Carcinoma cell culture and murine model of hepatic metastasis
MC38 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO Life Technologies, Grand Island, New York) containing 10% fetal bovine serum (GIBCO), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere containing 5% CO2. Confluent cultures were harvested by brief trypsinization (0.05% trypsin in 0.02% EDTA), and after centrifugation, single cell suspensions were prepared in physiological saline (106 cells/100 µl) for subsequent use. Male wild-type C57BL/6 mice (6 to 8 weeks old) were purchased from Vital River Laboratory Animal Technology Co. Ltd (Beijng, China), and housed under pathogen-free conditions. They were randomly assigned to various groups. Under isoflurane inhalation anesthesia, the abdominal cavity was open along the ventral midline. 106 MC38 cells were injected into the pulp of the anterior inferior pole of the spleen. Body temperature was maintained at 36.5°C to 37°C during the entire experiment. After 20 minutes, the spleen was removed to prevent intrasplenic tumor growth. Mice in the Sham group underwent the same procedure with intrasplenic injection of 100 µL of sterile PBS.
Murine model of hepatic IR injury
A partial hepatic ischemia was induced by placing an atraumatic vascular clamp across the three portal elements (hepatic arterial, portal vein and bile duct branch) of the left lateral lobe for 90 minutes. All these procedures were performed under aseptic conditions. In order to prevent dehydration, a small amount of saline was left in the abdominal cavity, which was covered with gauze. The animals were kept on a heated table to maintain a body temperature of 37°C. SHAM-operated splenectomized mice underwent the same procedure except for the clamping.
Inhibiting MagL in vivo/vitro
In vivo: The siMagL siRNA (Genomeditech, Shanghai) was coupled with a galactose-conjugated polymer (Polyplus transfection, France), and injected into the tail vein of mice 6 hours before the surgery to delivery to hepatocytes.
In vitro: AML cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO Life Technologies, Grand Island, New York) containing 10% fetal bovine serum (GIBCO), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere containing 5% CO2. They were pretreated with MJN110(1µM, MedChemExpress) for 6 hours before performing HR model.
Isolation and culture of hepatocytes (HCs) and nonparenchymal cells (NPCs) from mouse liver
The liver was perfused at 7 mL/min via the portal vein for 10 min with Gey’s balanced salt solution (GBSS). After the liver became completely white, the liver was perfused in situ with a solution containing 0.075% type Ⅳ collagenase (Sigma Aldrich, St. Louis, MO). After the two-step collagenase perfusion, the liver was excised, finely smashed by forceps in perfusion buffer, and the digested cells were filtered with a 70 µm cell strainer (Fisher-brand) to prepare a single-cell suspension. Hepatocytes and NPCs were isolated by a series of gradient centrifugation using Percoll.
Extraction of bone marrow-derived macrophages (BMDM)
Bone marrow cells were collected from the femur and tibia of mice. Filter through a 70 um filter and use red blood cell lysate to remove red blood cells. Cells were then cultured in DMEM containing 10% fetal bovine serum (GIBCO), penicillin (100 U/ml), and streptomycin (100 µg/ml), and stimulated with recombinant M-CSF (20 ng/ml, BioLegend) for 5 days for subsequent use.
Hypoxia-Reoxygenation (HR) Model and Co-culture system
AML cells were seeded into transwell inserts (24 mm in diameter and 0.4-µm pore size; Corning Inc., NY) and pretreated with MJN110 or control vehicle. The medium was replaced with Opti-MEM (GIBCO). Then AML cells were placed in a hypoxic environment (5% CO2, 94% N2 and 1% O2) at 37°C for 8 hours. After establishing hypoxia, the medium was replaced with DMEM containing 10% FBS, then cells were transferred to a 5% CO2 incubator for reoxygenation for 24 hours. After that, BMDMs were seeded into 6-well culture plates and co-cultured with AML cells.
Transwell migration and invasion assay
Both experiments were performed through a 24-well transwell system (8 µm pore size; Corning, USA). For the migration assay, MC38 cells were suspended in 200µl DMEM and seeded into the upper chamber, while NPCs isolated from siCtrl or siMagL IR livers were suspended in 600µl DMEM containing 20% FBS and seeded into the lower chamber. After incubation for 48 hours, the chamber was fixed with 4% paraformaldehyde and then stained with 0.5% crystal violet. Cells in the upper chamber were removed with a cotton swab. For the invasion assay, the upper chamber was coated with Matrigel (BD, USA) before inoculation. The remaining steps were the same as the migration assay.
