High-frequency irreversible electroporation is an effective tumor ablation strategy that induces immunologic cell death and promotes systemic anti-tumor immunity

Abstract

Background: Despite promising treatments for breast cancer, mortality rates remain high and treatments for metastatic disease are limited. High-frequency irreversible electroporation (H-FIRE) is a novel tumor ablation technique that utilizes high-frequency bipolar electric pulses to destabilize cancer cell membranes and induce cell death. However, there is currently a paucity of data pertaining to immune system activation following H-FIRE and other electroporation based tumor ablation techniques.

Methods: Here, we utilized the mouse 4T1 mammary tumor model to evaluate H-FIRE treatment parameters on cancer progression and immune system activation in vitro and in vivo.

Findings: H-FIRE effectively ablates the primary tumor and induces a pro-inflammatory shift in the tumor microenvironment. We further show that local treatment with H-FIRE significantly reduces 4T1 metastases. H-FIRE kills 4T1 cells through non-thermal mechanisms associated with necrosis and pyroptosis resulting in damage associated molecular pattern signaling in vitro and in vivo. Our data indicate that the level of tumor ablation correlates with increased activation of cellular immunity. Likewise, we show that the decrease in metastatic lesions is dependent on the intact immune system and H-FIRE generates 4T1 neoantigens that engage the adaptive immune system to significantly attenuate tumor progression.

Interpretation: Cell death and tumor ablation following H-FIRE treatment activates the local innate immune system, which shifts the tumor microenvironment from an anti-inflammatory state to a pro-inflammatory state. The non-thermal damage to the cancer cells and increased innate immune system stimulation improves antigen presentation, resulting in the engagement of the adaptive immune system and improved systemic anti-tumor immunity.

Keywords: Breast cancer; IRE; Metastasis; Pyroptosis; Tumor microenvironment.

Fig. 1

High Frequency Irreversible Electroporation (H-FIRE) Induces Inflammatory Cell Death and Effectively Attenuates the Tumor Promoting Microenvironment In Vitro. A total of 200H-FIRE waveforms were delivered once per second with a uniform voltage of either 240 V, 800 V, or 1600 V across the 4 mm cuvette totaling 500, 2000, or 4000 V/cm, respectively. A. Schematic of H-FIRE delivery to 4 T1 cells in a 4 mm cuvette. B. Temperature during in vitro H-FIRE treatment. At 2000 V/cm, cell suspension temperature remains below 30 °C. C. Influence of electric pulse parameters on cell viability. Cell viability was determined via trypan blue (manual) and AO/PI staining (automated); the average percent viability shown. LDH Activity was utilized to evaluate cell death at 2000 V/cm over time. D-H. Real time PCR-based gene expression arrays were utilized to evaluate the expression of 162 genes associated with cancer and inflammation. D. Heat map of gene expression changes at 2, 8, and 24 h following H-FIRE treatment with 2000 V/cm. E. Ingenuity Pathway Analysis (IPA) of gene expression data revealed significant shifts in pathways associated with inflammation, injury/repair, and cell death. F. Gene expression analysis further indicates that necrosis and pyroptosis are the dominant forms of cell death 24 h post-H-FIRE treatment. The z-score reflects the activation of the pathway at the time point shown. G. IPA identified significant changes in pathways associated with reactive oxygen species, adenosine triphosphate, and HMGB1 signaling. All studies were repeated at least 3 times. *p < 0.05; ***p ≤ 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

H-FIRE Treatment Results in Significant Tumor Ablation in the In Vivo 4 T1 Mammary Tumor Model. Mice were injected with 1.2 × 10⁶ 4 T1 cells into the mammary fat pad of wild type BALB/c mice. On day 11, mice were anesthetized and needle electrodes were inserted into the tumor to deliver 200H-FIRE waveforms once per second at 2500 V/cm (voltage-to-distance ratio). Animals were monitored a least 3 times/week for clinical parameters and tumor progression. A. Schematic illustrating treatment strategy and electrode placement. B. Tumor diameter changes following H-FIRE reveals significant decreases in tumor diameter and progression. C. Images of tumors from representative animals 15 days post-H-FIRE treatment. 4 T1 tumor/H-FIRE sham treatment (top panels); 4 T1 tumors and H-FIRE treatment (middle panels); no tumor/no H-FIRE treatment (bottom panels). D. A zoomed-in plot of accelerometer output at 2000 V/cm (voltage-to-distance ratio) IRE. E. A zoomed-in plot of accelerometer output at 2000 V/cm (voltage-to-distance ratio) H-FIRE. n = 3–10 mice in each experimental group. ***p ≤ 0.001.

