Rapid dramatic alterations to the tumor microstructure in pancreatic cancer following irreversible electroporation ablation

Abstract

Aim: NanoKnife(®) (Angiodynamics, Inc., NY, USA) or irreversible electroporation (IRE) is a newly available ablation technique to induce the formation of nanoscale pores within the cell membrane in targeted tissues. The purpose of this study was to elucidate morphological alterations following 30 min of IRE ablation in a mouse model of pancreatic cancer.

Materials & methods: Immunohistochemistry markers were compared with diffusion-weighted MRI apparent diffusion coefficient measurements before and after IRE ablation.

Results: Immunohistochemistry apoptosis index measurements were significantly higher in IRE-treated tumors than in controls. Rapid tissue alterations after 30 min of IRE ablation procedures (structural and morphological alterations along with significantly elevated apoptosis markers) were consistently observed and well correlated to apparent diffusion coefficient measurements.

Discussion: This imaging assay offers the potential to serve as an in vivo biomarker for noninvasive detection of tumor response following IRE ablation.

Keywords: MRI biomarker; NanoKnife®; apoptosis; irreversible electroporation; pancreatic cancer; therapy response.

Figure 1. Representative hematoxylin and eosin slides from control and following 30 min of irreversible electroporation-treated tumors

(A) Control tumor and (B) irreversible electroporation (IRE)-treated tumor at low magnification (4×). Post-IRE tumor specimens demonstrated acute coagulative necrosis. (C) Control tumor and (D) IRE-treated tumor at high magnification (200×). Apoptotic cells within the post-IRE tumor specimen were identified according to morphological criteria including chromatin condensation and margination, and apoptotic bodies. Significant levels of microvessel thrombosis were observed in post-IRE specimens.

Figure 2. Representative transmission electron microscopy slides from the control and following 30 min of irreversible electroporation-treated tumor specimens

Morphological characteristics of apoptosis were not readily observed in (A & B) control tumor specimens. In irreversible electroporation-treated specimens, marked chromatin condensation was observed with chromatin abutment to the nuclear envelope (arrows); apoptotic bodies (dashed arrows) were clearly observed throughout (C & D) treated specimens. (E) Transmission electron microscopy permitted direct observation of the significant alterations of the cell membrane and organelles along with nanoscale defects and pore formation in both cellular and nuclear membranes. (C & F) At high magnification, morphologic hallmarks of apoptosis were also observed within microvessel endothelial cells (asterisks). N: Nucleus.

Figure 3. Transmission electron microscopy apoptosis index measurements for the control and following 30 min of irreversible electroporation-treated tumor specimens

Six sections were observed from each tumor. Significantly increased apoptosis index was observed for both tumor cells and endothelial cells within IRE-treated tumor specimens (both p = 0.002). Error bars represent the standard deviations. IRE: Irreversible electroporation; TEM: Transmission electron microscopy.

Figure 4. TUNEL and caspase 3 slides from both control and irreversible electroporation-treated tumors

IRE-induced significant levels of apoptosis were verified by TUNEL assay ([A] the control tumor and [C] the IRE-treated tumor) and cleaved caspase 3 ([B] the control tumor and [D] the IRE-treated tumor). (E) Al was significantly increased in the IRE-treated tumor specimens compared with the control as assessed with both immunohistochemistry methods (TUNEL, p = 0.001; caspase 3, p = 0.008). Error bars represent the standard deviations. (F) A strong correlation was observed between ΔAI (difference between control and treated tumor markers in each rat) calculated from TUNEL and caspase 3 measurements, R = 0.71. Al: Apoptosis index; IRE: Irreversible electroporation; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling.

Figure 5. Representative diffusion-weighted magnetic resonance images

(A & B) Axial diffusion-weighted magnetic resonance images (b = 500 s/mm²) of PANC-1 xenograft tumors (arrows) in two representative animals (rat A and rat B) (A,i & B,i) before and (A,ii & B,ii) after IRE. Notice reduction in signal intensity within diffusion-weighted images following IRE for both tumors indicative of diffusion (water mobility) increases with these tissues immediately following IRE ablation. Quantitative apparent diffusion coefficient values were significantly higher in these tumor tissues following IRE ablation (p = 0.001); (C) there was no significant difference between ADC values measured in control tumors and treatment group tumors immediately prior to IRE (p = 0.05). Error bars represent the standard deviations. ADC: Apparent diffusion coefficient; IRE: Irreversible electroporation.

Figure 6. Comparison between immunohistochemistry markers of tumor apoptosis (TUNEL and caspase 3) and apparent diffusion coefficient changes following irreversible electroporation ablation procedures in PANC-1 xenograft tumors changes

The apoptosis index was evaluated by the detection of different molecular pathway methods including (A) caspase 3 and (B) TUNEL; however, a strong correlation was observed between these ADC and IHC measurements (caspase 3 assay: R = 0.83; and TUNEL: R = 0.78). ADC: Apparent diffusion coefficient; AI: Apoptosis index; IRE: Irreversible electroporation; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick-end labeling.