Nano-Pulse Stimulation for the Treatment of Pancreatic Cancer and the Changes in Immune Profile

Abstract

A Pancreatic cancer is a notorious malignant neoplasm with an extremely poor prognosis. Current standard of care is rarely effective against late-stage pancreatic cancer. In this study, we assessed nanopulse stimulation (NPS) as a local treatment for pancreatic cancer in a syngeneic mouse Pan02 pancreatic cancer model and characterized corresponding changes in the immune profile. A single NPS treatment either achieved complete tumor regression or prolonged overall survival in animals with partial tumor regression. While this is very encouraging, we also explored if this local ablation effect could also result in immune stimulation, as was observed when NPS led to the induction of immune-mediated protection from a second tumor challenge in orthotopic mouse breast and rat liver cancer models. In the Pan02 model, there were insufficient abscopal effects (1/10) and vaccine-like protective effects (1/15) suggesting that NPS-induced immune mechanisms in this model were limited. To evaluate this further, the immune landscape was analyzed. The numbers of both T regulatory cells (Tregs) and myeloid derived suppressor cells (MDSCs) in blood were significantly reduced, but memory (CD44⁺) T-cells were absent. Furthermore, the numbers of Tregs and MDSCs did not reduce in spleens compared to tumor-bearing mice. Very few T-cells, but large numbers of MDSCs were present in the NPS treated tumor microenvironment (TME). The number of dendritic cells in the TME was increased and multiple activation markers were upregulated following NPS treatment. Overall, NPS treatments used here are effective for pancreatic tumor ablation, but require further optimization for induction of immunity or the need to include effective combinational NPS therapeutic strategy for pancreatic cancer.

Keywords: T regulatory cells; ablation; abscopal effect; immune response; myeloid derived suppressor cells; nano-pulse stimulation; nanosecond pulsed electric fields; pancreatic cancer; tumor microenvironment; vaccine-like protection.

Figure 1

Kaplan-Meier survival curves of mice treated with NPS. Pan02 pancreatic tumors with the size of 4–7 mm (small) or 8–11 mm (large) were treated with NPS (200 ns, 30 kV/cm, 2 Hz with various pulses numbers) at day 11 or day 31 indicated by arrow. Ctr: mice with tumor but no treatment. (A) Dose response of NPS. 600p, 800p, 1000p or 1200p: treated with NPS with 600, 800, 1000 or 1200 pulses (n = 4 each group). (B) Different efficacy of NPS for small or large tumors treated. NPS-S: small tumors treated with NPS 800 to 1200 pulses using pinch electrodes (n = 8); NPS-L: large tumors treated with NPS 800 to 1200 pulses using needle electrodes (n = 11). (C) Survival curves of animals with partial regression. NPS-S (n = 4) or NPS-L (n = 8).

Figure 2

Growth curves of the second challenge tumors. Tumor-free animals after IRE or NPS treatment were challenged subcutaneously with 0.5 × 10⁶ Pan02 cells. IRE (n = 5): tumor-free mice after heat-assisted IRE treatment (100 µs, 1 Hz, pulse number 90 and electric fields 2100 V/cm). NPS (n = 15): tumor-free mice after NPS treatment using pinch electrode (200 ns, 30 kV/cm, 2 Hz with 800–1200 pulses). Each line represents a single tumor growth in one mouse.

Figure 3

Growth inhibition of the second distant tumor lesions after primary tumor treated with NPS. (A) The schedule of two tumors initiated and primary one treated. (B) Growth curves of the second distal tumors after primary tumors were removed with surgery (n = 9). (C) Growth curves of the second distant tumors after primary tumors were treated with NPS (n = 10): primary tumor treated with 200 ns, 30 kV/cm, 2 Hz, 1000 pulses using pinch electrodes. Each line represents one untreated tumor growth in a single mouse.

Figure 4

Various patterns of memory T cell responses after the second live tumor challenge. After NPS treatment mice with tumor free over 7 weeks (NPS, n = 13) or naïve control mice (Control, n = 6) were challenged subcutaneously with 0.5 × 10⁶ Pan02 cells. Blood was collected 2 days prior to challenge (pre-challenge) or 9 days after challenge (post-challenge). One representative flow cytometric graph for each pattern was shown here. (n = 6, 12…): numbers indicate how many animals shared the same pattern.

Figure 5

Different T cell responses after NPS treatment. In the abscopal effect experiment blood was collected 13 days after primary tumor was treated either with surgery (Surgery) or with NPS (NPS). One representative flow cytometric graph for the same pattern was shown. (n = 6, 1, 9): numbers indicate how many animals shared the same pattern.

Figure 6

Changes of the frequencies of immune suppressor cells in the blood and spleen after NPS treatment. Blood and spleens were collected from tumor-bearing animals (tumor, n = 4), or animals treated with NPS on day 2 (NPS-2, n = 5) or day 7 (NPS-7, n = 5) post-treatment. Regulatory T cells (Tregs) or myeloid-derived suppressor cells (MDSCs) were analyzed with flow cytometry. * p < 0.05. ** p < 0.01 for NPS-2 or NPS-7 vs. tumor by One Way ANOVA.

Figure 7

Activation of Dendritic cells (DCs) and changes of immune suppressor cells in tumor microenvironment (TME). Tumors were collected from tumor-bearing animals (tumor, n = 4), or animals treated with NPS on day 2 (NPS-2, n = 5) or day 7 (NPS-7, n = 5) post-treatment. Single cell suspension of tumor tissue was prepared and analyzed for the activation marker expression of DCs (A,B) and the frequencies of Tregs and MDSCs (C) by flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001 for NPS-2 or NPS-7 vs. tumor by One Way ANOVA.

Figure 8

Proposed mechanisms of the failed induction of immune response with NPS treatment in the Pan02 tumor. NPS: Nano-pulse stimulation; DCs: Dendritic cells; MDSCs: Myeloid derived suppressor cells; Tregs: T regulatory cells; TME: tumor microenvironment; Th1: type I help cells. CTL: cytotoxic T cells. Tm: memory T cells.