Fig. 3

H-FIRE Ablation Results in Significant Cell Death and Inflammation. Histopathology evaluation revealed significant signs of cell death in the 4T1 mammary tumor that was increased following H-FIRE. A. The architecture of the untreated 4T1 tumors remains intact, with aggressively proliferating neoplastic cells obliterating normal subcutaneous structures. B. The central core of these large tumors often contains abundant necrosis, and can be visually distinguished from the rest of the neoplasm (blue line). C. Following H-FIRE application, neoplastic cells in the treatment zone undergo cell death as evidenced by nuclear pyknosis and loss, cytoplasmic blanching, and disintegration of cellular architecture. D. Pathology assessments of the H-FIRE treated areas revealed a significant decrease in histologically identifiable mammary tumors. E. Gene expression arrays were utilized to evaluate 156 genes associated with cancer and inflammation. IPA analysis revealed that necrosis and pyroptosis are significantly increased and are the dominant pathways associated with cell death following H-FIRE treatment compared to the untreated 4T1 tumors in vivo. F. Typical 4T1 tumors are relatively immunosuppressive with minimal immune cell infiltration. G-H. Following H-FIRE, histopathology revealed increased inflammation. This inflammation could be further sub-classified as either (G) mild or (H) moderate. n = 3–10 mice in each group. *p ≤ 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Tumor Response to H-FIRE Ablation is Correlated with Increased Cellular Immunity. At necropsy 15 days post H-FIRE, tumor specimens were collected and total RNA was extracted for gene expression profiling. Data were analyzed based on the % of tumor ablation following H-FIRE (low response≤85% ablation; average response = 85%–94% ablation; high response≥95% ablation). A. The up-regulation of genes associated with cellular immunity are strongly correlated with tumor ablation. In tumors classified in the low-responder group, a significant number of genes associated with cellular immunity were down-regulated. Conversely, a significant number of genes were up-regulated in the animals that saw the highest levels of tumor ablation. B. Chemokine signaling networks were the most impacted by H-FIRE treatment, with highly responsive tumors demonstrating a significant up-regulation compared to the other groups. The z-scores shown reflect the level of signaling pathway activation between each group. C. Further pathway analysis identified 6 additional signaling networks that were significantly impacted by H-FIRE treatment response. We observed a significant decrease in breast cancer-associated gene expression and immunosuppression from untreated to high therapeutic response. Conversely, we observed a significant increase in gene expression associated with inflammation, cytotoxicity, and recruitment of leukocytes over the same treatment scale. We also observed a significant increase in IL-17 signaling and antigen presentation that was highest in the animals with average responses. n = 3–10 mice in each group.

Fig. 5

H-FIRE Treatment Significantly Alters Local Immune Cell Populations in the 4 T1 Tumor Microenvironment. Mice were treated with H-FIRE and tumors were harvested at 2 and 7 days post-treatment. Single cell suspensions were generated from each tumor and labeled for flow cytometry. A. 2 days after H-FIRE, the treated groups showed significant reduction in CD11b⁺LY6G⁺ cells, representing neutrophil populations, and CD11b⁺LY6G⁺Ly6C^loCD45⁻ cells, representing pMDSC populations. B. Significant decreases were observed in CD4⁺CD45⁺ T helper cells, while significant increases were observed in CD4⁺CD45⁺CD25^hiCD127^loFoxp3⁺ Treg population. C. CD11b⁺Ly6G⁻Ly6C⁻CD45⁺F4/80⁺ Tumor Associated Macrophage populations were significantly decreased 7 days post-treatment. n = 4 mice in each group. *p ≤ 0.05; **p ≤ 0.01.

Fig. 6

Local H-FIRE Treatment Attenuates Metastatic Lesions in Immunocompetent mice. Lung and blood metastasis is typical in the 4 T1 tumor model. A. Histopathology evaluation of the lungs revealed 4 T1 metastatic lesions in all of the animals that were not treated with H-FIRE. B. Pathologic enumeration of metastatic lesions revealed a significant decrease in lung metastases in animals 15 days post-H-FIRE treatment compared to untreated animals. C. Gene expression in the primary tumor post-H-FIRE revealed a significant decrease in signaling pathways associated with tumor metastasis in animals with tumors that were highly responsive to H-FIRE ablation. D-E. Metastasis attenuation depends on an intact immune system. NOD Scid Gamma (NSG) mice were injected with 1.2 × 10⁶ 4T1 cells into the mammary fat pad, treated with H-FIRE (2–5-2 waveform, 2500 V/cm), and tumor progression was monitored. D. A significant attenuation in tumor progression was observed in the NSG mice; however, no tumors reached complete ablation. E. Whole blood was collected from both NSG and BALB/c mice and plated under 6-thioguanine selection. A significant number of metastatic cells were observed in the NSG mice, regardless of H-FIRE treatment. In the BALB/c mice, a significant reduction in average circulating 4 T1 metastatic cells was observed in animals treated with H-FIRE. F-G. 4 T1 cells were treated with either H-FIRE (2–5-2 waveform, 2000 V/cm) or cryoablation (liquid Nitrogen to 37 °C, 3 freeze-thaw cycles). Cell-free lysates were generated and injected (i.v.) into wild type BALB/c mice. Mice were injected with 1.2 × 10⁶ 4T1 cells into the mammary fat pad 10 days post-injection of cell-free lysate. F. At necropsy, the calculated tumor diameter of mice treated with the H-FIRE lysate was significantly decreased compared to both untreated tumor only (19% reduction) and cryoablation lysate (17% reduction). G. Circulating blood 4 T1 metastatic cells were quantified at necropsy. Both cyroablation lysate and H-FIRE lysate treatment significantly attenuated 4T1 metastasis. n = 3–10 mice in each group. *p ≤ 0.05; **p ≤ 0.01.