Antitumor Effect and Immune Response of Nanosecond Pulsed Electric Fields in Pancreatic Cancer

Abstract

Nanosecond pulsed electric fields (nsPEFs) have emerged as a novel and effective strategy for the non-surgical and minimally invasive removal of tumors. However, the effects of nsPEFs treatment on the tumor immune microenvironment remain unknown. In this study, the changes in the morphology and function of pancreatic cancer cells after nsPEFs were assessed and the modifications in the immune profile in pancreatic cancer models were investigated. To this end, electrodes were inserted with different parameters applied to ablate the targeted tumor tissues. Tumor development was found to be inhibited, with decreased volumes post-nsPEFs treatment compared with control tumors (P < 0.05). Hematoxylin and eosin staining showed morphological changes in pancreatic cancer cells, Ki-67 staining confirmed the effects of nsPEFs on tumor growth, and caspase-3 staining indicated that nsPEFs caused apoptosis in the early stages after treatment. Three days after nsPEFs, positron emission tomography demonstrated little residual metabolic activity compared with the control group. Gene expression profiling identified significant changes in immune-related pathways. After treatment with nsPEFs, CD8⁺ T lymphocytes increased. We showed that nsPEFs led to a significant decrease in immune suppressive cells, including myeloid derived suppressor cells, T regulatory cells, and tumor-associated macrophages. In addition, the levels of TNF-α and IL-1β increased (P < 0.05), while the level of IL-6 was decreased (P < 0.05). NsPEFs alleviated the immunosuppressive components in pancreatic cancer stroma, including hyaluronic acid and fibroblast activation protein-α. Our data demonstrate that tumor growth can be effectively inhibited by nsPEFs in vivo. NsPEFs significantly altered the infiltration of immune cells and triggered immune response.

Keywords: ablation; immune response; nanosecond pulsed electric fields; pancreatic cancer; tumor microenvironment.

Figure 1

(A) Image of the nanosecond-pulsed tumor ablation system. (B) Schematic illustration of the treatment strategy. (C) Representative photograph showing nsPEFs electrode placement within the targeted tumor.

Figure 2

(A) Growth curve of pancreatic cancer tumor from PDX models post-nsPEFs treatment. Tumor volume was determined using calipers after treatment with different parameters. (B) Tumor volume comparison 14 days after treatment. The tumor volume of treated mice decreased significantly compared with control which had no nanosecond pulsed electric fields treatment (***p < 0.001). (C) PET/CT scans obtained at baseline and 3 days after nsPEFs treatment. Tumors had substantial initial FDG activity on the baseline fused FDG PET/CT images. FDG PET/CT scans obtained after nsPEFs showed limited metabolic activity. (D) Bar chart showing a significant difference in tumor uptake (ID%/g) between treated and untreated tumors 3 days after treatment (***p < 0.001).

Figure 3

Histopathology of pancreatic cancers after nsPEFs treatment. 0, 1, 3, and 14 days after treatment, the mice were euthanized and samples were harvested. Tumor cell structure and nuclear changes were analyzed by hematoxylin and eosin (H&E) staining. Samples were also prepared for immunohistochemistry with antibodies to Ki-67 and caspase-3. Magnification, 200×.

Figure 4

(A) Hierarchical clustering of differentially expressed mRNAs. Hierarchical clustering was performed using differentially expressed mRNAs between the nsPEFs-treated group and the control group. The gene expression values (log2-transformed intensities) are scaled and depicted in color code format (upregulated: red, downregulated: blue). (B) Bar chart of differentially expressed mRNAs between the nsPEFs-treated group and the control group (upregulated: red, downregulated: blue). (C) The expression profiles of the identified differentially expressed genes (DEGs). Red and green points represent the significant DEGs with p < 0.05 and log2(fold change) >1, and grey points show those without significance, respectively. Fold change refers to the values of FPKM change. (D) GO classification of DEGs. GO terms are summarized in three main categories: cellular component, molecular function, and biological process. (E) Top 20 pathways of KEGG functional enrichment among DEGs. The color of nodes changes from purple - blue - green - red, and the smaller the enrichment p-value, the greater the significance. The point size denotes the DEG number.

Figure 5

Profiling of T cells after nsPEFs treatment. The ratios of CD4^+/CD3⁺, CD8⁺/CD3⁺in response to nsPEFs treatment in the tumor tissue, spleen, and axillary lymph nodes on day 3 and 7. One representative flow cytometric graph for each pattern was shown here (n = 4). Data are presented as the mean ± SD. P-values were calculated based on a Student’s t-test (n = 4 per group).

Figure 6

Profiling of immune suppressive cells after nsPEFs treatment. (A) Frequency of Treg cells in response to nsPEFs treatment in the spleen on day 3 and 7. Analyses were performed on CD4⁺ cells. (B) Frequency of MDSCs (CD11b⁺Ly6G⁺ and CD11b⁺Ly6C⁺) and macrophages (CD11b⁺F4/80⁺) in the tumor on day 3 and 7. (C) Frequency of MDSC (CD11b⁺Ly6G⁺ and CD11b⁺Ly6C⁺) and macrophages (CD11b⁺F4/80⁺) in the spleen on day 3 and 7. (D) Frequency of MDSC (CD11b⁺Ly6G⁺ and CD11b⁺Ly6C⁺) and macrophages (CD11b⁺F4/80⁺) in the tumor on day 3 and 7. Data are presented as the mean ± SD. P-values were calculated based on a Student’s t-test (n = 4 per group). *p < 0.05, **p < 0.01.

Figure 7

The concentrations of immune cytokines and chemokines before and after nsPEFs treatment. (A) Expression levels of TNF-α in the blood. (B) Expression levels of IL-1β in the blood. (C) Expression levels of IL-6 in the blood. (D) Expression levels of CCL2 in the tumor. (E) Expression levels of CXCL9 in the tumor. Data are presented as the mean ± SD. P-values were calculated based on a Student’s t-test (n>3 independent experiments). *p < 0.05, ***p < 0.001.

Figure 8

IHC staining of viable tumor region at 3 and 7 days after the initiation of treatment. Representative micrographs of staining for α-SMA, FAP-α, HABP1. Five visual fields were randomly captured for each group. Magnification, 200×.