Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma

BACKGROUND: Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer. METHODS: Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD(50) assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated. RESULTS: Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect. CONCLUSIONS: Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation

Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors

The aim of the study was to explore the effectiveness of electrochemotherapy (ECT) during the treatment of melanoma patients with BRAF inhibitors. Its effectiveness was tested on BRAF mutated and non-mutated melanoma cells in vitro and in combination with BRAF inhibitors

Mcam Silencing With RNA Interference Using Magnetofection has Antitumor Effect in Murine Melanoma

The melanoma cell adhesion molecule (MCAM) is involved in melanoma development and its progression, including invasiveness, metastatic potential and angiogenesis. Therefore, MCAM represents a potential target for gene therapy of melanoma, whose expression could be hindered with posttranscriptional specific gene silencing with RNA interference technology. In this study, we constructed a plasmid DNA encoding short hairpin RNA against MCAM (pMCAM) to explore the antitumor and antiangiogenic effects. The experiments were performed in vitro on murine melanoma and endothelial cells, as well as in vivo on melanoma tumors in mice. The antiproliferative, antimigratory, antiangiogenic and antitumor effects were examined after gene therapy with pMCAM. Gene delivery was performed by magnetofection, and its efficacy compared to gene electrotransfer. Gene therapy with pMCAM has proved to be an effective approach in reducing the proliferation and migration of melanoma cells, as well as having antiangiogenic effect in endothelial cells and antitumor effect on melanoma tumors. Magnetofection as a developing nonviral gene delivery system was effective in the transfection of melanoma cells and tumors with pMCAM, but less efficient than gene electrotransfer in in vivo tumor gene therapy due to the lack of antiangiogenic effect after silencing Mcam by magnetofection

Radiosensitizing effect of intratumoral interleukin-12 gene electrotransfer in murine sarcoma

Abstract Background Interleukin-12 (IL-12) based radiosensitization is an effective way of tumor treatment. Local cytokine production, without systemic shedding, might provide clinical benefit in radiation treatment of sarcomas. Therefore, the aim was to stimulate intratumoral IL-12 production by gene electrotransfer of plasmid coding for mouse IL-12 (mIL-12) into the tumors, in order to explore its radiosensitizing effect after single or multiple intratumoral gene electrotransfer. Methods Solid SA-1 fibrosarcoma tumors, on the back of A/J mice, were treated intratumorally by mIL-12 gene electrotransfer and 24 h later irradiated with a single dose. Treatment effectiveness was measured by tumor growth delay and local tumor control assay (TCD50 assay). With respect to therapeutic index, skin reaction in the radiation field was scored. The tumor and serum concentrations of cytokines mIL-12 and mouse interferon γ (mIFNγ) were measured. Besides single, also multiple intratumoral mIL-12 gene electrotransfer before and after tumor irradiation was evaluated. Results Single intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral but not serum mIL-12 and mIFNγ concentrations, and had good antitumor (7.1% tumor cures) and radiosensitizing effect (21.4% tumor cures). Combined treatment resulted in the radiation dose-modifying factor of 2.16. Multiple mIL-12 gene electrotransfer had an even more pronounced antitumor (50% tumor cures) and radiosensitizing (86.7% tumor cures) effect. Conclusions Single or multiple intratumoral mIL-12 gene electrotransfer resulted in increased intratumoral mIL-12 and mIFNγ cytokine level, and may provide an efficient treatment modality for soft tissue sarcoma as single or adjuvant therapy to tumor irradiation.</p

Electrotransfer of Plasmid DNA Encoding an Anti-Mouse Endoglin (CD105) shRNA to B16 Melanoma Tumors with Low and High Metastatic Potential Results in Pronounced Anti-Tumor Effects

