Treatment of vulvar cancer recurrence with electrochemotherapy: a case-control study.

BACKGROUND: Electrochemotherapy (ECT) is a combined treatment method based on electroporation and simultaneous chemotherapy. In cases where radiotherapy has previously been used, surgery is often the only treatment option for vulvar cancer recurrence with potential resection of clitoris, vagina, urethra or anal sphincter. The unique advantage of ECT is its selectivity for cancer cells while sparing the surrounding healthy tissue. The aim of the study was to compare the ECT treatment of vulvar cancer recurrence for non-palliative purposes with surgical treatment. MATERIALS AND METHODS: Eleven patients with single vulvar cancer recurrence were treated with ECT and followed up for 12 months. As a control group, 15 patients with single vulvar cancer recurrence were treated with wide local excision. The following data were collected, analyzed and compared: Age, body mass index, comorbidities, histological type, location and size of vulvar cancer recurrence, treatment history, details of procedures and hospital stay. RESULTS: The probability curves for local tumor control did not differ between the ECT group and the surgical group (p = 0.694). The mean hospital stay and the mean duration of procedure were statistically significantly shorter in the ECT group (p < 0.001). There were no statistically significant differences between the ECT and surgical groups in terms of mean body mass index, associated diseases, previous treatments, presence of lichen sclerosus, p16 status, gradus, anatomical site of the tumor, and type of anesthesia. CONCLUSION: In this case-control study, treatment of vulvar cancer recurrence with ECT for non-palliative purposes was comparable to surgical treatment in terms of effectiveness. The results need to be confirmed in larger randomized trials.

Safety and Efficacy of IL-12 Plasmid DNA Transfection into Pig Skin: Supportive Data for Human Clinical Trials on Gene Therapy and Vaccination.

Gene electrotransfer (GET) of plasmids encoding interleukin 12 (IL-12) has already been used for the treatment of various types of tumors in human oncology and as an adjuvant in DNA vaccines. In recent years, we have developed a plasmid encoding human IL-12 (phIL12) that is currently in a phase I clinical study. The aim was to confirm the results of a non-clinical study in mice on pharmacokinetic characteristics and safety in a porcine model that better resembled human skin. The GET of phIL12 in the skin was performed on nine pigs using different concentrations of plasmid phIL12 and invasive (needle) or noninvasive (plate) types of electrodes. The results of our study demonstrate that the GET of phIL-12 with needle electrodes induced the highest expression of IL-12 at the protein level on day 7 after the procedure. The plasmid was distributed to all tested organs; however, its amount decreased over time and was at a minimum 28 days after GET. Based on plasmid copy number and expression results, together with blood analysis, we showed that IL-12 GET is safe in a porcine animal model. Furthermore, we demonstrated that pigs are a valuable model for human gene therapy safety studies.

HPV-positive murine oral squamous cell carcinoma: development and characterization of a new mouse tumor model for immunological studies.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

European e-Delphi process to define expert consensus on electrochemotherapy treatment indications, procedural aspects, and quality indicators in melanoma.

BACKGROUND: Skin metastases are an important co-morbidity in melanoma. Despite broad adoption, electrochemotherapy implementation is hindered by a lack of treatment indications, uncertainty regarding procedural aspects, and the absence of quality indicators. An expert consensus may harmonize the approach among centres and facilitate comparison with other therapies. METHODS: An interdisciplinary panel was recruited for a three-round e-Delphi survey. A literature-based 113-item questionnaire was proposed to 160 professionals from 53 European centres. Participants rated each item for relevance and degree of agreement on a five-point Likert scale, and received anonymous controlled feedback to allow revision. The items that reached concordant agreement in two successive iterations were included in the final consensus list. In the third round, quality indicator benchmarks were defined using a real-time Delphi method. RESULTS: The initial working group included 122 respondents, of whom 100 (82 per cent) completed the first round, thus qualifying for inclusion in the expert panel (49 surgeons, 29 dermatologists, 15 medical oncologists, three radiotherapists, two nurse specialists, two clinician scientists). The completion rate was 97 per cent (97 of 100) and 93 per cent (90 of 97) in the second and third rounds respectively. The final consensus list included 54 statements with benchmarks (treatment indications, (37); procedural aspects, (1); quality indicators, (16)). CONCLUSION: An expert panel achieved consensus on the use of electrochemotherapy in melanoma, with a core set of statements providing general direction to electrochemotherapy users to refine indications, align clinical practices, and promote quality assurance programmes and local audits. The residual controversial topics set future research priorities to improve patient care.

