Irreversible electroporation facilitates gene transfer of a GM-CSF plasmid with a local and systemic response

Abstract

Background: Electroporation uses an electric field to induce pores in the cell membrane that can transfer macromolecules into target cells. Modulation of electrical parameters leads to irreversible electroporation (IRE), which is being developed for tissue ablation. We sought to evaluate whether the application of IRE may induce a lesser electric field in the periphery where reversible electroporation may occur, facilitating gene transfer of a granulocyte macrophage colony-stimulating factor (GM-CSF) plasmid to produce its biologic response.

Methods: Yorkshire pigs underwent laparotomy, and IRE of the liver was performed during hepatic arterial infusion of 1 or 7 mg of a naked human GM-CSF plasmid. The serum, liver, lymph nodes, and bone marrow were harvested for analysis.

Results: Human GM-CSF level rose from undetectable to 131 pg/mL in the serum at 24 hours after IRE and plasmid infusion. The liver demonstrated an ablation zone surrounded by an immune infiltrate that had greater macrophage intensity than when treated with IRE or plasmid infusion alone. This dominance of macrophages was dose dependent. Distant effects of GM-CSF were found in the bone marrow, where proliferating myeloid cells increased from 14% to 25%.

Conclusion: IRE facilitated gene transfer of the GM-CSF plasmid and brought about a local and systemic biologic response. This technique holds potential for tumor eradication and immunotherapy of residual cancer.

Figure 1. Cells transfected with the isolated plasmid

Media of HCT8 cells transfected with the isolated plasmid via lipofectamine showed high levels of the GM-CSF cytokine whereas control transfection with no DNA resulted in no GM-CSF.

Figure 2. IRE ablation in the liver

Dual probes spaced 1.5 cm apart with a tip exposure of 1 cm were used to deliver the electric pulses of IRE at 1500 V/cm into the liver. A dark red area where IRE ablation has taken place can be seen grossly.

Figure 3. Human GM-CSF in the blood

After treatment with IRE and 7 mg of GM-CSF plasmid infusion, the levels of human GM-CSF levels rose and peaked at 24 hours then fell.

Figure 4. Immune infiltrate surrounding the ablation zone

(A) Normal untreated liver and (B) liver that received the GM-CSF plasmid infusion but not IRE did not have any immune infiltrate. (C) Liver that was treated with IRE but received saline infusion with no DNA showed an ablation zone (asterisk). (D) Liver with treatment with both IRE and GM-CSF plasmid infusion demonstrated an ablation zone (asterisk) that was surrounded by a prominent immune infiltrate (double asterisk). H&E stain. Scale bar represents 1 mm.

Figure 5. Macrophage response in the liver

(A) After treatment with IRE and GM-CSF plasmid infusion, an immune infiltrate surrounding the ablation zone was present and composed of few neutrophils (asterisk), few lymphocytes (hash mark), and florid macrophages which are large and mononuclear. Highlighted is a macrophage (arrowhead) which has phagocytosed necrotic debris. H&E stain. Scale bar represents 20 μm. (B) When graded on a scale from 0=none to 4=severe, the macrophage intensity in the immune infiltrate was highest in livers treated with IRE and infusion of 7 mg GM-CSF plasmid.

Figure 6. Flow cytometry of lymph nodes for immune cells

(A) Representative image from treatment with IRE and GM-CSF plasmid infusion after 6 hours showed an increase in the CD172a⁺, high-granularity population representing neutrophils, and the CD172a⁺, low-granularity population representing monocytes and dendritic cells. (B) The fold change from pre-treatment to post-treatment of the immune cells in the lymph nodes is depicted over time. An early rise in the CD172a⁺, high-granularity population, which is consistent with neutrophils appearing first in an inflammatory response, is followed by a rise in the CD172a⁺, low-granularity population which would represent the appearance of monocytes and dendritic cells. In control pigs receiving IRE but no plasmid, the 48 hour response has an only 1.4 fold increase in both the CD172a⁺, high-granularity and CD172a⁺, low-granularity populations.

Figure 7. Bone marrow response

(A) Bone marrow smears after treatment with IRE and GM-CSF plasmid infusion exhibited myeloblasts (hash mark), promyelocytes (arrowhead) and myelocytes (asterisk). Wright-Giemsa stain. Scale bar represents 20 μm. (B) The pool of proliferating myeloid cells increased after combined treatment with IRE and plasmid infusion but remained about the same with IRE only.