Endoglin overexpression is associated with highly proliferative tumor endothelium and also with some tumors, including melanoma. Its targeting has anti-tumor effectiveness, which can also be obtained by RNA interference. The aim of our study was to explore the anti-tumor effectiveness of endoglin silencing by electrotransfer of plasmid DNA encoding short hairpin RNA against endoglin in two murine B16 melanoma variants with different metastatic potential on cells, spheroids and subcutaneous tumors in mice. The results demonstrate that endoglin silencing with gene electrotransfer reduces the proliferation, survival and migration of melanoma cells and also has anti-tumor effectiveness, as the therapy resulted in a high percentage of tumor cures (23% and 58% on B16F1 and B16F10 tumors, respectively). The effectiveness of the therapy correlated with endoglin expression in melanoma cells; in vitro the effects were more pronounced in B16F1 cells, which express more endoglin than B16F10. However, the opposite was observed in vivo in tumors, where there was a higher expression of endoglin and better anti-tumor effectiveness in the B16F10 tumor. In conclusion, targeting endoglin for the treatment of melanoma seems to be a concept worthy of further exploration due to the increased therapeutic effect of the therapy based on simultaneous vascular targeting and its direct effect on tumor cells

Transfection efficacy in tumor and endothelial cells <i>in vitro</i>.

<p>(A) Percent of transfected cells after gene electrotransfer (GET) of CON plasmid (pEGFP-N1) with statistically significant differences (*p<0.05) observed when comparing B16F1 and 2H11 to all of the other cell lines. (B) Percent of transfected cells after GET of TS plasmid (pET-EGFP) was statistically significant ($ p<0.05) when comparing HMEC-1 to all of the other cell lines. In general, higher transfection efficacy was observed with CON plasmid in comparison to TS plasmid. The results represent three independent experiments, n = 3 or more in each group in each experiment. The data represent AM ± SEM.</p

The tumor growth of smaller and bigger tumors was affected after the therapy.

<p>The growth of TS/A tumors exposed to intratumoral injection of endotoxin-free water alone (control group; CTRL) or therapeutic plasmids combined with the application of electric pulses (GET of TS plasmid; GET of CON plasmid), after triple GET of plasmids, was more affected in (A) smaller avascular tumors than in (B) bigger well vascularized tumors. The results represent two independent experiments, n = 6–11 mice for each experimental group in each experiment. The data represent AM ± SEM. N.S. represents statistically non-significant difference between the therapeutic groups and (*p<0.05) in both of the therapeutic groups (GET of CON and TS plasmid) <i>vs</i>. CTRL. Due to the clarity of the results, only main groups are shown.</p

Gene electrotransfer into skin using noninvasive multi-electrode array for vaccination and wound healing.

Skin is an attractive target for gene electrotransfer due to its easy accessibility and its interesting immune properties. Since electrodes are often invasive and frequently induce discomfort during pulse application, there is a fundamental need for non-invasive electrodes for skin delivery. We developed circular pin non-invasive multi-electrode array (MEA), suitable for different clinical applications. MEA was first employed to deliver a luciferase reporter gene. Then, it was used to deliver a DNA vaccine coding for ovalbumin or a plasmid encoding hCAP-18/LL-37 for promoting wound healing. The results demonstrated a strong gene expression and an efficient delivery of both, DNA vaccine and wound healing agent, dependent on the pulses applied. The use of MEA to deliver the ovalbumin plasmid demonstrated a strong immune response, as evidenced by the presence of antibodies in sera, the IFN-gamma response and the delayed tumor growth when the mice were subsequently challenged with B16-OVA cells. The delivery of a plasmid encoding hCAP-18/LL-37 significantly accelerated wound closure. The easy applicability and non-invasiveness of MEA make it suitable for various clinical applications that require gene electrotransfer to skin. Specifically, by adapting electric pulses to the expected action of a transgene, non-invasive MEA can be employed either for vaccination or for wound healing

Endoglin silencing reduced tube formation of 2H11 endothelial cells <i>in vitro</i>.

<p>(A) Original images of tubular complexes in control and therapeutic groups; with addition of endotoxin-free water alone (control group; CTRL) or in combination with the application of electric pulses (EP group), addition of plasmid pET-antiCD105 (TS group), pU6-antiCD105 (CON group) or pU6-SCR (SCR group) alone or combined with the application of electric pulses (GET of TS plasmid; GET of CON plasmid; GET of SCR plasmid); scale bar = 400 μm. (B) The number of complexes normalized on the control group (CTRL); GET of CON plasmid ($ p<0.05) <i>vs</i>. all the other groups except GET of TS plasmid; GET of TS plasmid (* p<0.05) <i>vs</i>. CTRL, TS and EP group. The results represent three independent experiments, n = 3 or more in each group in each experiment for (B) and representative images for (A). The data represent AM ± SEM. N.S. represents statistically non-significant difference between the therapeutic groups.</p