Lung Ablation with Irreversible Electroporation Promotes Immune Cell Infiltration by Sparing Extracellular Matrix Proteins and Vasculature: Implications for Immunotherapy

Abstract

Background: This study investigated the sparing of the extracellular matrix (ECM) and blood vessels at the site of lung irreversible electroporation (IRE), and its impact on postablation T cell and macrophage populations. Materials and Methods: Normal swine (n = 8) lung was treated with either IRE or microwave ablation (MWA), followed by sacrifice at 2 and 28 days (four animals/timepoint) after treatment. En bloc samples of ablated lung were stained for blood vessels (CD31), ECM proteins (Collagen, Heparan sulfate, and Decorin), T cells (CD3), and macrophages (Iba1). Stained slides were analyzed with an image processing software (ImageJ) to count the number of positive staining cells or the percentage area of tissue staining for ECM markers, and the statistical difference was evaluated with Student's t-test. Results: Approximately 50% of the blood vessels and collagen typically seen in healthy lung were evident in IRE treated samples at Day 2, with complete destruction within MWA treated lung. These levels increased threefold by Day 28, indicative of post-IRE tissue remodeling and regeneration. Decorin and Heparan sulfate levels were reduced, and it remained so through the duration of observation. Concurrently, numbers of CD3⁺ T cells and macrophages were not different from healthy lung at Day 2 after IRE, subsequently increasing by 2.5 and 1.5-fold by Day 28. Similar findings were restricted to the peripheral inflammatory rim of MWA samples, wherein the central necrotic regions remained acellular through Day 28. Conclusion: Acute preservation of blood vessels and major ECM components was observed in IRE treated lung at acute time points, and it was associated with the increased infiltration and presence of T cells and macrophages, features that were spatially restricted in MWA treated lung.

Keywords: immune response; irreversible electroporation; microwave ablation.

FIG. 1.

Schematic illustrations of IR and CNR in IRE and MWA of normal swine lung. CNR, central necrotic regions; IR, inflammatory regions; IRE, irreversible electroporation; MWA, microwave ablation.

FIG. 2.

(A, B) Gross histology images show two regions in IRE treated lung (*, normal lung; **, IR). White arrow in Day 2 sample shows blood filled tract through which IRE applicator was inserted. Box and insert show high magnification of alveoli in the central IRE treated region of Day 2 sample. Alveolar space (white asterisk) is filled with red blood cells and inflammatory cells. A small number of cells on alveolar wall show pyknosis of nuclei (white arrowheads) and central necrotic zone was not remarkable. Scale bar in the insert shows 50 μm. (C, D) Gross histology images show three regions in MWA treated lung (*, normal lung; **, IR; ***, CNR). White arrow in Day 2 sample shows a hole in which MWA antenna was inserted. Box and insert show high magnification of alveoli in CNR of Day 2 sample. Cell necrosis was identified based on shrinkage and pyknosis of nuclei. Scale bar in the insert shows 100 μm.

FIG. 3.

Result of CD31 immunohistochemistry study. (A) Blood vessels within the IR were preserved, but the CNR of MWA was avascular. White arrows show CD31 positive stained vessels (brown). (B, C) On Day 2, the number of CD31 positive vessels was fewer in IR of both IRE and MWA. By Day 28, increased numbers of blood vessels were observed when compared to normal lung. (D) There was no CD31 positive vessels in CNR of MWA treated lung at both time points. (E) On Day 2, there was no significant difference between each region. (F) On Day 28, the number of CD31 positive vessels in IR of IRE and MWA was significantly higher than that in CNR of MWA. In addition, the number of CD31 positive vessels in IR of IRE was also significantly higher than that of MWA. Dashed bars in the graph show the mean number of CD31 positive stained vessels in untreated normal lung (*, ** and **** mean p < 0.05, p < 0.01, and p < 0.0001, respectively. Scale bar shows 50 μm). FOV, field of view.

FIG. 4.

