HPV infection renders oropharyngeal squamous cell carcinomas more radiosensitive, which results in a favorable prognosis for HPV-positive patients treated with radiation alone or with concurrent ...platinum-based chemotherapy. The degree of radiosensitivity in fractionated regimens has not yet been fully explored; therefore, in this study, the radiosensitivity of HPV-negative tumors (FaDu) was compared to that of HPV-positive tumors (2A3) subjected to concurrent cisplatin chemotherapy and fractionated versus isoeffective single-dose tumor irradiation in immunodeficient mice. HPV-positive tumors were approximately 5 times more radiosensitive than HPV-negative tumors, irrespective of the irradiation regimen. In both tumor models, concurrent cisplatin chemotherapy and the fractionated regimen induced significant tumor radiosensitization, with a 3- to 4-fold increase in the tumor growth delay compared to that of single-dose irradiation. Furthermore, the degree of radiosensitization induced by cisplatin chemotherapy concurrent with the fractionated irradiation regimen was much higher in HPV-positive tumors, where a synergistic antitumor effect was observed. Specifically, after combined therapy, a 26% higher survival rate was observed in mice with HPV-positive tumors than in mice with HPV-negative tumors. These data suggest that HPV-positive tumors are more radiosensitive to fractionated regimen than to single-dose irradiation with concurrent cisplatin chemotherapy acting synergistically to irradiation.
Triple-negative breast cancer (TNBC) accounts for about 10-20% of all breast cancer cases and is associated with an unfavorable prognosis. Until recently, treatment options for TNBC were limited to ...chemotherapy. A new successful systemic treatment is immunotherapy with immune checkpoint inhibitors, but new tumor-specific biomarkers are needed to improve patient outcomes. Cannabinoids show antitumor activity in most preclinical studies in TNBC models and do not appear to have adverse effects on chemotherapy. Clinical data are needed to evaluate efficacy and safety in humans. Importantly, the endocannabinoid system is linked to the immune system and immunosuppression. Therefore, cannabinoid receptors could be a potential biomarker for immune checkpoint inhibitor therapy or a novel mechanism to reverse resistance to immunotherapy. In this article, we provide an overview of the currently available information on how cannabinoids may influence standard therapy in TNBC.
Infection with high-risk human papillomavirus (HPV) strains is one of the risk factors for the development of oral squamous cell carcinoma (OSCC). Some patients with HPV-positive OSCC have a better ...prognosis and respond better to various treatment modalities, including radiotherapy or immunotherapy. However, since HPV can only infect human cells, there are only a few immunocompetent mouse models available that enable immunological studies. Therefore, the aim of our study was to develop a transplantable immunocompetent mouse model of HPV-positive OSCC and characterize it in vitro and in vivo.
Two monoclonal HPV-positive OSCC mouse cell lines were established by inducing the expression of HPV-16 oncogenes E6 and E7 in the MOC1 OSCC cell line using retroviral transduction. After confirming stable expression of HPV-16 E6 and E7 with quantitative real-time PCR and immunofluorescence staining, the cell lines were further characterized in vitro using proliferation assay, wound healing assay, clonogenic assay and RNA sequencing. In addition, tumor models were characterized in vivo in C57Bl/6NCrl mice in terms of their histological properties, tumor growth kinetics, and radiosensitivity. Furthermore, immunofluorescence staining of blood vessels, hypoxic areas, proliferating cells and immune cells was performed to characterize the tumor microenvironment of all three tumor models.
Characterization of the resulting MOC1-HPV cell lines and tumor models confirmed stable expression of HPV-16 oncogenes and differences in cell morphology, in vitro migration capacity, and tumor microenvironment characteristics. Although the cell lines did not differ in their intrinsic radiosensitivity, one of the HPV-positive tumor models, MOC1-HPV K1, showed a significantly longer growth delay after irradiation with a single dose of 15 Gy compared to parental MOC1 tumors. Consistent with this, MOC1-HPV K1 tumors had a lower percentage of hypoxic tumor area and a higher percentage of proliferating cells. Characteristics of the newly developed HPV-positive OSCC tumor models correlate with the transcriptomic profile of MOC1-HPV cell lines.
In conclusion, we developed and characterized a novel immunocompetent mouse model of HPV-positive OSCC that exhibits increased radiosensitivity and enables studies of immune-based treatment approaches in HPV-positive OSCC.
Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin ...12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.
Objective: Pelvic exenteration in women with recurrent vulvar carcinoma is associated with high morbidity and mortality and substantial treatment costs. Because pelvic exenteration severely affects ...the quality of life and can lead to significant complications, other treatment modalities, such as electrochemotherapy, have been proposed. The aim of this study was to evaluate the feasibility and suitability of electrochemotherapy in the treatment of recurrent vulvar cancer. We aimed to analyze the treatment options, treatment outcomes, and complications in patients with recurrent vulvar cancer of the perineum. Methods: A retrospective analysis of patients who had undergone pelvic exenteration for vulvar cancer at the Institute of Oncology Ljubljana over a 16-year period was performed. As an experimental, less mutilating treatment, electrochemotherapy was performed on one patient with recurrent vulvar cancer involving the perineum. Comparative data analysis was performed between the group with pelvic exenteration and the patient with electrochemotherapy, comparing hospital stay, disease recurrence after treatment, survival after treatment in months, and quality of life after treatment. Results: We observed recurrence of disease in 2 patients with initial FIGO stage IIIC disease 3 months and 32 months after pelvic exenteration, and they died of the disease 15 and 38 months after pelvic exenteration. Two patients with FIGO stage IB were alive at 74 and 88 months after pelvic exenteration. One patient with initial FIGO stage IIIC was alive 12 months after treatment with electrochemotherapy with no visible signs of disease progression in the vulvar region, and the lesions had a complete response. The patient treated with electrochemotherapy was hospitalized for 4 days compared with the patients with pelvic exenteration, in whom the average hospital stay was 19.75 (± 1.68) days. Conclusion: Our experience has shown that electrochemotherapy might be a less radical alternative to pelvic exenteration, especially for patients with initially higher FIGO stages.
