The 12-LOX/12-HETE/GPR31 metabolic pathway promotes tumor-associated macrophage M2 polarization mediated pancreatic cancer development

Clinical samples

The human serum and cancer tissue samples used in this study were obtained from patients undergoing pancreatic resection surgery due to pancreatic ductal adenocarcinoma. Blood samples were collected before the excision of cancer tissue samples. Informed consent was signed by all blood donors or their relatives. All samples were used only for experimental purposes and research procedures were conducted according to the principles outlined in the Helsinki Declaration. All procedures involving human samples were approved by the Ningxia Medical University General Hospital Scientific Research Ethics Committee (Approval No. KYLL-2022-0045).

Cell lines

PANC-1 and THP-1 cells were purchased from the Chinese Academy of Sciences Cell Bank (Beijing, China). PANC02 cell was obtained from Cellverse (Shanghai, China). PANC-1 and PANC02 were cultured in H-DMEM (Gibco, Waltham, MA, USA), while THP-1 was cultured in RPMI 1640 (Gibco) containing 0.05 mM β-mercaptoethanol. All media were supplemented with 10% FBS and 1% penicillin/streptomycin. All experiments were conducted within six months of establishing stable cell lines.

Mouse model

The 7–8-week-old C57BL/6 male mice were obtained from the Experimental Animal Center of Ningxia Medical University. The mice were housed in the animal facility (humidity 40%–60%, 12-hour light/dark cycle, temperature 20–24 °C), with six mice in a cage measuring 465*300*155 mm. All mice had ad libitum access to water and food. Lidocaine gel was applied to the skin under the left axilla of the mice, followed by the rapid subcutaneous injection of 5 × 10⁶ PANC02 cells using an insulin needle. On the 5th-day post cell suspension injection, the mice were randomly divided into two groups: ML355 was administered via intraperitoneal injection (3 mg/kg/day (Sun et al., 2022)), referred to as the ML355 group; blank solution served as the solvent control, referred to as the vehicle group (a small amount of lidocaine gel was applied to the injection site before each injection). Based on previous studies (Arifin & Zahiruddin, 2017), each group consisted of six mice, with a total of 12 mice used in each experiment. The formula for calculating xenograft tumor volume was V = π × L × W × H/6 (L: length, W: width, H: height) (Sun et al., 2022). Tumor size and mouse weight were monitored every 5 days. The experiment was terminated after 35 days: all mice were anesthetized with 2% isoflurane inhalation, blood was collected from the orbital sinus, and then all mice were euthanized by intraperitoneal injection of pentobarbital sodium at 150∼200 mg/kg, followed by a collection of subcutaneous tumors and other organs. During the study, if a mouse’s tumor diameter exceeded 15 mm, the mouse was euthanized according to the aforementioned method and excluded from the final analysis. All animal experiments were approved by the Ethics Committee of Ningxia Medical University (Approval No: IACUC-NYLAC-2023-007). All procedures were conducted following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and regulations. All methods were performed according to relevant guidelines and regulations.

Cell isolation

Subcutaneous tumors from mice were surgically excised, minced, exposed to a working solution of 1 mg/ml collagenase (Sigma-Aldrich, St. Louis, MI, USA), and dissociated using a gentleMACS™ dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Finally, they were filtered through a 70 µm cell strainer to obtain a cell suspension.

Co-culture

THP-1 monocytes were differentiated into macrophage-like cells (M0) by incubating with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; Solarbio, Beijing, China) for 48 h. Morphological observations were performed under a microscope to confirm differentiation into macrophages.

M0 macrophages were digested with trypsin and co-cultured with PANC-1 cells (3 × 10⁴ cells/cm²) at a 1:1 ratio for 48 h. M0 macrophages were seeded in Transwell permeable cell culture inserts (Corning, Corning, NY, USA; growth area 4.67 cm²; pore size 0.4 µm), while PANC-1 cells were seeded in Costar^® multiwell cell culture plates (Corning). Intervention with ML355 (30 × 10⁻³ µmol/ml) was provided, with a blank solution used as a solvent control.

Small interfering RNA knockdown

Lipofectamine 2000 (Invitrogen, Waltham, MA, USA) was used to transfect PANC-1 cells with 12-LOX-targeting small interfering RNA (siRNA) or control siRNA (Shanghai Jikai Gene Chemical Technology Co., Shanghai, China) following the manufacturer’s protocol. Knockdown efficiency was verified by RT-PCR analysis at 24 h post-transfection to assess 12-LOX expression levels.

RNA extraction and real-time quantitative PCR analysis

Total mRNA was extracted from tissue samples or cultured cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription was done using PrimeScript RT Master Mix (Takara Bio, Kusatsu, Japan) according to the manufacturer’s instructions. Real-time quanitative PCR (RT-PCR) was performed using TB Green Premix Ex Taq (Takara Bio) and CFX96 Real-Time PCR Detection System. Primers used are listed in Table 1.