Result of Masson's Trichrome staining for collagen. (A) Collagen (blue) was preserved after IRE and the percentage increased in IR because of new collagen deposit. On the other hand, collagen deposit occurred only in IR after MWA. There was no Masson's Trichrome positive staining in the CNR after MWA. (B, C) A slight reduction of collagen positive area was observed in IR of both IRE and MWA. On Day 28, collagen positive area in IR recovered to higher levels than untreated normal lung. (D) No positive staining was observed in CNR of MWA treated lung at both timepoints. (E) On Day 2, more Masson's Trichrome positive area was observed in IR of both modalities than the CNR of MWA. In addition, IR of IRE had larger region of positive staining than the IR of MWA. (F) On Day 28, more Masson's Trichrome positive area was observed in IR than CNR of MWA. Dashed bars in quantification results show mean percentage of Masson's Trichrome positive tissue area in untreated normal lung (*** and **** mean p < 0.001 and p < 0.0001. Scale bar shows 50 μm).

FIG. 5.

Result of Heparan sulfate immunohistochemistry (A) Heparan sulfate (brown staining) was preserved in IR after IRE and was restricted in IR of MWA but was absent in the CNR of MWA. (B, C) Compared to surrounding normal lung, Heparan sulfate positive area was reduced, but relatively preserved in IR after IRE and MWA. (D) At both the time points, there was no positive staining for Heparan sulfate in CNR after MWA. (E) On Day 2, more Heparan sulfate positive area was observed in IR of both modalities than the CNR of MWA. (F) On Day 28, findings mirrored that of the earlier time point. Dashed bars in quantification results show mean percentage of positive tissue are with Heparan sulfate in untreated normal lung (**, ***, and **** mean p < 0.01, p < 0.001, and p < 0.0001, respectively. Scale bar shows 50 μm).

FIG. 6.

Result of Decorin immunohistochemistry. (A) Decorin (brown staining) was preserved in IR after IRE. These was no Decorin positive stained tissue after MWA in both IR and CNR. (B) Compared to surrounding normal lung, Heparan sulfate positive area was reduced, but relatively preserved in IR after IRE. (C, D) No positive staining was observed in IR and CNR of MWA treated lung at both timepoints. (E) On Day 2, more Decorin positive area was observed in the IR of IRE than the IR and CNR of MWA. (F) On Day 28, findings mirrored that of the earlier time point. Dashed bars in quantification results show mean percentage of positive tissue are with Decorin in untreated normal lung (*** and **** mean p < 0.001 and p < 0.0001, respectively. Scale bar shows 50 μm). IR, inflammatory regions.

FIG. 7.

Result of CD3 immunohistochemistry study. (A) On Day 2, CD3 positive cells (brown) were observed in IR after IRE and MWA but not in CNR after MWA. On Day 28, CD3 positive stained T cells increased in IR after IRE and MWA. (B, C) T cells were less than normal lung tissue, but relatively preserved. The number of T cells was significantly higher on Day 28 than on Day 2 in IR of IRE and MWA. (D) At Day 2, only a few T cells were observed in CNR but, at Day 28, no T cells were observed in the CNR of MWA treated lung. (E) On Day 2, there were more T cells in the IR of both treatment modalities than the CNR of MWA. (F) On Day 28, greater number of T cells were observed in IR of IRE than the IR or CNR of MWA samples. Dashed bars in quantification results show the mean number of CD3 positive stained T cell (brown) in untreated normal lung (*, **, ***, and **** mean p < 0.05, p < 0.01, p < 0.001, and p < 0.0001, respectively. Scale bar shows 50 μm).

FIG. 8.

Result of Iba1 immunohistochemistry study. (A) Macrophages (brown) penetration was seen in IR after IRE and MWA on Day 2 while no positive cells were observed in CNR after MWA. On Day 28, macrophage populations increased in IR after IRE and MWA. (B, C) On Day 2, the number of macrophages was reduced but maintained comparing with normal lung tissue. On Day 28, macrophages population increased in IR after IRE and MWA, higher than normal lung tissue. (D) After MWA, there were no Iba1 stained macrophages in CNR. (E) On Day 2, there were more macrophages in the IR of both treatment modalities than the CNR of MWA. In addition, more macrophages were observed in the IR of IRE when compared to the IR of MWA. (F) On Day 28, more macrophages were observed in IR of MWA than the IR of IRE. Dashed bars in quantification results show the mean number of Iba1 positive stained macrophages in untreated normal lung (**, ***, and **** means p < 0.01, p < 0.001, and p < 0.0001, respectively. Scale bar shows 50 μm).