number of neoplastic cells, particularly those in regressing or stable tumours with a marked lymphoplasmacytic infiltrate. This suggests that the expression of Class II by neoplastic cells is associated with the effective immune response and regression of CTVT.

Detection of immune complexes in sera of dogs with canine transmissible venereal sarcoma (CTVS) by a conglutinin-binding assay.

The canine transmissible venereal sarcoma (CTVS) is capable of extended growth in an allogeneic host. Since immune complexes can enhance allograft survival in other animal models, we used an enzyme-linked conglutinin-binding assay to determine the presence and amount of circulating immune complexes in dogs with CTVS. With the conglutinin-binding assay, 23 of 64 dogs (36 per cent) bearing CTVS had concentration of immune complexes 3 standard deviations greater than those detected in normal canine serum. When dogs with circulating immune complexes were separated into 3 groups based on whether the size of CTVS had increased (progressor), decreased (regressor) or remained the same (steady-state) during the week before collection of serum, no significant difference was found in the amounts of immune complexes in sera from progressor dogs. In 3 regressor dogs that received a second transplant of CTVS, the mean concentration of circulating immune complex was significantly greater than the mean for progressor dogs. In progressor dogs, amounts of immune complexes decreased with increasing tumour volume. Following sucrose density gradient ultracentrifugation analysis of sera from a normal and progressor dog, 27S complexes containing both IgG and IgM were detected in serum from a progressor dog. Thus, it appears that the conglutinin-binding assay is a useful and sensitive method for detecting immune complexes in canine serum.

Canine transmissible venereal tumour: cytogenetic origin, immunophenotype, and immunobiology. A review.

Canine transmissible venereal tumour (CTVT) is the only known naturally occurring tumour that can be transplanted as an allograft across major histocompatibility (MHC) barriers within the same species, and even to other members of the canine family, such as foxes, coyotes and wolves. The progression of this tumour is unique in that, it follows a predictable growth pattern. In natural and experimental cases, the growth pattern includes progressive growth phase, static phase and regression phase, and this is followed by transplantation immunity in immunocompetent adults, while metastasis occurs in puppies and immunosuppressed dogs. Because of the uniqueness of CTVT transmission and progression, experimental investigations of various aspects of the biology of CTVT have been used to provide clues to the immunobiology of both animal and human tumours. This review examines the current state of knowledge of the aspects of the cytogenetic origin, immunophenotype, immunobiology and immunotherapy of CTVT.

Canine transmissible venereal tumor (TVT): a review.

The occurrence, transmission, clinical appearance, histological findings, chromosome studies, immunity, different methods of treatment and the prevention of canine transmissible venereal tumour are reviewed.