Electrochemotherapy is an effective locoregional therapy for skin metastases from melanoma, a problem faced by almost half of patients with metastatic disease. The lack of comparative studies and the heterogeneity of its clinical application among centres make it challenging to support consistent, evidence-based recommendations. To address this unmet need, a three-round online survey was conducted to establish a consensus on treatment indications, standard operating procedures, and quality indicators. In the survey, a panel of 100 European melanoma experts agreed on 56 statements that can be used to improve patient selection, homogenize treatment application, and monitor outcomes.

An effective validation of analytical method for determination of a polar complexing agent: the illustrative case of cytotoxic bleomycin.

The effectiveness of highly polar agents in cancer treatment is well recognized, but their physicochemical properties make their analytical determination a demanding task. Their analysis requires peculiar sample preparation and chromatographic separation, which heavily impacts the precision of such an analytical method. As a case study, we chose a polar cytotoxic bleomycin, which is a mixture of complexing congeners with relatively high molecular mass, a fact that creates an added challenge in regard to its detection via electrospray mass spectrometry. These issues combined lead to a deprived method performance, so the aim of this study is manifold, i.e., to optimize, validate, and establish quality performance measures for determination of bleomycin in pharmaceutical and biological specimens. Quantification of bleomycin is done at diametrically different concentration levels: at the concentrations relevant for analysis of pharmaceutical dosage forms it is based on a direct reversed-phase HPLC-UV detection, involving minimum sample pretreatment. On the contrary, analysis of bleomycin in biological specimens requires phospholipid removal and protein precipitation followed by HILIC chromatography with MS/MS detection of bleomycin A2 and B2 copper complexes being the predominant species. This study further attempts to solve the traceability issue in the absence of certified reference standards, determines measurement uncertainty, investigates BLM stability and method performance characteristics, and, last but not least, provides an explanatory example of how a method quality assurance procedure should be established in case of an exceedingly complex analytical method.

Tumor Radiosensitization by Gene Electrotransfer-Mediated Double Targeting of Tumor Vasculature.

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor ß co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors.

Electrochemotherapy of Melanoma Cutaneous Metastases in Organ Transplant Recipients: A Systematic Review of Preclinical and Clinical Studies.

Cutaneous melanoma is a highly aggressive form of skin cancer. The development of immune checkpoint inhibitors (ICIs) has revolutionized the management of advanced melanoma, led to durable responses, and improved overall survival. However, the success of ICIs in melanoma treatment is influenced by the tumor microenvironment (TME) which plays a critical role in regulating the immune response to the tumor. Understanding the mechanisms underlying this interaction is crucial to optimizing the efficiency of ICIs. Electrochemotherapy (ECT) has been shown to enhance the efficacy of ICIs in melanoma treatment by inducing tumor cell death and facilitating the release of tumor antigens which can subsequently be recognized and targeted by the immune system. Moreover, ECT has been reported to modulate the TME, leading to increased infiltration of immune cells and a more favorable immunological profile. In this review, we summarize the available knowledge of changes in TME after ECT of melanoma cutaneous metastasis and highlight the differences in tumor-infiltrating immune cells between immunocompetent and immunosuppressed organisms. In addition, we showed that ECT can be an effective and safe procedure for organ transplant recipients. Furthermore, repeated ECT may enhance immune activation and probably induce a bystander effect by trained immunity.

Gene Immunotherapy of Colon Carcinoma with IL-2 and IL-12 Using Gene Electrotransfer.

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.

Prospektive Kohortenstudie von InspECT zur Sicherheit und Wirksamkeit der Elektrochemotherapie bei Hauttumoren und Metastasen in Abhängigkeit von Ulzeration.