Tumor analysis
After harvesting the liver, they were calculated total tumor volume per liver [volume (mm3) = (long diameter × short diameter2)/2] and visible tumor numbers per liver. Intrahepatic tumor load was scored as the hepatic replacement area (HRA), the percentage of hepatic tissue that has been replaced by tumor cells. On 3 nonsequential H&E stained sections per liver, 30 random fields (objective magnification 100×) were selected and were used to calculate the ratio of tumor cells versus normal hepatocytes plus necrotic cells. The average percentage of tumor tissue of all the fields was expressed by HRA.
Biochemical analysis
The serum levels of alanine aminotransferase (ALT) were examined using the corresponding kit (S03030) to export the results after the automatic biochemical analyzer (Rayto Life and Analytical Sciences Co., Ltd. Chemray 240).
qRT-PCR
Total RNA was isolated from liver tissues or cells using Trizol reagent. Then total RNA was reverse-transcribed into cDNA using SuperScriptTM Ⅲ First-Strand Synthesis System (Invitrogen, Carlsbad, CA). The samples were then subjected to real-time quantitative qPCR measurement (Roche, Indiana, USA) with SYBR Green fluorescent dye. The expression level and results of the target gene are standardized for the β-actin expression. The 2−ΔΔCt method was used to calculate the relative transcription level of target genes. Sequences for all primer pairs are summarized in Supplementary Table 1.
Western blot assay
Total protein extracted from liver tissues or cells were subjected to electrophoresis separation. Then, the protein on the gel were transferred to the PVDF nitrocellulose membrane. The membrane was incubated with primary antibodies against β-actin (Cell Signaling Technology, 3700), MagL (Abcam, ab77398), Arg1 (Abcam, ab96183), CX3CR1 (Affinity, DF7096), TGF-β(Abcam, ab215715) at 4℃ overnight. Horseradish peroxidase-labeled secondary antibody of goat anti-rabbit IgG (Abcam, ab6721) or goat anti-mouse IgG (Abcam, ab6789) was added for another 1-h of culturing with membrane. The membrane was immersed in an enhanced chemiluminescence reaction solution (BIO-RAD, 1705062) for development.
Flow cytometry
The obtained NPCs lysed red blood cells were added to cell staining buffer (Biolegend, 420201) to make a single-cell suspension. Fc receptor blocker was added to reduce non-specific staining. Cell staining involved the combination of fluorochrome-coupled antibodies to CD45 (Biolegend, 103144), CD11b (Biolegend, 101206), CX3CR1 (Biolegend, 149048), Ly6C (Biolegend, 128012) and CCR2 ༈Biolegend, 150603༉ for 30 mins at room temperature in the dark. They were processed with a flow cytometer (Beckman, Gallios) after incubation. The data were analyzed with Flow Jo.
Histology and immunohistochemistry
Fix liver tissue samples in 4% paraformaldehyde, embed in paraffin, slice into 4um-thick sections and then perform (hematoxylin-eosin) H & E staining, Sirius red staining, and masson staining. The degree of liver IRI tissue damage was blindly evaluated from 0 to 4 points by the standard scores established by Suzuki.
For immunohistochemistry, sections were incubated with the primary antibody for MagL (Abcam, ab77398) or Ly6G (Abcam, ab238132), then incubated with the Dako ChemMate™ EnVision System (Dako, Glostrup, Denmark) for 30 minutes. Staining was visualized with use of diaminobenzidine and counterstaining with hematoxylin.
Immunofluorescence
The paraffin-embedded sections were blocked with normal goat serum blocking solution for 20 minutes at room temperature, followed by reaction with different combination of diluted primary antibodies against MagL (Abcam, ab77398), HNF4 (Abcam, ab201460), Ly6G (Cell Signaling Technology, 88876), MPO (Abcam, ab208670), CD206 (Cell Signaling Technology, 24595T), Arg1 (Abcam, ab96183), Mertk (Invitrogen, 2202635), CX3CR1 (Affinity Biosciences, DF7096), TGF-β (Abcam, ab215715), α-SMA (Abcam, ab124964) at 4℃ overnight. After washing with PBS, the sections were incubated with secondary antibody IgG with different fluorescent labels at room temperature for 1h. Subsequently, the nuclei were stained with 4′,6-diamidino-2-phenylindole (D9542, Sigma-Aldrich, USA). For the cells, they were fixed with 4% formaldehyde for 10 minutes. The remaining steps were the same.
Elisa
The level of PGE2 was estimated in the liver or the supernatant by an ELISA kit (R&D Systems) according to the manufacturer’s instructions.
Statistical analysis
Representative results are shown, and data are presented as mean ± SD. Statistical significance was analyzed using a Student’s unpaired t test or one-way ANOVA followed by Bonferroni’s method for post hoc pair-wise multiple comparisons. Two-tailed P values less than 0.05 were considered statistically significant.