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15 Pulsed electric field therapy increases immune response and improves murine 4T1 breast tumor local and metastatic outcomes relative to radiofrequency ablation
  1. Ebtesam Nafie1,
  2. Chiara Pastori2,
  3. Mukta Wagh2,
  4. Ella Goldschmidt2 and
  5. Robert Neal2
  1. 1Galvanize Therapeutics Inc., San Carlos, CA, USA
  2. 2Galvanize Therapeutics Inc., Redwood City, CA, USA
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Pulsed Electric Field (PEF) ablation kills cells via non-thermal processes, offering improved safety and distinct immunomodulatory effects versus Radiofrequency (RFA) thermal ablation. A proprietary PEF system with an optimized immunostimulatory waveform was modified for murine treatments and previously shown to induce local and systemic anti-tumor immunity in conjunction with aPD-1.1–3 This study compares the immune responses stimulated by this PEF versus RFA, particularly regarding innate and adaptive immune cell populations.

Methods PEF or RFA treatments with equivalent ablation volumes were delivered to 4T1 orthotopic mammary murine tumors, followed by tumor resection seven days later (figure 1). Sham mice did not receive energy but underwent resection at the same timepoint. Immune cell populations in the resected tumors, including dendritic cells (DCs), Natural Killer cells, monocytic-derived immune suppressor cells (mMDSCs), neutrophils, M1 macrophages, and M2 macrophages, were assessed using flow cytometry. Flow cytometry was also used to quantify circulating regulatory T-cells and to measure tumor-specific CD8+ T-cell activation using gp70 tetramers in blood samples taken 21 days after treatment. Survival from metastatic burden was monitored.

Results PEF-treated mice significantly suppressed tumor growth and increased survival compared to RFA-treated mice despite matched partial ablation volumes (figure 2). Relative to sham controls, PEF ablation induced significant increases in 7-day intratumoral DCs (p=0.009), NK cells (p=0.006), neutrophils (p=0.01), and M1 Macrophages, while decreasing the immunosuppressive mMDSCs (p=0.001) and M2 macrophages (p=0.05) (figure 3). Conversely, RFA did not influence any of these cell populations relative to sham. At 21-days post-treatment, circulating T-cells increased in the PEF group relative to sham and RFA groups (figure 4A). Further, gp70 tetramer assay showed a significant increase in Tet+CD8+ T cells in PEF versus sham and RFA mice (figure 4B). Finally, PEF did not significantly affect regulatory T-cells, while RFA substantially increased this immunosuppressive cell population (figure 4C). Examination of the lungs in mice euthanized for burden confirmed lung metastases, while those surviving to the terminal endpoint confirmed the absence of metastasis, indicating systemic immune anti-cancer protection (figure 5).

Conclusions PEF with a proprietary immunostimulatory waveform significantly increased innate and adaptive anti-cancer immune cell populations in both short- and long-term timeframes. In contrast, RFA thermal ablation had minimal impact and even increased certain immunosuppressive cell populations. As a result, PEF treatment enhanced tumor response and improved survival compared to RFA treatment. These findings indicate that the PEF employed in this study may induce a beneficial immunostimulatory profile and better outcomes in cancer patients versus thermal ablation.


  1. C Pastori, M Wagh, E Nafie, F Murad, R Neal. Pulsed Electric Field (PEF) Ablation Invokes Different Immune Cytokine Profile and Tumor Response than Radiofrequency Thermal Ablation for Matched Ablation Volumes in the EMT6 Mouse Model. SIR 2023, Phoenix, AZ.

  2. E Nafie, C Pastori, M Wagh, F Murad, M Trikha, R Neal. Pulsed electrical fields in combination with anti-PD1 and survival of mice with TNBC (EMT6) murine breast tumor. Journal of Clinical Oncology 2023;41(16_suppl):540–540

  3. E Nafie, C Pastori, M Wagh, M Trikha, R Neal. Pulsed electric fields combined with anti-PD1 prolongs survival and triggers an adaptive immune response in an IO-non-responsive orthotopic mouse model. Cancer Research 2023;83(7_Supplement):6638.

Abstract 15 Figure 1

Preclinical Study on 4T1 Murine Mouse Model – Ablation Modalities Comparison. Schematic representation of the experimental design. PEF (Pulsed Electric Field) and RFA (Radiofrequency) ablation were applied at Treatment Day 0 (12 days after tumor inoculation). Tumors were resected at Treatment Day 7 (including sham controls). Flow cytometry was performed on resected tumor samples and in blood at Treatment Day 21. Mouse survival was monitored for 80 days post-treatment.

Abstract 15 Figure 2

Survival of 4T1 murine model. Kapan-Meier of mouse survival for each treatment group. All mice underwent primary tumor resection on Treatment Day 7. Thus mortality reflects systemic metastatic burden.

Abstract 15 Figure 3

Innate immune response in 4T1 Tumor microenvironment, 7 days post treatment. Intratumoral Innate Immune Cell Populations. Flow cytometry on tumors resected at Treatment Dat7. (A) Dendritic cells (CD11c+/MHC-11+), (B) Natural killer cells (NKp46+), (C) Neutrophils (CD11b+ Ly6G+), (D) Monocytic myeloid-derived suppressor cells (mMDSCs) (CD11b+ Ly6G- Ly6C high), (E) Macrophages M1 (CD11b+ F4/80+ CD11c+), (F) Macrophages M2 (CD11b+ F4/80+ CD206+). Statistical significance is indicated by p-values: *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

Abstract 15 Figure 4

Adaptive immune response in 4T1 Tumor microenvironment, 21 days post treatment. Systemic Adaptive Immune Response. Flow cytometry of blood at Treatment Day 21. (A) Percentage of activated T-cells (CD28+), (B) Antigen-specific T cells (gp70+/CD8+), and (C) Regulatory T cells were quantified. Statistical significance is indicated by p-values: *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001.

Abstract 15 Figure 5

PEF treatment reduces metastasis in the lung. Representative scan of H&E-stained lungs. (A) Untreated group: Despite tumor resection on day 0, extensive lung metastatis was observed on day 44, indicating significant tumor progression. (B) Sham group: Lung metastatic lesions were present at the time of death on day 26, suggesting that the surgical procedure alone did not prevent the development of lung metastasis. (C) RFA group: Lung metastasis was still evident at the time of death on day 58, indicating that radiofrequency ablation treatment did not fully eliminate metastatic spread to the lungs by that point. (D&E) Complete response: Absence of metastasis in euthanized mice from PEF group at time of death day 80. Indicating successful prevention of metastasis through Partial Electrical Fields treatment.

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