HINTERGRUND: Elektrochemotherapie (ECT) ist eine wirksame lokale Behandlung von Hauttumoren. Ziel dieser Studie war es, die Wirksamkeit der ECT bei ulzerierten gegenüber nichtulzerierten Tumoren zu vergleichen und den Effekt auf tumorassoziierte Symptome zu untersuchen. METHODIK: 20 Krebszentren des International Network for Sharing Practices on Electrochemotherapy (InspECT) sammelten prospektiv Daten. Die ECT wurde nach dem ESOPE-Protokoll durchgeführt. Das Therapieansprechen wurde anhand der Entwicklung der Läsionsgröße bewertet. Zusätzlich wurden Schmerzen, Symptome, Leistungsstatus (ECOG-Index) und Gesundheitszustand (EQ-5D-Fragebogen) untersucht. ERGEBNISSE: 716 Patienten mit ulzerierten (n = 302) und nichtulzerierten (n = 414) Hauttumoren und Metastasen wurden eingeschlossen (Mindest-Nachsorge 45 Tage). Nicht-ulzerierte Läsionen sprachen besser auf die ECT an als ulzerierte Läsionen (vollständiges Ansprechen: 65 % gegenüber 51 %, p = 0,0061). Nur 38 % (115/302) der Patienten mit ulzerierten Läsionen vor der ECT wiesen bei der letzten Nachuntersuchung ulzerierte Läsionen auf. Patienten mit ulzerierten Läsionen berichteten über stärkere Schmerzen und schwerere Symptome im Vergleich zu Patienten mit nichtulzerierten Läsionen, die sich nach der ECT signifikant und kontinuierlich besserten. Bei Patienten mit nichtulzerierten Läsionen hingegen nahmen die Schmerzen während der Behandlung vorübergehend zu. Es wurden keine schwerwiegenden Nebenwirkungen beobachtet. SCHLUSSFOLGERUNGEN: Die ECT ist eine sichere und wirksame lokale Behandlung von Hauttumoren. Während die ECT die Symptome insbesondere bei Patienten mit ulzerierten Läsionen verbessert, sollte auf Basis der Daten die Implementation eines perioperativen Schmerzmanagements besonders bei nichtulzerierten Läsionen während der ECT erwogen werden.

Prospective cohort study by InspECT on safety and efficacy of electrochemotherapy for cutaneous tumors and metastases depending on ulceration.

BACKGROUND: Electrochemotherapy (ECT) is an effective local treatment for cutaneous tumors. The aim of this study was to compare the effectiveness of ECT in ulcerated vs. non-ulcerated tumors and investigate the effect on tumor-associated symptoms. METHODS: Twenty cancer centers in the International Network for Sharing Practices on Electrochemotherapy (InspECT) prospectively collected data. ECT was performed following ESOPE protocol. Response was evaluated by lesion size development. Pain, symptoms, performance status (ECOG-Index) and health status (EQ-5D questionnaire) were evaluated. RESULTS: 716 patients with ulcerated (n = 302) and non-ulcerated (n = 414) cutaneous tumors and metastases were included (minimum follow-up of 45 days). Non-ulcerated lesions responded to ECT better than ulcerated lesions (complete response 65 % vs. 51 %, p = 0.0061). Only 38 % (115/302) with ulcerated lesions before ECT presented with ulcerated lesions at final follow-up. Patients with ulcerated lesions reported higher pain and more severe symptoms compared to non-ulcerated lesions, which significantly and continuously improved following ECT. In non-ulcerated lesions however, pain spiked during the treatment. No serious adverse events were reported. CONCLUSIONS: ECT is a safe and effective local treatment for cutaneous tumors. While ECT improves symptoms especially in patients with ulcerated lesions, data suggest the implementation of a perioperative pain management in non-ulcerated lesions during ECT.

Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation.

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.

Antitumor in situ vaccination effect of TNFα and IL-12 plasmid DNA electrotransfer in a murine melanoma model.

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.

The General Explanation Method with NMR Spectroscopy Enables the Identification of Metabolite Profiles Specific for Normal and Tumor Cell Lines.

Machine learning models in metabolomics, despite their great prediction accuracy, are still not widely adopted owing to the lack of an efficient explanation for their predictions. In this study, we propose the use of the general explanation method to explain the predictions of a machine learning model to gain detailed insight into metabolic differences between biological systems. The method was tested on a dataset of 1 H NMR spectra acquired on normal lung and mesothelial cell lines and their tumor counterparts. Initially, the random forests and artificial neural network models were applied to the dataset, and excellent prediction accuracy was achieved. The predictions of the models were explained with the general explanation method, which enabled identification of discriminating metabolic concentration differences between individual cell lines and enabled the construction of their specific metabolic concentration profiles. This intuitive and robust method holds great promise for in-depth understanding of the mechanisms that underline phenotypes as well as for biomarker discovery in complex diseases.

