Pancreatic adenocarcinoma up-regulated factor (PAUF) enhances the accumulation and functional activity of myeloid-derived suppressor cells (MDSCs) in pancreatic cancer

Pancreatic cancer is characterized by an immunosuppressive tumor microenvironment (TME) with a profound immune infiltrate populated by a significant number of myeloid-derived suppressor cells (MDSCs). MDSCs have been increasingly recognized for their role in immune evasion and cancer progression as well as their potential as a target for immunotherapy. However, not much is known about the mechanisms regulating their behavior and function in the pancreatic TME. Here we report that pancreatic adenocarcinoma up-regulated factor (PAUF), a soluble protein involved in pancreatic tumorigenesis and metastasis, plays a role as an enhancer of tumor-infiltrating MDSC and its functional activity. We show that PAUF enhanced the accumulation of MDSCs in the spleen and tumor tissues of PAUF-overexpressing tumor cell-injected mice. In addition, PAUF was found to enhance the immunosuppressive function of MDSCs via the TLR4-mediated signaling pathway, which was demonstrated by PAUF-induced increased levels of arginase, nitric oxide (NO), and reactive oxygen species (ROS). The role of PAUF in modulating the functional properties of MDSCs was further demonstrated by the use of a PAUF-neutralizing antibody that caused a decreased number of tumor-infiltrating MDSCs and reduced MDSC immunosuppressive activity. The observations made in mice were confirmed in human pancreatic cancer patient-derived MDSCs, supporting the clinical relevance of our findings. Collectively, we conclude that the PAUF is a powerful and multifunctional promoter of tumor growth through increase and functional activation of MDSCs, suggesting therapeutic potential for targeting PAUF in pancreatic cancers.


Generation of MDSCs from bone marrow progenitors
Tibias and femurs were removed from C57BL/6 mice using sterile techniques and BM cells were flushed with syringe, followed by depletion of RBCs using ACK Lysing Buffer (Lonza). To induce differentiation of MDSCs from BMs in vitro, 5 x 10 5 cells were plated in 6-well plates in 3 ml of RPMI 1640 supplemented with 10% FBS, 40 ng/ml GM-CSF, and 40 ng/ml IL-6, as described in [1]. Cells were maintained at 37˚C in a humidified atmosphere containing 5% CO 2 for 4 days. For cell cycle analysis, MDSCs were seeded at 5 x 10 5 cells per well in 6-welll plates and treated with rPAUF (0.5 µg/ ml) for 0-16 hour(s). Cell cycle profile was determined by propidium iodide (PI) staining of nuclear DNA using CycleTEST PLUS kit (BD Biosciences), followed by flow cytometry on FACSCalibur (BD Biosciences). Data were analyzed using FlowJo software (FlowJo, Ashland, OR, USA). In vitro experiments were carried out using the whole MDSC population (Gr-1 + CD11b + cells), monocytic (MO) MDSCs (Ly6G -Ly6C high CD11b + cells), and/or polymorphonuclear (PMN) MDSCs (Ly6G + Ly6C low CD11b + cells), which were isolated using the Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi Biotec) and purified using streptavidin-conjugated MicroBeads with MidiMACS separators (Miltenyi Biotec). Cell viability was assessed by trypan blue exclusion, and cell purity, which was > 95%, was determined by flow cytometry.

MDSC isolation and in vitro characterization
CXCR4 expression on MDSCs was examined by flow cytometry after 16-hour treatment with rPAUF (0.5 µg/ml). For inhibition of ERK signaling, MDSCs were treated with PD98059 (20 μM) or vehicle (dimethylsulfoxide) and incubated for 2 hours before rPAUF (0.5 µg/ml) treatment for 30 minutes for qRT-PCR analysis or for 0-60 minute(s) for western blotting analysis. To examine PAUF-TLR4 interaction, MDSCs were treated for 1 hour with TLR4-neutralizing antibody at a concentration of 10 µg/ml or CLI-095 from InvivoGen (San Diego, CA, USA) at a concentration of 1 µg/ml before addition of rPAUF (0.5 µg/ml) for 30 minutes.
Human blood samples were obtained from patients with pancreatic ductal adenocarcinoma (PDAC) according to the protocols approved by the Institutional Review Board (IRB) of the Asan Medical Center in Korea. All patients provided informed consent. Peripheral blood samples were collected in BD Vacutainer K2 EDTA 5.4mg (BD Diagnostics, Franklin Lakes, NJ, USA), and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation. These cells were then washed with 0.5% BSA in PBS, and purified using anti-CD33 and anti-CD11b MicroBeads with MiniMACS separators (Miltenyi Biotec) according to the manufacturer's protocol. Cell purity, determined by flow cytometry, was > 95%.

Real-time cell migration analysis
MDSC migration was evaluated using the xCELLigence System (Roche Applied Science, Mannheim, Germany), which allows label-free, realtime monitoring of cell migration. More details of this system are described elsewhere [2]. For a migration assay, the lower and upper chambers were filled with cell-free complete medium and serum-free medium containing none-or rPAUF-treated MDSCs at a density of 3 x 10 4 cells/well, respectively. Cell index (CI) values, which reflect electrical impedance changes caused by cell migration from the upper to the lower chambers, were obtained at 15-minute intervals from the time of plating until the end of the experiment for a total of 5.5 hours. The data obtained were analyzed using the Real-Time Cell Analyzer software (Roche Applied Science).

Quantitative real-time RT-PCR (qRT-PCR)
Total RNA was extracted from MDSCs isolated from BM using the RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) and extracted RNA was quantified by measuring absorbance at 260 nm using an ND-1000 spectrophotometer ( Real-time RT-PCR data were obtained in the form of threshold cycle (Ct) values, and target gene expression was normalized to beta-actin expression. Relative expression levels of target genes (Arg1, Cox2, Nos2, and Cybb) were calculated by the comparative Ct (2 -ΔΔCt ) method as previously described [3,4].