Flow cytometry

To detect macrophage subtypes, the following antibodies were used for cell staining: Anti-Mouse F4/80 (E-AB-F0995J; Elabscience Biotechnology, Houston, TX, USA), Anti-Mouse CD11b (E-AB-F1081C; Elabscience Biotechnology), Anti-Mouse CD86 (E-AB-F0994D; Elabscience Biotechnology), Anti-Mouse CD206 (E-AB-F1135E; Elabscience Biotechnology), and Anti-Human CD206 (E-AB-F1161E; Elabscience Biotechnology). Blank controls were run in parallel. Flow cytometry was conducted using BD FACSCelesta™ Multicolor Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Western blot

Protein was extracted from tissues and cells. The following antibodies were used: anti-GPR31 (1:1000, ab75579; Abcam, Cambridge, UK), anti-ALOX12 (1:1000, ab168384; Abcam), anti-β-Actin (1:5000, 20536-1-1AP, Proteintech, Rosemont, Illinois, USA), and anti-Tubulin (1:5000, 11224-1-AP; Proteintech). Following 1-hour incubation with an horseradish peroxidase (HRP)-conjugated α-rabbit secondary antibody (1:10000, SA00001-2; Proteintech), protein bands were detected using ECL substrate.

Immunohistochemistry

Tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, and sectioned. Deparaffinization was carried out in xylene followed by ethanol gradient hydration. Tissue sections were heated in boiling citrate buffer in a pressure cooker for 20 min. Sections were then treated with 3% H₂O₂ at room temperature for 10 min, followed by incubation with anti-GPR31 (1:150, ab150633; Abcam) and goat anti-rabbit IgG H&L (HRP) (1:20000, ab205718; Abcam). A DAB staining kit (Thermo Fisher Scientific) with HRP labeling was used according to the manufacturer’s instructions. Samples were mounted and observed under a microscope.

Enzyme-linked immunosorbent assay

According to the manufacturer’s instructions, enzyme-linked immunosorbent assay (ELISA) assay kits (FuSheng and JingMei, China) were used to measure the 12-LOX activity and 12-HETE levels in supernatants from tissue samples or cell cultures.

Transwell migration and invasion assays

Cells were seeded in RPMI-1640 with or without matrix gel. The culture medium supplemented with 10% FBS was placed in the lower chamber of Transwell permeable cell culture chambers (Corning; growth area 0.33 cm²; pore size 8 µm). After 24 h of incubation, cells were fixed on the underside of the membrane with 4% paraformaldehyde and stained with 0.1% crystal violet. Migration and invasion cell counts were recorded after capturing stained images under a microscope.

5-Ethynyl-2′-deoxyuridine assay

PANC-1 cells were seeded in six-well plates and cultured for 24 h. Following the manufacturer’s instructions, cells were cultured with EdU-488 Cell Proliferation Assay Kit (BeyoClic, China), fixed, and then photographed under a fluorescence microscope.

Statistical analysis

Statistical analysis and processing were performed using GraphPad Prism (version 9.5.0; GraphPad Software, Boston, MA, USA) software, with quantitative data presented as mean ± standard deviation (SD). Statistical comparisons between groups were performed using a two-tailed Student’s t-test for normally distributed data, whereas the Mann–Whitney U test (nonparametric) was applied to datasets with skewed distributions. P < 0.05 was considered statistically significant.

The expression of the 12-LOX/12-HETE/GPR31 metabolic pathway is active in PC

We collected serum specimens from patients who underwent pancreaticoduodenectomy at the General Hospital of Ningxia Medical University due to pancreatic ductal adenocarcinoma (PDAC), as well as pancreatic tumor tissues and their normal tissues. Total proteins were extracted from tumor and normal tissues, revealing low levels of 12-LOX expression in tumor tissues compared to normal tissues (Fig. 1A). However, ELISA testing indicated higher enzyme activity of 12-LOX in tumor tissue (Fig. 1B). In both tumor tissues and the serum of tumor patients, the metabolic product 12-HETE of 12-LOX (Guo et al., 2011b) is elevated (Fig. 1D). Furthermore, western blot (WB) experiments indicated an upregulation of the membrane receptor GPR31 for 12-HETE in tumor tissue (Fig. 1A). The immunohistochemistry (IHC) results showed that the mean expression of GPR31 in tumor tissue is higher than the adjacent normal tissue, however, this statistical difference was not significant, possibly due to the small sample size (Fig. 1C).

In conclusion, these results suggested that the expression of the 12-LOX/12-HETE/GPR31 metabolic pathway was active in PDAC patients, which might be related to the occurrence and development of tumors.