Inhibition of the Innate Immune Receptors for Foreign DNA Sensing Improves Transfection Efficiency of Gene Electrotransfer in Melanoma B16F10 Cells.

Gene electrotransfer upregulate DNA pattern recognition receptors or DNA sensors, which are part of the innate immune system. In this study, we tested if addition of the cocktail of innate immune system inhibitors to the cells during gene electrotransfer (GET) can increase transfection efficiency and cell survival. The results indicate that this cocktail can decrease cytosolic DNA sensors expression after GET, and consequently increase cell survival and transfection efficiency in B16 cells, but only in highly metastatic B16F10 subtype. We demonstrated that DNA sensors expression during the transfection methods needs to be downregulated if higher transfection efficiency and better cells' survival is needed. The inhibition of the receptors of the innate immune system can improve the transfection efficiency also for GET of malignant melanoma B16 cells, but only of highly metastatic subtype.

Updated standard operating procedures for electrochemotherapy of cutaneous tumours and skin metastases.

Electrochemotherapy is now in routine clinical use to treat cutaneous metastases of any histology, and is listed in national and international guidelines for cutaneous metastases and primary skin cancer. Electrochemotherapy is used by dermatologists, surgeons, and oncologists, and for different degrees and manifestations of metastases to skin and primary skin tumours not amenable to surgery. This treatment utilises electric pulses to permeabilize cell membranes in tumours, thus allowing a dramatic increase of the cytotoxicity of anti-cancer agents. Response rates, often after only one treatment, are very high across all tumour types. The most frequent indications are cutaneous metastases from malignant melanoma and breast cancer. In 2006, standard operating procedures (SOPs) were written for this novel technology, greatly facilitating introduction and dissemination of the therapy. Since then considerable experience has been obtained treating a wider range of tumour histologies and increasing size of tumours which was not originally thought possible. A pan-European expert panel drawn from a range of disciplines from dermatology, general surgery, head and neck surgery, plastic surgery, and oncology met to form a consensus opinion to update the SOPs based on the experience obtained. This paper contains these updated recommendations for indications for electrochemotherapy, pre-treatment information and evaluation, treatment choices, as well as follow-up.

Electrochemotherapy with Bleomycin of Different types of Cutaneous Tumours in a Ferret (Mustela Putorius Furo).

BACKGROUND: Mast cell tumour, sebaceous gland adenoma, and less common squamous papilloma are skin tumours in ferrets (Mustela putorius furo), and early excisional surgery is usually the treatment of choice. The aim of our study was to investigate the effectiveness of electrochemotherapy (ECT), a new, minimally invasive non-surgical method, as first treatment option of different types of ferret skin tumours located on surgically difficult sites. MATERIALS AND METHODS: A 5-year-old castrated male ferret with two cutaneous masses, presenting 4 months apart and a 7-year-old spayed female ferret with two cutaneous masses, that appeared simultaneously on two locations are presented. In the first patient, both masses were diagnosed as mast cell tumours, and in the second patient, squamous papilloma and sebaceous adenoma were diagnosed. One session of ECT with bleomycin injected intratumourally was applied in all tumours. RESULTS: Complete response (CR) of all tumours was obtained, without recurrence during observation period of 15 months after ECT for first tumour and 11 months after ECT of the tumour located on the right hock in first patient, and 8 months after treatment for the second patient. CONCLUSIONS: In present study, ECT with bleomycin proved to be safe and effective against different cutaneous tumours in ferrets. Due of good results, low cost and relatively easy procedure, ECT could be the treatment of choice instead of surgery for the selected skin tumours in ferrets.

Tailor-made fibroblast-specific and antibiotic-free interleukin 12 plasmid for gene electrotransfer-mediated cancer immunotherapy.

Electrotransfer mediated delivery of interleukin-12 (IL-12) gene, encoded on a plasmid vector, has already been demonstrated to have a potent antitumor efficacy and great potential for clinical application. In the present study, our aim was to construct an optimized IL-12-encoding plasmid that is safe from the regulatory point of view. In light of previous studies demonstrating that IL-12 should be released in a tumor localized manner for optimal efficacy, the strong ubiquitous promoter was replaced with a weak endogenous promoter of the collagen 2 gene, which is specific for fibroblasts. Next, to comply with increasing regulatory demands for clinically used plasmids, the expression cassette was cloned in a plasmid lacking the antibiotic resistance gene. The constructed fibroblast-specific and antibiotic-free IL-12 plasmid was demonstrated to support low IL-12 expression after gene electrotransfer in selected cell lines. Furthermore, the removal of antibiotic resistance did not affect the plasmid expression profile and lowered its cytotoxicity. With optimal IL-12 expression and minimal transgene non-specific effects, i.e., low cytotoxicity, the constructed plasmid could be especially valuable for different modern immunological approaches to achieve localized boosting of the host's immune system.