Abstract
Electrochemotherapy with bleomycin (ECT BLM) is an effective antitumor treatment already used in clinical oncology. However, ECT alone is still considered a local antitumor therapy because ...it cannot induce systemic immunity. When combined with adjuvant gene electrotransfer of plasmid DNA encoding IL-12 (GET pIL-12), the combined therapy leads to a systemic effect on untreated tumors and distant metastases. Although the antitumor efficacy of both therapies alone or in combination has been demonstrated at both preclinical and clinical levels, data on the predictors of efficacy of the treatments are still lacking. Herein, we evaluated the results of dynamic contrast-enhanced ultrasound (DCE-US) as a predictive factor for ECT BLM and GET pIL-12 in murine melanoma. Melanoma B16F10 tumors grown in female C57Bl/6NCrl mice were treated with GET pIL-12 and ECT BLM. Immediately after therapy, 6 h and 1, 3, 7 and 10 days later, tumors were examined by DCE-US. Statistical analysis was performed to inspect the correlation between tumor doubling time (DT) and DCE-US measurements using semilinear regression models and Bland–Altman plots. Therapeutic groups in which DCE-US showed reduced tumor perfusion had longer tumor DTs. It was confirmed that the DCE-US parameter peak enhancement (PE), reflecting relative blood volume, had predictive value for the outcome of therapy: larger PE correlated with shorter DT. In addition, perfusion heterogeneity was also associated with outcome: tumors that had more heterogeneous perfusion had faster growth, i.e., shorter DTs. This study demonstrates that DCE-US can be used as a method to predict the efficacy of electroporation-based treatment.
Display omitted
•Electrochemotherapy affects the early C2C12 myogenesis.•Electrochemotherapy has little effect on C2C12 myotubes.•Electrochemotherapy influenced differentiation but not proliferation ...of myoblasts.•Electrochemotherapy prevents formation of nAChR clusters.
Electrochemotherapy (ECT) is a local ablative therapy for the treatment of different skin and subcutaneous tumors and certain tumors in internal organs. Skeletal muscle represents a major tumor- surrounding tissue, exposed to side effects of ECT. At the cellular level, side-effects of ECT on skeletal muscle and underlying mechanisms have not been examined yet. Thus, we aimed to determine the effect of ECT in the mouse muscle cell line C2C12 during in vitro myogenesis. We evaluated the electroporation efficiency and viability of C2C12 myotubes at increasing voltages (200–1300 V/cm) using propidium iodide (PI). Permeabilization of PI into the cells was voltage-dependent accounting up to 97 % efficiency at the highest voltage. High cell viability and myotube integrity were maintained until 4 days after electroporation. ECT with the cytostatic drugs bleomycin and cisplatin decreased the viability of C2C12 myoblasts and myotubes in a dose-dependent manner. However, myoblasts were more sensitive to ECT than myotubes. Increased secretion of IL-6, observed 3 days after ECT, confirming its effects on early myogenesis. Only minor effects of ECT were observed in treated myotubes. These results contribute to the safety profile of ECT in tumor treatment.
DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim ...of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer (GET)-mediated delivery of plasmid DNA into skin and muscle tissues on a model of COVID-19 vaccine. Plasmids encoding the SARS-CoV-2 proteins spike (S) and nucleocapsid (N) were used as the antigen source, and a plasmid encoding interleukin 12 (IL-12) was used as an adjuvant. Vaccination was performed in the skin or muscle tissue of C57BL/6J mice on days 0 and 14 (boost). Two weeks after the boost, blood, spleen, and transfected tissues were collected to determine the expression of S, N, IL-12, serum interferon-γ, the induction of antigen-specific IgG antibodies, and cytotoxic T-cells. In accordance with prior in vitro experiments that indicated problems with proper expression of the S protein, vaccination with S did not induce S-specific antibodies, whereas significant induction of N-specific antibodies was detected after vaccination with N. Intramuscular vaccination outperformed skin vaccination and resulted in significant induction of humoral and cell-mediated immunity. Moreover, both boost and adjuvant were found to be redundant for the induction of an immune response. Overall, the study confirmed the feasibility of the GET for DNA vaccination and provided valuable insights into this approach.
The contactless high intensity pulsed electromagnetic field (HI-PEMF)-induced increase of cell membrane permeability is similar to conventional electroporation, with the important difference of ...inducing an electric field non-invasively by exposing a treated tissue to a time-varying magnetic field. Due to the limited number of studies in the field of electroporation induced by HI-PEMF, we designed experiments to explore the feasibility of such a contactless delivery technique for the gene electrotransfer of nucleic acids in tissues in vivo. By using HI-PEMF for gene electrotransfer, we silenced enhanced green fluorescent protein (EGFP) with siRNA molecules against EGFP in B16F10-EGFP tumors. Six days after the transfer, the fluorescent tumor area decreased by up to 39% as determined by fluorescence imaging in vivo. In addition, the silencing of EGFP to the same extent was confirmed at the mRNA and protein level. The results obtained in the in vivo mouse model demonstrate the potential use of HI-PEMF-induced cell permeabilization for gene therapy and DNA vaccination. Further studies are thus warranted to improve the equipment, optimize the protocols for gene transfer and the HI-PEMF parameters, and demonstrate the effects of HI-PEMF on a broader range of different normal and tumor tissues.
PDF
