Calcium electroporation induces tumor eradication, long-lasting immunity and cytokine responses in the CT26 colon cancer mouse model

Abstract

Electroporation is used in cancer treatment because of its ability to increase local cytotoxicity of e.g. bleomycin (electrochemotherapy) and calcium (calcium electroporation). Calcium electroporation is a novel anticancer treatment that selectively kills cancer cells by necrosis, a cell death pathway that stimulates the immune system due to high release of antigens and "danger signals." In this exploratory study, we aimed to investigate whether calcium electroporation could initiate an anticancer immune response similar to electrochemotherapy. To this end, we treated immunocompetent balb/c mice with CT26 colon tumors with calcium electroporation, electrochemotherapy, or ultrasound-based delivery of calcium or bleomycin. High treatment efficiency was observed with 100% complete remission in all four groups (12/12 with complete remission in each treatment group). In addition, none of the surviving mice from these groups formed new tumors when re-challenged with CT26 cancer cells 100-d post treatment, whereas mice challenged with different cancer cells (4T1 breast cancer) all developed tumors. Treatment of immunodeficient mice with calcium electroporation and electrochemotherapy showed no long-lasting tumor response. Calcium electroporation and electrochemotherapy was associated with a release of High Mobility Group Box 1 protein (HMGB1) in vitro (p = 0.029) and a significant increase of the overall systemic level of pro-inflammatory cytokines in serum from the treated mice (p < 0.003). These findings indicate that calcium electroporation as well as electrochemotherapy could have a role as immune stimulators in future treatments.

Keywords: CT26 colon cancer model; Calcium electroporation; cytokines; electrochemotherapy; immunity.

Figures

Figure 1.

Tumor response over time. Immunocompetent CT26 balb/c mice randomized into treatment groups, CaEP = calcium electroporation, CaUS = calcium ultrasound, BleoEP = electrochemotherapy, BleoUS = bleomycin ultrasound, Ca = calcium alone, Bleo = bleomycin alone, EP = electroporation alone, US = ultrasound alone. After one treatment tumor volume was measured over time, n = 12 (means + SD). (A) Calcium electroporated compared with control groups p < 0.001 (B) Calcium ultrasound compared with control groups p < 0.001 (C) electrochemotherapy compared with control groups p < 0.001 (D) Bleomycin ultrasound compared with control groups p < 0.001.

Figure 2.

Survival curves after re-challenge. Surviving mice and naïve mice were re-challenged with the same tumor cell type (CT26) or a different tumor cell type (4T1) and observed for 50 d after inoculation. CT26 CaEP /4T1 CaEp = mice treated with calcium electroporation, inoculated with CT26 and 4T1, respectively; CT26 CaUS/4T1 CaUS = mice treated with calcium ultrasound, inoculated with CT26 and 4T1, respectively; CT26 BleoEP/4T1 BleoEP = mice treated with electrochemotherapy, inoculated with CT26 and 4T1, respectively; CT26 BleoUS/4T1 BleoUS = mice treated with bleomycin ultrasound, inoculated with CT26 and 4T1, respectively; CT26 naïve/4T1 naïve = naïve mice inoculated with CT26 and 4T1, respectively. N = 5 in each group. (A) Calcium electroporation compared with control groups, p = 0.004–0.008 (B) Calcium ultrasound compared with control groups, p = 0.006–0.008 (C) electrochemotherapy compared with control groups, p = 0.004–0.009 (D) Bleomycin ultrasound compared with control groups, p = 0.002–0.007.

Figure 3.

Heat map demonstrating the level of proinflammatory cytokines in serum 3 and 7 d after treatment. The color bar on the left represent values fold increase relative to untreated control. Values above 4-fold are represented as white. (1) Calcium electroporation showed no difference in cytokine level day 3 (p = 0.5) but a significant increased level day 7 (p<0.0001). (2) Electrochemotherapy showed a significant increase in cytokine level day 3 (p = 0.003) but no difference at day 7 (p = 1). (3) Electroporation alone showed a significant increase in cytokines both day 3 (p<0.0001) and day 7 (p<0.0001) and a noticeable increase at day 3 of G-CSF. (4) Calcium alone and bleomycin alone showed no difference in cytokine level day 3 (p = 1) but a significantly increased level day 7 (p<0.0001). (5) treatment with ultrasound showed no significant difference day 3 or 7. More detailed graph is shown in supplementary 1.

Figure 4.

(A) Tumor response over time. NMRI-Foxn1nu mice randomized into treatment groups, CaEP = calcium electroporation, BleoEP = electrochemotherapy, Ca = calcium alone, Bleo = bleomycin alone, EP = electroporation alone, n = 11–12 (means + SD). After one treatment tumor volume was measured over time. The individual graphs terminates when n < 4. The difference in tumor volume between the six groups was not statistical significant (p = 0.07–1.0). (B) Survival curves from the same treatment groups show no statistical difference in survival between untreated mice and the five treatment groups (p = 0.06–0.8).

Figure 5.

HMGB1 release measured in the supernatant of CT26 cells treated with CaEP = calcium electroporation, BleoEP = electrochemotherapy, Ca = calcium alone, Bleo = bleomycin alone, EP = electroporation alone, n = 4 (normalized means + SD). The supernatants was collected 30 min and 72 hafter treatment. After 30 min there no significant difference in extracellular HMGB1 concentration between the groups. At 72 h an 7-fold increase is observed for Calcium electroporation, electrochemotherapy and electroporation alone (p = 0.029) there was no statistical increase in calcium and bleomycin alone (p = 0.2).