Gene Electrotransfer of Plasmid-Encoding IL-12 Recruits the M1 Macrophages and Antigen-Presenting Cells Inducing the Eradication of Aggressive B16F10 Murine Melanoma.

Cancer immunotherapy is currently one of the leading approaches in cancer treatment. Gene electrotransfer of plasmids encoding interleukin 12 (IL-12) into the cells leads to the production of IL-12, which drives immune cell polarization to an antitumoral response. One of the cell types that shows great promise in targeting tumor cells under the influence of IL-12 cytokine milieu is that of macrophages. Therefore, the aim of this study was to evaluate gene electrotransfer of antibiotic resistance-free plasmid DNA-encoding murine IL-12 (mIL-12) in mice bearing aggressive B16F10 murine melanoma. IL-12 electrotransfer resulted in the complete long-term eradication of the tumors. Serum mIL-12 and murine interferon γ (mIFNγ) were increased after IL-12 gene electrotransfer. Further on, hematoxylin and eosin (HE) staining showed increased infiltration of immune cells that lasted from day 4 until day 14. Immunohistochemistry (IHC) staining of F4/80, MHCII, and CD11c showed higher positive staining in the IL-12 gene electrotransfer group than in the control groups. Immune cell infiltration into the tumors and the high density of MHCII- and CD11c-positive cells suggest an antitumor polarization of macrophages and the presence of antigen-presenting cells that contributes to the important antitumor effectiveness of IL-12.

Electrotransfer of plasmid DNA radiosensitizes B16F10 tumors through activation of immune response.

BACKGROUND: Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. MATERIALS AND METHODS: The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. RESULTS: Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. CONCLUSIONS: The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response.

In Vitro and in vivo Evaluation of Electrochemotherapy with trans-platinum Analogue trans-[PtCl2(3-Hmpy)2].

BACKGROUND: Cisplatin is used in cancer therapy, but its side effects and acquired resistance to cisplatin have led to the synthesis and evaluation of new platinum compounds. Recently, the synthesized platinum compound trans-[PtCl2(3-Hmpy)2] (3-Hmpy = 3-hydroxymethylpyridine) (compound 2) showed a considerable cytotoxic and antitumour effectiveness. To improve compound 2 cytotoxicity in vitro and antitumour effectiveness in vivo, electroporation was used as drug delivery approach to increase membrane permeability (electrochemotherapy). MATERIALS AND METHODS: In vitro, survival of sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 moderately sensitive) was determined using a clonogenic assay after treatment with compound 2 or cisplatin electrochemotherapy. In vivo, the antitumour effectiveness of electrochemotherapy with compound 2 or cisplatin was evaluated using a tumour growth delay assay. In addition, platinum in the serum, tumours and platinum bound to the DNA in the cells were performed using inductively coupled plasma mass spectrometry. RESULTS: In vitro, cell survival after treatment with compound 2 electrochemotherapy was significantly decreased in all tested sarcoma cells with different intrinsic sensitivity to cisplatin (TBLCl2 sensitive, TBLCl2Pt resistant and SA-1 moderately sensitive). However, this effect was less pronounced compared to cisplatin. Interestingly, the enhancement factor (5-fold) of compound 2 cytotoxicity was equal in cisplatin-sensitive TBLCl2 and cisplatin-resistant TBLCl2Pt cells. In vivo, the growth delay of subcutaneous tumours after treatment with compound 2 electrochemotherapy was lower compared to cisplatin. The highest antitumour effectiveness after cisplatin or compound 2 electrochemotherapy was obtained in TBLCl2 tumours, resulting in 67% and 11% of tumour cures, respectively. Compound 2 induced significantly smaller loss of animal body weight compared to cisplatin. Furthermore, platinum amounts in tumours after compound 2 or cisplatin electrochemotherapy were approximately 2-fold higher compared to the drug treatment only, and the same increase of platinum bound to DNA was observed. CONCLUSIONS: The obtained results in vitro and in vivo suggest compound 2 as a potential antitumour agent in electrochemotherapy.