Inhibition of 12-LOX enzyme activity suppresses the development of PC

To investigate the impact of the 12-LOX/12-HETE/GPR31 metabolic pathway on the occurrence and development of PC, we intervened in this pathway using the 12-LOX enzyme inhibitor ML355 in a mouse model of pancreatic cancer. On the 5th day after inoculation of PANC02 cell suspension, ML355 or blank solvent was administered. Mice receiving ML355 intervention were designated as the ML355 group, the control group used blank solvent and was labeled as the Vehicle group (Fig. 2A). Results showed that the tumor size and growth rate in the ML355 group were lower compared to the Vehicle group (Figs. 2B and 2C). Histopathological evaluation using hematoxylin and eosin (H&E) staining and pan-cytokeratin (panCK) IHC demonstrated that the mouse xenograft tissue exhibited characteristic features of carcinosarcoma (Fig. 2D). This suggested that the mouse model can somewhat simulate the development of PC.

By embedding and staining the tumor tissues of mice, we also observed a lower level of GPR31 IHC staining in the ML355 group (Fig. 3A). In addition, ELISA analysis and WB showed that the expression and activity of 12-LOX, the expression of 12-HETE and GPR31 in the tumor tissues of mice in the ML355 group were all suppressed by ML355 (Figs. 3B–3D). Previous studies have demonstrated that the 12-LOX/12-HETE/GPR31 metabolic pathway regulates macrophage infiltration (Zhang et al., 2018; Chung et al., 2019). Moreover, macrophages have been shown to promote pancreatic cancer progression through the secretion of diverse cytokines (Liou et al., 2017; Von Itzstein et al., 2020). To examine changes in TAMs within this context, we selected CD86 and CD206 as markers for M1 and M2 macrophages, respectively.

We utilized flow cytometry to detect the subtypes of macrophages in tumor primary cells from different groups of mice. The results indicated that the proportion of CD86⁺ cells in the ML355 group increased, while the proportion of CD206⁺ cells decreased, with statistical significance (Fig. 3E). In addition, we also measured a series of polarization markers to identify the types of macrophages in tumor tissues. M1 macrophages express interleukin-12A (IL-12a), TNF, and nitric oxide synthase (NOS2), while M2 macrophages express transforming growth factor beta (TGF-β), ARG-1, and Fizz-1 (Hansakon et al., 2019; Brenot et al., 2018). RT-PCR analysis showed that inhibiting 12-LOX suppressed the expression of anti-inflammatory factors TGF-β, ARG-1, and Fizz-1, but the expression of M1 macrophage markers showed both increases and decreases without statistical significance (Fig. 3F).

The above results indicated that intervention of 12-LOX enzyme could intervene in the 12-LOX/12-HETE/GPR31 metabolic pathway, inhibit the growth of xenografts in mice, and also suppress TAMs polarization towards M2 macrophages. This intervention might promote TAMs polarization towards M1 macrophages, but further research is still needed.

Inhibiting 12-LOX enzyme activity reduces the proliferation, invasion, and migration capabilities of PC cells when co-cultured with macrophages

To investigate whether the involvement of TAMs plays a role in the impact of the 12-LOX/12-HETE/GPR31 metabolic pathway on the development of PC tissue, we co-cultured PC cells with M0 cells and conducted in vitro analyses. The cells were divided into four groups: PC group, PC+ML355 group, PC+M0 group, and PC+M0+ML355 group. Results from 5-Ethynyl-20-deoxyuridine (EdU) assays and Transwell assays measuring cell migration and invasion showed no difference in proliferation, migration, and invasion capabilities between the PC group and the PC+ML355 group. The proliferation, migration, and invasion capabilities of the PC co-cultured with M0 cells were significantly enhanced compared to the PC group. However, compared to the PC+M0 group, the proliferation, migration, and invasion capabilities of the PC+M0+ML355 group were weakened (Figs. 4A and 4B). Flow cytometry analysis revealed a reduction in CD206⁺ macrophage proportion within the ML355 treatment group (Fig. 4C). Furthermore, RT-PCR demonstrated coordinated downregulation of TGF-β, interleukin-10 (IL-10), and Fizz-1 (canonical M2 macrophage markers (He et al., 2022; Gong et al., 2022) in this group (Fig. 4D). To further investigate whether the polarization of M0 macrophages toward the M2 phenotype in the co-culture system is directly influenced by ML355, this study employed siRNA to knock down 12-LOX expression in PANC-1 cells before co-culturing them with M0 macrophages. Flow cytometry analysis revealed no statistically significant difference in CD206⁺ cell numbers between the groups treated with and without ML355 (Fig. S1C).

Therefore, we speculated that the 12-LOX/12-HETE/GPR31 metabolic pathway may exhibit a macrophage-dependent pro-tumor effect in the TME.