Fig 1.
Study workflow and associated analyses.
(A) Mice were inoculated on their right flank with CT26 cells and allowed to grow for 14 days. Tumors were then locally ablated. After 3 and 7 days post-treatment, the mice were sacrificed and tumors harvested for immunohistochemistry analysis. Serum was also collected at each time point for conducting cytokine analyses. (B) Mice were inoculated on both flanks with CT26 cells. After 14 days, the primary tumor was ablated. Primary and contralateral tumors harvested after 7 days post-treatment for immunohistochemical analysis. Created with Biorender.com.
Table 1.
Overview of ablation treatments used in each experiment and relevant endpoints.
Fig 2.
Pro-inflammatory cytokine expression induced by EC-ethanol ablation.
The fold-change of (A) IL-6 and (B) KC/GRO over a period of 7 days (n = 8-10). Error bars are standard error of the mean (SEM). *P < 0.10, **P < 0.05, ***P < 0.01, ****P < 0.001.
Fig 3.
Visualization of the tumor ablation zone induced by EC-ethanol.
Representative H&E-stained tumors treated by (A,E) EC-ethanol at 3 and 7 days post-ablation or (C,G) sham treatments at 3 and 7 days post-ablation. Scale bars = 2 mm. Magnified inserts of the necrotic regions at 20x for tumors treated by (B,F) EC-ethanol at 3 and 7 days post-ablation or (D,H) sham treatments at 3 and 7 days post-ablation are shown to illustrate the patterns of coagulative (red star) and liquefactive (green arrow) necrosis. Scale bars = 200 µm. (I) Quantification and comparison of the amount of necrosis within the tumor between EC-ethanol and sham treatment groups (n = 5-8). Error bars are standard error of the mean (SEM). *P < 0.10, **P < 0.05, ***P < 0.01, ****P < 0.001.
Fig 4.
Increased infiltrating cytotoxic T-cells into the ablated tumor after EC-ethanol ablation.
Representative IHC-stained tumors for CD8a treated by (A,B) EC-ethanol at 3 and 7 days post-ablation or (C,D) sham treatments at 3 and 7 days post-ablation. UT = untreated tumor. Scale bars = 2 mm. (E) Quantification and comparison of the amount of CD8a + CTLs within the tumor and (F) within the ablation zone only (denoted as the region bordered in yellow dashed lines) between EC-ethanol and sham treatment groups (n = 3-5). *P < 0.10, **P < 0.05, ***P < 0.01, ****P < 0.001.
Fig 5.
Substantial accumulation of cytotoxic T-cells around the margins of the ablation zone.
Closeups of the EC-ethanol ablation zone margins after (A) 3 days (black box) and (B) 7 days (red box), with the margins (region denoted with black arrow and boundary delineated with yellow and green dashed lines) defined as 400 µm out from the ablation zone perimeter. Quantification and comparison of the amount of CD8a + CTLs within the margins at (C) 3 and (D) 7 days post-ablation (n = 3-5). Error bars are standard error of the mean (SEM). Scale bars = 300 µm. *P < 0.10, **P < 0.05, ***P < 0.01, ****P < 0.001.
Fig 6.
Increased infiltrating cytotoxic T-cells in the contralateral tumor after ablative treatments.
Representative IHC-stained contralateral tumors for CD8a treated by (A) sham, (B) EC-ethanol, (C) RFA, or (D) combination (EC-ethanol + RFA) at 7 days post-ablation. Scale bars = 400 µm. Quantification and comparison of the amount of CD8a + CTLs within (E) the entirety of the contralateral tumor and (F) within the margins of the tumor periphery (n = 5-6). Error bars are standard error of the mean (SEM). *P < 0.10, **P < 0.05, ***P < 0.01, ****P < 0.001.