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. 2023 May 25:14:1189960.
doi: 10.3389/fimmu.2023.1189960. eCollection 2023.

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent

Barbara Lisec  1 , Bostjan Markelc  1   2 , Katja Ursic Valentinuzzi  1   3 , Gregor Sersa  1   2 , Maja Cemazar  1   4
Affiliations

The effectiveness of calcium electroporation combined with gene electrotransfer of a plasmid encoding IL-12 is tumor type-dependent

Barbara Lisec et al. Front Immunol. .

Abstract

Introduction: In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT.

Methods: The effect of CaEP was tested in vitro as well as in vivo in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells.

Results: In vitro, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed in vivo; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature.

Conclusions: Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

Keywords: bleomycin (BLM); calcium; calcium electroporation; electrochemotherapy (ECT); gene electrotransfer (GET); interleukin 12 (IL12); murine tumor models.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Electric pulses application and treatment protocol. (A) Injection site for p.t. GET with non-invasive multi-electrode array (MEA) and distribution of electric pulses between the pins arranged in a circle. (B) i.t. injection of calcium, bleomycin and/or pIL-12 and distribution of electric pulses between two parallel stainless-steel electrodes. (C) Study design of IL-12 p.t. GET and i.t. CaEP or ECT. The treatment was performed when tumors reached ~40 mm3. For i.t. treatment, calcium/bleomycin/pIL-12 or mixture of calcium/bleomycin with pIL-12 was injected prior electroporation (*). For p.t. treatment, pIL-12 was injected in 4 places in tumor proximity prior electroporation.
Figure 2
Figure 2
Viability of B16-F10 and 4T1 cell lines after (A) CaEP and (B) ECT measured after 72 h. Dotted lines represent IC50 values. P value (P < 0.05) is color coded and calculated vs –EP or +EP control cells. The values are presented as AM ± SE. **P 0.001 – 0.01; ***P 0.0001 – 0.001; ****P <0.0001.
Figure 3
Figure 3
Effectiveness of CaEP or ECT with bleomycin is different in B16-F10 and 4T1 tumors. The response to the therapy is presented by (A, B) tumor growth delay and (C, D) Kaplan-Meier graphs. In (A, B) each dot represents one animal in violin plots showing distribution of data. ns P≥0.05; *P 0.01 – 0.05; **P 0.001 – 0.01; ***P 0.0001 – 0.001; ****P <0.0001.
Figure 4
Figure 4
Tumor growth delay and Kaplan-Meier graphs of B16-F10 tumors in response to different treatments. Survival of the animals is significantly prolonged after (A) IL-12 i.t. EP alone or in combination with IL-12 p.t. GET. ns P≥0.05; *P<0.05. Adjuvant IL-12 did not significantly contributed to (B) Ca injection without electric pulses, but IL-12 p.t. GET significantly prolonged survival of mice after (C) CaEP and resulted in tumor cures. (D) Combination of i.t. IL-12 and Ca with EP and IL-12 p.t. GET significantly contributed to CaEP with no tumor cures. (E) No tumor growth delay was observed after BLM injection, whereas significantly prolonged survival was observed after (F) ECT with IL-12 p.t. GET and (G) ECT with i.t. IL-12 and IL-12 p.t. GET.
Figure 5
Figure 5
Tumor growth delay and Kaplan-Meier graphs of 4T1 tumors in response to different treatments. Survival of the animals is significantly prolonged after (A) IL-12 i.t. EP and i.t. with IL-12 p.t. GET. (B) Ca injection resulted in tumor cures, with no contribution of adjuvant IL-12 p.t. GET, also after (C) CaEP with IL-12 p.t. GET and (D) CaEP with i.t. and p.t. IL-12 GET. ns P≥0.05; *P<0.05 (E) No tumor growth delay was observed after BLM injection, whereas significantly prolonged survival was obtained after (F) ECT with IL-12 p.t. GET and (G) ECT with i.t. IL-12 and IL-12 p.t. GET.
Figure 6
Figure 6
GET of IL-12 impacts infiltration of immune cells to B16-F10 tumors. Tumors were collected on day 3 after treatment for histological analysis. Frozen sections of tumor tissue were stained with (A) anti-CD4 (green, Alexa 488) and anti-F4/80 (red, Alexa 647), (B) anti-CD8 (green, Alexa 488) and anti-NKp46 (red, Alexa 647) and (C) anti-Ki-67 (orange, Cy3) and anti CD31 (red, Alexa 647). Nuclei (blue) were stained with Hoechst 33342. Scale bar: 100 μm.
Figure 7
Figure 7
GET of IL-12 impacts infiltration of immune cells to 4T1 tumors. Tumors were colected on day 3 after treatment for histological analysis. Frozen sections of tumor tissue were stained with (A) anti-CD4 (green, Alexa 488) and and anti-F4/80 (red, Alexa 647), (B) anti-CD8 (green, Alexa 488) and anti-NKp46 (red, Alexa 647) or (C) anti-Ki-67 (orange, Cy3) and anti CD31 (red, Alexa 647). Nuclei (blue) were stained with Hoechst 33342. Scale bar: 100 μm.
Figure 8
Figure 8
Statistical analysis and graphical presentation of immunfluorescence data from frozen tumor tissue sections. Graphs represent percentage of Ki-67 positive cells and percentage of tumor vessels after different treatments in (A, B) B16-F10 and in (C, D) 4T1 tumors. The values are presented with violin plots with data distribution. ns P≥0.05; *P 0.01 – 0.05; ****P <0.0001.
Figure 9
Figure 9
Statistical analysis and graphical presentation of immunfluorescence data from frozen tumor tissue sections. Graphs represent percentage of CD4 and CD8 positive cells, percentage of NKp46 and F4/80 positive area after different treatments in (A-D) B16-F10 and in (E-H) 4T1 tumors. The values are presented with violin plots with data distribution. ns P≥0.05; *P 0.01 – 0.05; **P 0.001 – 0.01; ****P <0.0001.
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