The use of existing mouse models in cancer research is of utmost importance as they aim to explore the casual link between candidate cancer genes and carcinogenesis as well as to provide models to develop and test new therapies. However, faster progress in translating mouse cancer model research into the clinic has been hampered due to the limitations of these models to better reflect the complexities of human tumors. Traditionally, immunocompetent and immunodeficient mice with syngeneic and xenografted tumors transplanted subcutaneously or orthotopically have been used. These models are still being widely employed for many different types of studies, in part due to their widespread availability and low cost. Other types of mouse models used in cancer research comprise transgenic mice in which oncogenes can be constitutively or conditionally expressed and tumor-suppressor genes silenced using conventional methods, such as retroviral infection, microinjection of DNA constructs, and the so-called “gene-targeted transgene” approach. These traditional transgenic models have been very important in studies of carcinogenesis and tumor pathogenesis, as well as in studies evaluating the development of resistance to therapy. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing approach has revolutionized the field of mouse cancer models and has had a profound and rapid impact on the development of more effective systems to study human cancers. The CRISPR/Cas9-based transgenic models have the capacity to engineer a wide spectrum of mutations found in human cancers and provide solutions to problems that were previously unsolvable. Recently, humanized mouse xenograft models that accept patient-derived xenografts and CD34+ cells were developed to better mimic tumor heterogeneity, the tumor microenvironment, and cross-talk between the tumor and stromal/immune cells. These features make them extremely valuable models for the evaluation of investigational cancer therapies, specifically new immunotherapies. Taken together, improvements in both the CRISPR/Cas9 system producing more valid mouse models and in the humanized mouse xenograft models resembling complex interactions between the tumor and its environment might represent one of the successful pathways to precise individualized cancer therapy, leading to improved cancer patient survival and quality of life.

Electrochemotherapy combined with peritumoral interleukin-12 (IL-12) gene electrotransfer was used for treatment of mast cell tumours in 18 client-owned dogs. Local tumour control, recurrence rate, as well as safety of combined therapy were evaluated. One month after the therapy, no side effects were recorded and good local tumour control was observed with high complete responses rate which even increased during the observation period to 72%. IL-12 gene electrotransfer resulted in 78% of patients with detectable serum IFN-and/or IL-12 levels. In the treated tumours vascular changes as well as minimal T-lymphocytes infiltration was observed. After 1 week, the plasmid DNA was not detected intra-or peritumorally and no horizontal gene transfer was observed. In summary, our study demonstrates high antitumour efficacy of electrochemotherapy combined with IL-12 electrotransfer, which also prevented recurrences or distant metastases, as well as its safety and feasibility in treatment of canine mast cell tumours.

Electrochemotherapy (ECT) is a local approach which is used for treating solid tumors of different histologies. Its mechanism is based on cell membrane permeabilization by means of "electroporation". To achieve the "electroporation" of the cells, electric pulses are generated by a generator and delivered to the target tissue by the use of electrodes. Electroporation is a physical method which is used to introduce molecules, like cytostatic drugs, into the cells that could not pass the cell membrane on their own. In electrochemotherapy, currently, cisplatin and bleomycin are clinically used. Electrochemotherapy antitumor effectiveness is high, for example up to 100% complete response of canine mast cell tumors smaller than 2 cm 3 was achieved. Additionally, electrochemotherapy can be used for the treatment of inoperable tumors. One of the important characteristics of electrochemotherapy is that it can be effective as a one-time treatment only. However, in the case of failure or partial tumor response it can be repeated several times with equal or improved effectiveness. Electrochemotherapy is already a standard treatment for cutaneous and subcutaneous tumors of various histologies in human and veterinary oncology. Furthermore, several clinical studies exploiting electrochemotherapy for deep-seated tumors are on-going.

The aim of this study was to evaluate the safety and efficacy of the combination of electrochemotherapy (ECT) with bleomycin and gene electrotransfer (GET) of plasmid encoding canine interleukin 12 (IL-12) for the treatment of canine oral malignant melanoma (OMM). Our focus was to determine the effect of the treatment on achieving local tumor control and stimulation of an antitumor immune response. Nine dogs with histologically confirmed OMM stage I to III were included in a prospective, nonrandomized study. The dogs were treated with a combination of cytoreductive surgery, ECT and IL-12 GET, which was repeated up to five times, depending on the clinical response to the treatment, evaluated according to the followup protocol (7, 14 and 28 days after, the last treatment). One month after treatment, the objective response (OR) rate was 67% (6/9). Median survival time (MST) was 6 months and, even though the disease progressed in 8/9 patients at the end of the observation period (2 to 22 months), four animals were euthanized due to tumor-unrelated reasons. In addition, we observed a decline in the percentage of regulatory T cells (Treg) in the peripheral blood in the course of the treatment, which could be attributed to a systemic antitumor response to IL-12 GET. The results of this study suggest that a combination of ECT and IL-12 GET may be beneficial for dogs with OMM, especially when other treatment approaches are not acceptable due to their invasiveness or cost.

