V príspevku se avtorica osredotoča na nekatera vprašanja o jeziku in spolu, ki se zastavljajo ob učnih gradivih za osnovno šolo. Ob tem se ustavi ob vprašanjih nastavljanja učencev in učenk v ...navodilih, rabi generičnega moškega spola v primerih in spolno stereotipnih vsebinah tako v verbalnih kot tudi neverbalnih delih sporočila.
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.
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.
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.
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.
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.
•HI-PEMF facilitated delivery of plasmid DNA in muscle, skin and tumors.•HI-PEMF induced less infiltration of inflammatory mononuclear cells compared to EP.•Antitumor effectiveness using HI-PEMF for ...silencing MCAM was demonstrated.
High-Intensity Pulsed Electromagnetic Fields (HI-PEMF) treatment is an emerging noninvasive and contactless alternative to conventional electroporation, since the electric field inside the tissue is induced remotely by external pulsed magnetic field. Recently, HI-PEMF was applied for delivering siRNA molecules to silence enhanced green fluorescent protein (EGFP) in tumors in vivo. Still, delivered siRNA molecules were 21 base pairs long, which is 200-times smaller compared to nucleic acids such as plasmid DNA (pDNA) that are delivered in gene therapies to various targets to generate therapeutic effect. In our study, we demonstrate the use HI-PEMF treatment as a feasible noninvasive approach to achieve in vivo transfection by enabling the transport of larger molecules such as pDNA encoding EGFP into muscle and skin. We obtained a long-term expression of EGFP in the muscle and skin after HI-PEMF, in some mice even up to 230 days and up to 190 days, respectively. Histological analysis showed significantly less infiltration of inflammatory mononuclear cells in muscle tissue after the delivery of pEGFP using HI-PEMF compared to conventional gene electrotransfer. Furthermore, the antitumor effectiveness using HI-PEMF for electrotransfer of therapeutic plasmid, i.e., silencing MCAM was demonstrated. In conclusion, feasibility of HI-PEMF was demonstrated for transfection of different tissues (muscle, skin, tumor) and could have great potential in gene therapy and in DNA vaccination.
Electrochemotherapy of cutaneous tumor nodules requires local or general anesthesia. For multiple and larger nodules, general anesthesia is recommended by standard operating procedures. The choice of ...general anesthesia is at the discretion of the treating center. Continuous intravenous sedation is also an option. Our study aimed to elucidate the tolerability, safety and possible advantages of continuous intravenous sedation in comparison to general anesthesia in patients undergoing electrochemotherapy.
In the prospective study, 27 patients undergoing electrochemotherapy were either under general anesthesia or under continuous intravenous sedation. Evaluated were different endpoints, such as feasibility and safety, duration of anesthesia and compliance with the patients.
Ten patients were treated under general anesthesia, and 17 patients were under continuous intravenous sedation. The comparison of the approaches indicated that continuous intravenous sedation required a lower overall dosage of propofol, a shorter duration of anesthesia, a shorter time to reach an Aldrete score >8, and greater satisfaction of the patients with the procedure compared to general anesthesia.
The results indicate the feasibility and safety of continuous intravenous sedation for patients undergoing electrochemotherapy of cutaneous tumor nodules. This proved the preferred choice of anesthesia due to its shorter duration and better compliance with the patients compared to general anesthesia.
Pulsed electromagnetic field (PEMF) induces pulsed electric field, which presumably increases membrane permeabilization of the exposed cells, similar to the conventional electroporation. Thus, ...contactless PEMF could represent a promising approach for drug delivery.
Noninvasive electroporation was performed by magnetic field pulse generator connected to an applicator consisting of round coil. Subcutaneous mouse B16F10 melanoma tumors were treated with intravenously injection of cisplatin (CDDP) (4 mg/kg), PEMF (480 bipolar pulses, at frequency of 80 Hz, pulse duration of 340 μs) or with the combination of both therapies (electrochemotherapy - PEMF + CDDP). Antitumor effectiveness of treatments was evaluated by tumor growth delay assay. In addition, the platinum (Pt) uptake in tumors and serum, as well as Pt bound to the DNA in the cells and Pt in the extracellular fraction were measured by inductively coupled plasma mass spectrometry.
The antitumor effectiveness of electrochemotherapy with CDDP mediated by PEMF was comparable to the conventional electrochemotherapy with CDDP, with the induction of 2.3 days and 3.0 days tumor growth delay, respectively. The exposure of tumors to PEMF only, had no effect on tumor growth, as well as the injection of CDDP only. The antitumor effect in combined treatment was related to increased drug uptake into the electroporated tumor cells, demonstrated by increased amount of Pt bound to the DNA. Approximately 2-fold increase in cellular uptake of Pt was measured.
The obtained results in mouse melanoma model in vivo demonstrate the possible use of PEMF induced electroporation for biomedical applications, such as electrochemotherapy. The main advantages of electroporation mediated by PEMF are contactless and painless application, as well as effective electroporation compared to conventional electroporation.
Bacteriophages, prokaryotic viruses, hold great potential in genetic engineering to open up new avenues for vaccine development. Our study aimed to establish engineered M13 bacteriophages expressing ...MAGE-A1 tumor peptides as a vaccine for melanoma treatment. Through in vivo experiments, we sought to assess their ability to induce robust immune responses. Using phage display technology, we engineered two M13 bacteriophages expressing MAGE-A1 peptides as fusion proteins with either pVIII or pIIII coat proteins. Mice were intraperitoneally vaccinated three times, two weeks apart, using two different engineered bacteriophages; control groups received a wild-type bacteriophage. Serum samples taken seven days after each vaccination were analyzed by ELISA assay, while splenocytes harvested seven days following the second boost were evaluated by ex vivo cytotoxicity assay. Fusion proteins were confirmed by Western blot and nano-LC-MS/MS. The application of bacteriophages was safe, with no adverse effects on mice. Engineered bacteriophages effectively triggered immune responses, leading to increased levels of anti-MAGE-A1 antibodies in proportion to the administered bacteriophage dosage. Anti-MAGE-A1 antibodies also exhibited a binding capability to B16F10 tumor cells in vitro, as opposed to control samples. Splenocytes demonstrated enhanced CTL cytotoxicity against B16F10 cells. We have demonstrated the immunogenic capabilities of engineered M13 bacteriophages, emphasizing their potential for melanoma immunotherapy.
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