br In this article we aimed to
In this article, we aimed to explore the therapeutic use of DNT cells and their mechanism of action in pancreatic cancer. Here, we used a novel method to efficiently expand human DNT cells ex vivo, performed DNT cell cytotoxicity and Fas/FasL pathway blocking experiments and established a nude mouse model of pancreatic cancer.
Materials and methods
Cells and reagents
To enrich the DNT cell population, we used the “antibody adsorption” method [7,13,14]. In this method, 10 ml peripheral blood was obtained from healthy volunteers, added to a CD4 Depletion Cocktail and CD8 Depletion Cocktail (Stemcell Technol-ogies, Canada), and incubated for 10 min. Then, we used human blood lymphocyte separation medium (TianJinHaoYang, China) to extract lymphocytes. To further purify DNT cells, we incubated collected cells with an anti-CD3 antibody in complete medium supplemented with interleukin-2 (IL-2) and IL-4 (Zhongshan Golden Bridge Biotechnology, China). On day 12, we collected cells from wells, counted the cells and verified the phenotype by flow cytometry. Pancreatic cancer cell lines Panc-1 and SW1990 cells were cultured in Roswell Memorial Park Institute medium 1640 (RPMI 1640) supplemented with 10% fetal bovine serum (FBS, HyClone, USA). This research was approved by the Human Scientific Ethics Committee of the Affiliated Provincial Hospital of Anhui
Primers used for Fas, caspase-8 and GAPDH detection.
Medical University (Hefei, China).
DNT cells were first stained with CD4-phycoerythrin (PE), CD8-allophycocyanin (APC) and CD3-fluorescein isothiocyanate (FITC) AUY922 (Biolegend, USA). Next, these cells were detected by flow cytometry. Cell phenotype was analyzed by FlowJo software.
The cytotoxicity of DNT cells was assessed by a cell counting kit-8 (CCK-8) assay (BestBio, China). Panc-1 or SW1990 cells, which were used as the target cells, were seeded into 96-well plates. The number of cells per well was 1 103. DNT cells were inoculated with target cells with or without the blocking agent decoy receptor 3 (DcR3, 20 ng, R&D Systems, USA) . Importantly, DNT cells were cocultured with DcR3 for 2 h before they were seeded into 96-well plates. The DNT/target ratio was 1:1. As a control, groups of target cells with or without DcR3 treatment were established. After cells were cultured for 12, 24, 36 or 48 h, a CCK-8 assay was per-formed. The spectrophotometric absorbance of each sample was measured at 450 nm by a microplate reader (Thermo Fisher Sci-entific, USA). Finally, the optical density (OD) values of each group were compared and analyzed.
Establishment of animal models
A total of 24 BALB/c male immunodeficiency mice (4e5 weeks) were used to establish an animal model as previously described . Panc-1 cells were injected subcutaneously into the underarms of BALB/c mice (2 106 cells/mouse, Slac Laboratory Animal Co. Ltd, China). After subcutaneous tumors reached >0.5 cm in diameter, the possibility of tumor suppression was tested by injecting DNT cells into the tail vein of 8 mice every 2 days (5 106, 0.1 ml). As controls, 8 mice received no treatment, and another 8 mice received an intravenous injection of gemcitabine every 2 days
Forward primer Reverse primer
Fas 50 - TTGCTTAGGGTTCCCTCCTG-30 50 -AAACTGGAGAGCAGACAGCA-30 Caspase-8 50 -CGTCTATGGAACGGATGGGA-30 50 -TACTTCCTTGGCAAGCCTGA-30 GAPDH 50 -CAAGGCTGTGGGCAAGGT-30 50 -GGAAGGCCATGCCAGTGA-30
Fig. 3. DNT cells decreased the tumor size in animal models. A and B, Image of a tumor in the mouse pancreatic cancer model. C, Volume of tumor tissues in the three groups. D, Weight of tumor tissues in the three groups. ***P < 0.001.
(50 mg/kg) after the tumor size reached >0.5 cm. The growth and development of mice and their tumors were closely monitored each day. Nude mice were sacrificed at 50 days, and the weight and volume of tumor tissues were measured and analyzed. The tumor
volume was determined by (length width2)/2. This experiment was approved by the Animal Scientific Ethics Committee of the Affiliated Provincial Hospital of Anhui Medical University (Hefei, China).
Fig. 4. DNT cells inhibited pancreatic cancer growth via the Fas pathway in vivo. A, Western blot analysis of Fas, caspase-8 and cleaved caspase-8 protein expression in tumor tissues from the three groups. B, qPCR analysis of Fas and caspase-8 expression in tumor tissues from the three groups. ***P < 0.001.
After 48 h of coculture with Panc-1 and SW1990 cells, the su-pernatant was discarded, cells were washed 3 times with phosphate-buffered saline (PBS), and suspended DNT cells were removed. Then, proteins were extracted from Panc-1 and SW1990 cells. In addition, proteins were extracted from murine tumor tissues. After full denaturation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), each sample was transferred to a polyvinylidene membrane. After the addition of skim milk for an appropriate dilution of anti-Fas, anti-caspase-8, anti-cleaved caspase-8 and anti-cleaved caspase-3 (1:500, Abcam, UK) antibodies, the membrane was incubated at room temperature for 2 h. After the membrane was washed, the secondary antibody was added at a dilution of 1:10,000, and the membrane was incu-bated in this solution at room temperature for 1.5 h. After another wash, detection reagent was added. Then, the membrane was exposed, developed in the dark and postfixed.