CD3⁺ Immunohistochemsistry of primary (T1) tumors.

<p>CD3⁺ staining, indicative for T-cell presence, performed for (A,C) untreated and (B,D) treated initial T1 tumors between (A,B) ID nude and (C,D) IC BALB/c mice. There is no notable difference observed in CD3⁺ infiltration for ID nude mice between (A) untreated and (B) treated tumors. For the IC BALB/c mice, a robust increase in CD3⁺ (T-cell) infiltration is observed in some treated tumors (D) relative to untreated T1 controls (C). Increased T-cell presence in treated T1 IC mice was also more robust than for both groups for nude mice (A,B). All scale bars 200 µm. Panels (A,C,D) 200x, panel (B) 400x magnification.</p

Original inoculation tumor volume curves.

<p>(A) Imputed median primary tumor volumes (mm³) for each group, showing significantly smaller median tumor size in the treated immunocompetent (IC) BALB/c mice. (B–E) Individual trial tumor comparisons between (B,D) immunodeficient (ID) nude and (C,E) immunocompetent mouse strains for (B,C) sham and (D,E) pulsed treatment groups, showing a clear improvement in progressive disease free survival for the treated IC mice (E) relative to both controls and treated ID mice (B–D). Treated ID mice show an initial pause in tumor volume for the first 12 days from the treatment, followed by progressive growth, a result of selecting a sub-optimal IRE pulsing protocol for this study. All endpoints in data are a result of euthanasia due to tumor reaching 18 mm in any dimension.</p

Haematoxylin and Eosin staining of various treatment groups.

<p>Histology of different inoculation conditions for (A–D) immunodeficient (ID) Nude mice and (E–H) immunocompetent (IC) BALB/C mice. (A,E) untreated initial tumors, (B,F) matrigel controls, (C,G), treated initial tumors (T1), and (D,H) untreated rechallenge tumors (T2). There is little overall immune reaction present in (A,B,E,F), the untreated T1 tumors and untreated matrigel controls, with no appreciable immunocyte presence difference between ID and IC groups. ID treated T1 (C) and untreated T2 (D) tumors had a relatively low immune response, with some neutrophils present. Arrows in (C,G) denote transition between dead and viable tumor cells in both group treated T1s. Treated IC T1 tumors (G) had most immune reaction at the transition zone between dead and viable tumor. Untreated T2 rechallenge tumors in the IC mice (H) show the presence of lymphocytes (arrowheads) and polymorphonuclear leukocytes (arrows). All scale bars 200 µm. Panels (A,B,C,E,F,G) 200x and (D, H) 400x magnification.</p

Kaplan-Meier survival plot.

<p>Survival of the different experimental groups based on euthanasia timepoints resulting from excessive tumor burden, when tumors reached 18 mm in any given dimension. The total experimental period was 34 days post-IRE treatment. Where 70% of the treated immunocompetent (IC) BALB/c mice survived until the endpoint of the study, nearly all control IC mice and both experimental immunodeficient (ID) nude mice groups were euthanized prior to reaching the endpoint due to excessive tumor burden.</p

Primary and rechallenge BALB/c tumor growth curves.

<p>Growth curves for immunocompetent BALB/c mice for tumors grown after the (A) first inoculation and (B) rechallenge inoculation, which occurred 18 days after treatment of the first tumor. Average time for first inoculation tumors to reach minimum protocol treatment size of approximately 5×5 mm across and 4 mm deep was 9.7 days. Rechallenge inoculation tumors showed significantly delayed or completely inhibited growth of contralateral rechallenge tumors from the second inoculation.</p

Schematic of experimental setup.

<p>Plate electrodes are placed on either side of the subcutaneous murine tumor, with a highly conductive gel facilitating improved current delivery into the tumor. Two small electrodes are inserted directly into the tumor perpendicular to the plate electrodes to measure the voltage drop between them, ensuring adequate electric field distribution through the center of the tumor. Treated groups had IRE electric pulses delivered through the plate electrodes, while controls had no pulse delivery.</p

Primary tumor volume means and standard deviations.

<p>Mean tumor volume for all surviving mice at days 10, 15, 20, and 25 post-treatment. Mann-Whitney statistical significance was calculated between immunodeficient (ID) nude mice and immunocompetent (IC) mice that received treatment or sham control, showing statistically significant difference in mean tumor size for the groups at different timepoints, where treated ID mice show a difference at days 10 and 15, while treated IC mice show a significant difference for all time points considered. *0.05>p>0.01; **0.01>p>0.001; ***p<0.001.</p