Illustration of FD leakage from blood vessels surrounding the tumor and their constriction after EP and ECT.

<p>Images were acquired at 20×magnification. Control – mice without treatment, Bleomycin – mice treated with bleomycin only, EP – mice treated with EP, ECT – mice treated with ECT. (<b>A</b>) Illustration of FD leakage from blood vessels surrounding the tumor, when FD was injected <i>i.o.</i> before the therapy. Arrows indicate the position of the tumor. Scale bar is 2 mm. (<b>B</b>) Illustration of the constriction of blood vessels surrounding the tumor visualized by FD. The enlarged sections of images were taken at 20×magnification. Scale bar is 500 µm.</p

Timeline of the decrease in FVD and increase in D_V after EP and ECT.

<p>Control – mice without treatment, Bleomycin – mice treated with bleomycin only, EP – mice treated with EP, ECT – mice treated with ECT. (<b>A</b>) The changes in FVD within the tumors are presented as a function of time. (<b>B</b>) The changes of D_V within the tumors are presented as a function of time. n = 3–6. **p<0.05 compared to all other groups, *p<0.05 compared to control and bleomycin groups. Error bars indicate SEM.</p

Quantification of FD leakage kinetics from tumor blood vessels.

<p>Relative mean fluorescence intensity changes as a function of time in the tumor tissue, outside the tumor blood vessels. Control – mice without treatment, Bleomycin – mice treated with bleomycin only, EP – mice treated with EP, ECT – mice treated with ECT. n = 3–5. **p<0.05 compared to all other groups, *p<0.05 compared to control and EP groups. Error bars indicate SEM.</p

Imaging of the leakage of FD from tumor blood vessels into the tumor tissue.

<p>Tumor blood vessels were visualized by fluorescence microscopy at 80×magnification. Control – mice without treatment, Bleomycin – mice treated with bleomycin only, EP – mice treated with EP, ECT – mice treated with ECT. Tumors are marked with a dashed line. Scale bar is 500 µm.</p

Illustration of a “vascular lock” and re-perfusion of tumor blood vessels in the first hour and 4–24 h after EP and ECT.

<p>Tumor blood vessels were visualized by fluorescence microscopy. Control – mice without treatment, EP – mice treated with EP, ECT – mice treated with ECT. (<b>A</b>) FD was injected <i>i.o.</i> 1 min after EP, and images were acquired at 20×magnification at designated times. Arrows indicate the position of the tumor. Scale bar is 2 mm. (<b>B</b>) RhD was injected <i>i.o.</i> at 4, 8 and 24 h after the therapy, and images were acquired 5 min after the injection at 80×magnification. The images are representative of different tumors. Tumors are marked with a dashed line. Scale bar is 500 µm.</p

Characterization of the DWC model.

<p>(<b>A</b>) Filling of tumor blood vessels after <i>i.o.</i> injection of FD and increase of fluorescence intensity in the tumor tissue in control mice. Mean fluorescence intensities were expressed as a percentage of the maximum mean fluorescence intensity (Imax) reached in the observation period. The first image in the series was acquired ∼10 s after the <i>i.o.</i> injection of FD. (<b>B</b>) The timeline of the protocol used in the experiments.</p

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

Skin damage observed under different plasma flow rates.

<p>A) Representative images of the skin damage in the treated region. Treated skin regions were observed under stereomicroscope. Images were taken under visible light (VIS) and the fluorescence was observed under RGB (red, green, blue) parameters. Scale bar: 5 mm. B) The area of the direct skin damage was evaluated from RGB images. Error bars indicate SEM. *P value < 0.05 between the selected group and the group treated under the highest plasma flow rate (treated 4 min under 5 L/min). C) Percentage of indirect skin damage calculated from the total damage (direct and indirect) measured 48 h post-treatment. Damage was statistically evaluated from RGB image. Error bars indicate SEM.</p

Increased tumor necrosis and reduced tumor vascularization after GET of therapeutic plasmids <i>in vivo</i>.

<p>Histologically stained and analyzed sections after treatments of TS/A tumors: intratumoral injection of endotoxin-free water alone (control group; CTRL) or in combination with the application of electric pulses (EP group), injection 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). Triple GET of CON or TS plasmid increased necrosis (HE) and reduced the number of blood vessels (CD105) in TS/A tumors. The percentage of necrosis (upper graph) was statistically significantly increased (*p<0.05) in both of the therapeutic groups (GET of CON and TS plasmid) <i>vs</i>. all the pertinent control groups, with no difference between therapies, also seen in histological sections. The reduced number of blood vessels (CD105) was observed in histological sections of both therapeutic groups that were (lower graph) non-statistically significant to each other after analysis, although the reduction was statistically significant (*p<0.05) <i>vs</i>. all the pertinent control groups. The results represent one experiment, n = 3–4 mice for each experimental group and at least 5 analyzed fields of view for each mouse. The data represent AM ± SEM. N.S. represents statistically non-significant difference between the therapeutic groups. Scale bar = 100 μm.</p

Skin damage observed under different treatment times at constant gas flow rate of 5 L/min.

<p>A) Representative images of the skin damage in the treated region. Treated skin regions were observed under stereomicroscope. Images were taken under visible light (VIS) and the fluorescence was observed under RGB (red, green, blue) parameters. Scale bar: 5 mm. B) The area of the direct skin damage was measured 3 h post-treatment and statistically evaluated from RGB images. Error bars indicate SEM. *P value < 0.05 between the selected group and the group exposed to the longest treatment time (treated 4 min under 5 L/min). C) Percentage of indirect skin damage quantified from the total damage (direct and indirect), measured 48 h post-treatment. Damage was statistically evaluated from RGB image. Error bars indicate SEM.</p