An Ionic Liquid Ablation Agent for Local Ablation and Immune Activation in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma rapidly acquires resistance to chemotherapy, remaining a fatal disease. Immunotherapy is one of the breakthroughs in cancer treatment, which includes immune checkpoint inhibitors, chimeric antigen receptor T‐cell immunotherapy, and neoantigen vaccines. However, immunotherapy has not achieved satisfactory results in the treatment of pancreatic cancer. Immunogenic death comprises proinflammatory cell death, which provides a way to enhance tumor immunogenicity and promote an immune response in solid tumors. Herein, an ionic liquid ablation agent (LAA), synthesized from choline and geranic acid, which triggers necrosis‐induced immunotherapy by remodeling an immunosuppressive “cold” tumor to an immune activated “hot” tumor is described. The results indicate that LAA‐treated tumor cells can enhance immunogenicity, inducing dendritic cell maturation, macrophage M1 polarization, and cytotoxic T lymphocyte infiltration. The results of the present study provide a novel strategy for solid tumor immunotherapy.

Pdx1-Cre mouse model, was a kind gift from the laboratory of Prof. Raghu Kalluri (MD Anderson Cancer Center, Houston, TX, USA). Luciferase-expressing KPC cell line (KPC-Luc) was isolated from KPC cell transfected with firefly luciferase report gene. Pancreatic stellate cell line (PSC) was obtained from American Type Culture Collection (ATCC). The KPC cells were cultured in McCoy's 5A (Modified) Medium (Thermo Fisher Scientific) supplemented with 10% FBS (Biological Industries 04-001-1) and 1% Penicillin/Streptomycin (Cienry CR-15140) in a humidified cell incubator with 5 % CO2. PSC were cultured in Stellate Cell Medium.

Isolation of BMDM and BMDC from C57BL/6 mouse.
After sacrificing the mouse, we disinfect the skin of the mouse with 70% alcohol and remove the skin and muscles from the legs down to the hip bone, cut the epiphyses of the bones and flush the marrow into a 15 mL centrifuge tube using a 1 mL syringe. The resulting cells were lysed with red blood cell lysis buffer (Beyotime Institute of Biotechnology) and filtered through nylon mesh filters (70 μm). Centrifuge at 600g and 4°C for 5 min. Dilute the cells at a concentration of 1 × 10 6 cells/mL, add M-CSF at a final concentration of 20 ng/mL (BMDM) or 10 ng/ mL GM-CSF and 5 ng/mL IL-4 (BMDC), and seed 10 mL per 10 cm cell culture-treated petri dish. Incubate at 37 °C with 5% CO2 for 3 days. Replace the medium and incubate for another 3 days.

Isolation CD8 + T cells.
CD8 + T cells were isolated from the spleen of C57BL/6 mouse using a CD8 + T cell isolation kit, an LS Column and a MidiMACSTM Separator. Briefly, Spleen was mechanically dissociated into small pieces using a grind rod. The digested tissues were milled into single cells using a 70-μm cell strainer and washed with PBS for 3 times. Red blood cell lysis buffer was used to remove red blood cells. Then the CD8 + T cell were purified using a mice CD8 + T cell isolation kit according the manufacturer's instructions.

Establishment of pancreatic cancer model.
Male nude mice (4-6 weeks) and Male C57BL/6 mice (4-6 weeks) were purchased from the Model Animal Research Center of Nanjing University, China. All animals were maintained in a pathogen-free environment under controlled humidity and temperature. The animal experiments were performed in accordance with the China Animal Protection Law. Animal suffering was minimized or prevented at all time to improve their welfare. Nude and C57BL/6 mice were subcutaneously inoculated with 2 × 10 5 KPC cancer cells in subcutaneous model experiment of pancreatic cancer. Pancreatic tail of C57BL/6 mice were inoculated with 2 × 10 5 KPC cancer cells in orthotopic model experiment of pancreatic cancer. 25 μL 25% LAA was injected intratumorally in all animal experiments.

In vivo Biocompatibility assay.
At the end of the LAA treatment in vivo, the peripheral blood (1mL per mouse) was collected under anesthesia for blood biochemistry detection. The biochemistry indicators including Alanine Transaminase (ALT), Aspartate Transaminase (AST), UREA, Creatinine (CREA), were the liver and kidney function index. The liver and kidney function tests were performed to investigate the biocompatibility of the LAA treatment.
And the major organs (hearts, livers, spleens, lungs and kidneys) of the mice were collected to evaluate the toxicity of the LAA treatment by H&E staining. For the H&E assay, the organs were fixed in 10% neutral buffered formalin for 24 h. After paraffin embedding, they were cut into 4 μm-thick sections and deparaffinized after being baked at 68°C for 90 min. The sections were dehydrated in graded ethanol and immersed in hematoxylin staining solution for 5 min. The color of the sections changed from blue to red 2 seconds after adding 50 uL 1% hydrochloric acid ethanol. The sections were then immersed in eosin staining solution for 5 min and washed with ddH2O. Finally, the representative images were captured by ImageScope software.

CyTOF analysis.
CyTOF experiment and analysis are supported by Zhejiang Puluoting Health Technology Co.
Ltd. The information of antibody was showed in Table S1. Figure S1. Photograph of neat ionic liquid synthesized by choline and geranic acid. Figure S2. Images of C57BL/6 mice orthotopic pancreatic cancer model at 24 hours following injection of 5%, 10%, 25% or 50% LAA.