Background: Electrochemotherapy (ECT) is emerging as a powerful strategy to induce immunogenic cell death and profoundly reshape the tumor microenvironment. However, the specific immunomodulatory role of electric pulses, independently of chemotherapeutic agents, remains to be fully elucidated. This study investigates the potential of high-voltage electroporation, as used in ECT protocols, to stimulate immune responses in breast cancer. Materials and Methods: A 3D hydrogel-based culture system was used to generate uniform spheroids from HCC1954 breast cancer cells. Electroporation was applied with increasing electric field strengths (0–1000 V/cm) to modulate membrane permeability. Spheroids were then co-cultured with phytohemagglutinin-stimulated Jurkat cells or primary human CD4+/CD8+ T lymphocytes. Electroporation efficiency was assessed using propidium iodide (PI) staining. T cell-mediated cancer cell mortality was assessed through cancer cell viability assay via flow cytometry. Expression of Interleukin-2 (IL-2), Tumour Necrosis Factor alpha (TNF-α), and Interferon gamma (IFN-γ) was evaluated to assess T cell immune activation. Results: Electroporation at 600 and 1000 V/cm caused marked structural changes in the spheroids. At 1000 V/cm, a necrotic core was observed, surrounded by a layer of viable but stressed cells, whereas 600 V/cm resulted in a favorable proportion of successfully electroporated cells while preserving overall cell viability. These heterogeneous responses promoted differential immune recognition and cytotoxic targeting of cancer cells in co-culture experiments with immune cells. In co-culture, spheroids treated at 600 V/cm increased T cell activation and promoted targeted tumor cell killing. These effects were confirmed using both Jurkat cells and primary human lymphocytes. Gene expression analysis revealed upregulation of pro-inflammatory cytokines, indicating enhanced immunogenicity following high-voltage electroporation. Conclusions: High-voltage electroporation alone can increase the immunogenic potential of breast cancer cells and promote T cell-mediated anti-tumor activity. These findings support its application as a standalone immunomodulatory strategy and lay the groundwork for its integration into combined immunotherapeutic approaches.
Electroporation of 3D-Cultured Breast Cancer Cells Elicits T Lymphocyte-Mediated Killing
Sieni E.;
2025-01-01
Abstract
Background: Electrochemotherapy (ECT) is emerging as a powerful strategy to induce immunogenic cell death and profoundly reshape the tumor microenvironment. However, the specific immunomodulatory role of electric pulses, independently of chemotherapeutic agents, remains to be fully elucidated. This study investigates the potential of high-voltage electroporation, as used in ECT protocols, to stimulate immune responses in breast cancer. Materials and Methods: A 3D hydrogel-based culture system was used to generate uniform spheroids from HCC1954 breast cancer cells. Electroporation was applied with increasing electric field strengths (0–1000 V/cm) to modulate membrane permeability. Spheroids were then co-cultured with phytohemagglutinin-stimulated Jurkat cells or primary human CD4+/CD8+ T lymphocytes. Electroporation efficiency was assessed using propidium iodide (PI) staining. T cell-mediated cancer cell mortality was assessed through cancer cell viability assay via flow cytometry. Expression of Interleukin-2 (IL-2), Tumour Necrosis Factor alpha (TNF-α), and Interferon gamma (IFN-γ) was evaluated to assess T cell immune activation. Results: Electroporation at 600 and 1000 V/cm caused marked structural changes in the spheroids. At 1000 V/cm, a necrotic core was observed, surrounded by a layer of viable but stressed cells, whereas 600 V/cm resulted in a favorable proportion of successfully electroporated cells while preserving overall cell viability. These heterogeneous responses promoted differential immune recognition and cytotoxic targeting of cancer cells in co-culture experiments with immune cells. In co-culture, spheroids treated at 600 V/cm increased T cell activation and promoted targeted tumor cell killing. These effects were confirmed using both Jurkat cells and primary human lymphocytes. Gene expression analysis revealed upregulation of pro-inflammatory cytokines, indicating enhanced immunogenicity following high-voltage electroporation. Conclusions: High-voltage electroporation alone can increase the immunogenic potential of breast cancer cells and promote T cell-mediated anti-tumor activity. These findings support its application as a standalone immunomodulatory strategy and lay the groundwork for its integration into combined immunotherapeutic approaches.
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