AbstractBackgroundElectrochemotherapy is an effective treatment of colorectal liver metastases and hepatocellular carcinoma (HCC) during open surgery. The minimally invasive percutaneous approach of electrochemotherapy has already been performed but not on HCC. The aim of this study was to demonstrate the feasibility, safety and effectiveness of electrochemotherapy with percutaneous approach on HCC.Patient and methodsThe patient had undergone the transarterial chemoembolization and microwave ablation of multifocal HCC in segments III, V and VI. In follow-up a new lesion was identified in segment III, and recognized by multidisciplinary team to be suitable for minimally invasive percutaneous electrochemotherapy. The treatment was performed with long needle electrodes inserted by the aid of image guidance.ResultsThe insertion of electrodes was feasible, and the treatment proved safe and effective, as demonstrated by control magnetic resonance imaging.ConclusionsMinimally invasive, image guided percutaneous electrochemotherapy is feasible, safe and effective in treatment of HCC.

Skin is an attractive target for gene electrotransfer. It consists of different cell types that can be transfected, leading to various responses to gene electrotransfer. We demonstrate that these responses could be controlled by selecting the appropriate electrotransfer parameters. Specifically, the application of low or high electric pulses, applied by multi-electrode array, provided the possibility to control the depth of the transfection in the skin, the duration and the level of gene expression, as well as the local or systemic distribution of the transgene. The influence of electric pulse type was first studied using a plasmid encoding a reporter gene (DsRed). Then, plasmids encoding therapeutic genes (IL-12, shRNA against endoglin, shRNA against melanoma cell adhesion molecule) were used, and their effects on wound healing and cutaneous B16F10 melanoma tumors were investigated. The high-voltage pulses resulted in gene expression that was restricted to superficial skin layers and induced a local response. In contrast, the low-voltage electric pulses promoted transfection into the deeper skin layers, resulting in prolonged gene expression and higher transgene production, possibly with systemic distribution. Therefore, in the translation into the clinics, it will be of the utmost importance to adjust the electrotransfer parameters for different therapeutic approaches and specific mode of action of the therapeutic gene.

Gene electrotransfer (GET) is one of the most efficient non-viral gene therapy approaches for the localized transfer of multiple genes into tumors in vivo; therefore, it is especially promising for delivering different cytokines that are toxic if administered systemically. In this study, we used concomitant intratumoral GET of two cytokines: tumor necrosis factor alpha (TNFα), a potent cytotoxic cytokine to induce in situ vaccination, and interleukin 12 (IL-12), an immunostimulatory cytokine to boost the primed local immune response into a systemic one. After performing GET in murine melanoma tumors, both TNFα and IL-12 mRNA levels were significantly increased, which resulted in a pronounced delay in tumor growth of 27 days and a prolonged survival time of mice. An antitumor immune response was confirmed by extensive infiltration of immune cells in the tumor site, and expansion of the effector immune cells in the sentinel lymph nodes. Furthermore, the effect of in situ vaccination was indicated by the presence of vitiligo localized to the treatment area and resistance of the mice to secondary challenge with tumor cells. Intratumoral GET of two cytokines, one for in situ vaccination and one for an immune boost, proved feasible and effective in eliciting a potent and durable antitumor response; therefore, further studies of this approach are warranted.

Electroporation is a method of inducing an increase in permeability of the cell membrane through the application of an electric field and can be used as a delivery method for introducing molecules of interest (e.g., chemotherapeutics or plasmid DNA) into cells. Electroporation-based treatments (i.e., electrochemotherapy, gene electrotransfer, and their combinations) have been shown to be safe and effective in veterinary oncology, but they are currently mostly recommended for the treatment of those solid tumors for which clients have declined surgery and/or radiotherapy. Published data show that electroporation-based treatments are also safe, simple, fast and cost-effective treatment alternatives for selected oral and maxillofacial tumors, especially small squamous cell carcinoma and malignant melanoma tumors not involving the bone in dogs. In these patients, a good local response to treatment is expected to result in increased survival time with good quality of life. Despite emerging evidence of the clinical efficacy of electroporation-based treatments for oral and maxillofacial tumors, further investigation is needed to optimize treatment protocols, improve clinical data reporting and better understand the mechanisms of patients' response to the treatment.