Tumor-associated macrophages promote PD-L1 expression in tumor cells by regulating PKM2 nuclear translocation in pancreatic ductal adenocarcinoma

In many types of cancer, tumor cells prefer to use glycolysis as a major energy acquisition method. Here, we found that the 18fluoro-deoxyglucose (FDG) positron emission tomography (PET)/computed tomography (CT)-based markers were positively associated with the expression of programmed cell death ligand 1 (PD-L1), pyruvate kinase M2 (PKM2), both of which indicate poor prognosis in patients with pancreatic ductal adenocarcinoma (PDAC). However, the regulatory mechanism of PD-L1 remains elusive. In this study, we confirmed that transforming growth factor-beta1 (TGF-β1) secreted by tumor-associated macrophages (TAMs) was a key factor contributing to the expression of PD-L1 in PDAC cells by inducing the nuclear translocation of PKM2. Using co-immunoprecipitation and chromatin immunoprecipitation assays, we demonstrated that the interaction between PKM2 and signal transducer and activator of transcription 1 (STAT1) was enhanced by TGF-β1 stimulation, which facilitated the transactivation of PD-L1 by the binding of PKM2 and STAT1 to its promoter. In vivo, PKM2 knockdown decreased PD-L1 expression in PDAC cells and inhibited tumor growth partly by promoting natural killer cell activation and function, and the combination of PD-1/PD-L1 blockade with PKM2 knockdown limited tumor growth. In conclusion, PKM2 significantly contributes to TAM-induced PD-L1 overexpression and immunosuppression, providing a novel target for immunotherapies for PDAC.

Additional details are provided in Supplementary Material (Supplementary Table 2).

Animal
Male 6-8-week-old nude mice and NOD/SCID mice were purchased from the Model Animal Research Center of Nanjing University. Mice experiments were conducted according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The study procedures were performed with the approval of the Institutional Animal Care and Use Committee of Animal Core Facility of Nanjing Medical University.

Tumor model experiment
For xenograft mice models, BxPC-3 and Capan-2 cell lines were infected with the scrambled or PKM2 knockdown lentivirus, nude mice were injected subcutaneously with 3 × 10 6 scrambled or sh-PKM2 BxPC-3/Capan-2 cells in 100-μL PBS on the right axilla, respectively (n = 10 per group). Tumor size was measured once every other day using a vernier caliper. Tumor volume was calculated based on the two perpendicular measurements and using the following formula: volume = (length × width 2 )/2. When the tumor volumes reached the endpoint criteria (diameter, 10-15 mm), all mice were euthanized, and the tumors were collected for further fluorescence-activated cell sorting (FACS) and IHC analysis.

Human NK cell sorting and adoptive transfer experiments
A FACS Aria cell sorter (BD Biosciences) was used to purify NK cells (CD3-CD56+) from PBMCs. The purity of the sorted NK cell populations was more than 95%, as verified by post-sort flow cytometry. For the infusion of human NK cell experiment, after receiving subcutaneous injections of 3 × 10 6 scrambled or sh-PKM2 BxPC-3/Capan-2 cells in 100-μL PBS on the right axilla, respectively, mice were treated with anti-PD-1 mbA or IgG mbA (10 mg/kg, 3 of 7 days). After being irradiated to destroy the immune system, NOD/SCID mice were infused with human NK cells (1 × 10 6 /mice) on day 5. Ten mice were included in each group. Tumor volumes were assessed as the aforementioned tumor model experiment. When the tumor volumes reached the endpoint criteria (diameter, 10-15 mm), all mice were euthanized, and the tumors were collected for further FACS and IHC analysis.

IHC analysis
Tumor tissues were fixed in 4% paraformaldehyde for at least 48 h and then embedded in paraffin. The samples were sectioned at 5-μm thickness. These sections were rehydrated and then boiled in 10-mM citric acid buffer and pH 6.0 at 95°C for antigen retrieval, and endogenous peroxidases were quenched with 3% hydrogen peroxide. The slides were blocked with 5% goat serum for 1 h at room temperature and then incubated with an optimal dilution of a primary rabbit antibody overnight at 4°C. After three 5-min washes with PBS, the samples were stained with fluorescent secondary antibodies for 1 h at room temperature, followed by DAB staining for nuclei visualization. Images were acquired and analyzed using a Zeiss fluorescence microscope with the Axiovision image analysis software.

qRT-PCR
Following the manufacturer's instructions, total RNAs were extracted using TRIzol reagent (Invitrogen, USA). RNA samples were reverse-transcribed into cDNA using HiScript ® Q Select RT SuperMix. qRT-PCR was performed using an AceQ ® qPCR SYBR Green Master Mix (High ROX Premixed) Kit (Thermo Fisher Scientific, USA) in a 7500 Fast Real-Time PCR System (Applied Biosystems, USA). The results were standardized to control β-actin values.
The primers used in these assays are presented in Supplementary Table 3.

Short hairpins, plasmids, and lentiviral transduction and infection
PKM2-specific shRNAs originate from the "MISSIONshRNA Library" designed and developed by the TRCat the Broad Institute of MIT and Harvard. One PKM2 hairpin (NM_182471.1-1706s1c1-#) that showed high-efficacy knockdown was selected. High-titer virus-containing supernatants of HEK293FT cells after transient co-transfection of lentiviral vectors with pMD2.G and psPAX2 packaging vectors were used for lentiviral-mediated transduction of cancer cells.

Western blotting
Cells were collected and washed twice using cold PBS. Cell extracts were collected and quantified using the BCA Protein Assay Kit (Thermo Fisher Scientific, USA). Equal amounts of the proteins were electrophoresed on SDS polyacrylamide gels for separation, transferred to PVDF membranes (Millipore, USA), and blocked with 5% non-fat dry milk in PBS (PBST) at room temperature for 1 h. The membranes were incubated with primary antibodies at 4 ℃ overnight and then incubated with an alkaline phosphatase-conjugated goat anti-mouse or anti-rabbit IgG secondary antibody (Jackson, 1:5000 dilution) for 1 h at room temperature. An enhanced chemiluminescence detection system was used to detect the immunoreactive protein bands according to the manufacturer's instructions. Each experiment was repeated at least three times.

Apoptosis assays
Adherent and nonadherent cells were harvested and washed three times with cold PBS. Cell apoptosis was determined by staining with PE Annexin V and 7-AAD. Cells that were considered viable were PE Annexin V-and 7-AAD-negative; cells that were in early apoptosis were PE Annexin V-positive and 7-AAD-negative; and cells that are in late apoptosis or already dead are both PE Annexin V-positive and 7-AAD-positive. The percentage of apoptotic cells was analyzed using the FlowJo software.

Flow cytometry analysis
The single cells prepared from the collected tumor of mice or the treated NK cells isolated from PBMCs were harvested and labeled with indicated antibodies. In detail, the subcutaneous tumors were digested and homogenized by crushing using the head of a 1-mL syringe in a Petri dish,

Immunofluorescence
BxPC-3 cells in a confocal dish were fixed with 4% paraformaldehyde at room temperature for 30 min. The coverslips were washed three times with PBS, and the fixed cells were treated with 0.3% Triton X-100 at room temperature for 10 min to permeabilize the plasma membrane and washed three times with PBS. The cells in the confocal dish were blocked overnight at 4°C with 1% w/v bovine serum albumin (BSA) in PBS. Primary antibodies were diluted in 1% w/v BSA. Following staining, the coverslips were washed three times with PBS.
Secondary antibodies were diluted with PBS containing 1% w/v BSA.
The immunofluorescence was imaged using a Zeiss LSM 800 confocal microscope, and analyzed using the line profile tool in the LSM 800 META ZEN software package (Carl Zeiss). Multiplex immunofluorescence was performed using three kits (B40922/B40923/B40926; Invitrogen, CA, USA) according to the reagent manufacturer's instructions. Briefly, slides were sequentially stained with specific antibodies, positively stained regions of all images were extracted, aligned, overlaid, and segmented using Image-Pro plus 6.0.

Macrophage and tumor cell co-culture
The co-cultivation of macrophages and tumor cells was performed using the noncontact co-culture transwell system. Then, 2 x 10 5 polarized macrophages were seeded in 1.0-μm pore inserts with a fresh medium for 24 h. The inserts containing M1 or M2 polarized macrophages were transferred to 6-well cell culture plates seeded with BxPC-3 or Capan-2 cells (1 x 10 5 cells per well) in advance and co-cultured for another 48 h. Then, the inserts were discarded, and the BxPC-3 or Capan-2 cells were washed with cold PBS three times, and the supernatant medium was collected for further antibody array analysis.

Subcellular fractionation
The fractionation of nuclear and cytosolic extracts was performed by using the PARIS™ Kit (Thermo Fisher Scientific, USA) according to the manufacturer's instructions.

Antibody array
RayBio ® Human Angiogenesis Antibody Array 1 (G-Series, RayBiotech), allowing simultaneous detection of 20 angiogenic-related proteins, was used to assess secretion profiles in conditioned media of BxPC-3 cells according to the manufacturer's instructions. Arrays were visualized using the ImageQuant LAS4000 software (GE Healthcare) and analyzed using ImageJ (National Institutes of Health).
The qPCR primers were used as follows: Pd-l1 promoter:

Promoter reporter assay
Putative STAT1-binding domains in PD-L1 promoters were amplified and inserted into pGL3-based luciferase reporter plasmids, followed by transfection into 293T cells. Then, 5 million cells were collected 48 h after transfection, and luciferase activity was detected using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol.

Biochemical assays
The glucose uptake level was determined using a Glucose Assay Kit (Sigma, USA), as described by the manufacture's protocol. The lactate concentration of the cell supernatant or the tumor tissues were determined using a Lactate Testing Kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacture's protocol.

EdU assay
EdU experiments were performed using a Cell-Light EdU Apollo567 In Vitro Kit   Nuclear and cytosolic lysates were prepared from BxPC-3/Capan-2 shPKM2 cells pretreated with TEPP-46 or/and co-cultured with M2 macrophages, followed by Western blotting. All intensity values were caculated using ImageJ software and was normalized to β-actin. Data were shown as mean ± SEM from three independent experiments. Statistical significance was determined by Mann whitney U test; *P<0.05, **P<0.01,***P<0.001, NS=no significance. within PD-L1promoter was analyzed using ChIP-qPCR in Capan-2 cells following treatment with TGF-β1 (20ng/ml) for 12 hours.Data were shown as mean ± SEM from three independent experiments; Statistical significance was determined by Mann whitney U test; *P<0.05,**P<0.01,***P<0.001. NS=no significance.