Cell Thawing and Culture

All cells used for in vitro experiments are established cell lines stored at -80°C.

• Preheat the water bath to 37°C
• Remove the cryopreservation vial from the -80°C freezer and place it in the 37°C water bath. Gently shake for 1–2 minutes to accelerate thawing. Note: Keep the vial sealed to prevent water contamination
• Spray the vial surface with 75% ethanol using a spray bottle, then transfer it to the laminar flow hood
• Transfer the cell suspension to a 15mL centrifuge tube. Add 5mL of complete medium. Centrifuge at 350g for 4 minutes
• Discard the supernatant. Add fresh medium and gently resuspend the cells. Transfer the cells to a culture dish
• Gently rock the culture dish back and forth to evenly distribute the cells. Place in a 37°C, 5% CO₂ incubator for culture and observation

Media Change

• Observe cultured cells under a microscope. When cell density reaches 60–70% confluence or the medium turns yellow, it indicates the need for media change
• Place the culture dish in a laminar flow hood, aspirate and discard the supernatant, then add fresh medium
• Gently swirl the cells and return the dish to the incubator for continued culture

Cell Passaging

• Dislodge cells by pipetting, transfer to a 15mL centrifuge tube. Digest with trypsin as needed
• Centrifuge at 350g for 4 minutes. Remove supernatant, resuspend cells in 4mL fresh medium. Aliquot 1mL into a large dish, top up with medium
• Gently swirl the flask to distribute cells evenly, then incubate

Cell Freeing

• Prepare freezing solution at a DMSO: serum ratio of 1:9 and set aside
• Resuspend cells in a 15mL centrifuge tube. Trypsinize cells if necessary
• Discard supernatant. Resuspend cells in freezing solution at 1mL per vial. Incubate at 4°C for 30 minutes, then transfer to 20°C for 2 minutes before final storage at -80°C

• Add 10 μL DPBS to each well of the prepared 12-well plate. Using forceps, hold a round coverslip and briefly heat it over an alcohol lamp before placing it onto the bottom of the well, ensuring it adheres to the well base
• Digest cultured Hepa1-6 hepatocellular carcinoma cells and LLC lung carcinoma cells. Aspirate supernatant, add 2 mL trypsin for 2 min digestion, then terminate digestion with complete medium (DMEM containing 10% serum). Centrifuge at 350g for 4 min
• Count cells and seed at 2 × 10⁶ cells per well into a 12-well plate. Add 2 mL complete medium (containing serum and dual antibiotics) and incubate
• After 24 hours, remove the 12-well plate. Aspirate the supernatant, wash three times with PBS, and add 100 μL 4% paraformaldehyde to fix cells for 15 minutes
• Wash three times with PBS, add 100μL fluorescent blocking solution and block for 30 min
• Remove blocking solution, wash three times with PBS, add Cy-3 labeled Anti-SLC17A2 scFv (Our team commissioned Xi'an Haina Biotechnology Co., Ltd. to prepare it) diluted in primary antibody dilution buffer (1:800 dilution), incubate overnight at 4°C
• Aspirate antibody. Wash three times with PBS for 5 min each. Add Hoechst stain diluted in secondary antibody diluent at 1:5000 dilution. Incubate at room temperature for 15 min
• Wash three times with PBS. Remove PBS, lift the slide using a needle, add 10 μL of 50% glycerol, mount the slide, and observe results under a fluorescence microscope

Cell Transfection

• Seed iBMDM cells into a 12-well plate and allow them to grow until they reach 70–80% confluency per wel
• Aspirate the culture medium, then add 500 μL per well of viral supernatant prepared by Xi’an Tsingke Biotechnology Co., Ltd. on behalf of our team. Add an additional 500 μL of serum-free DMEM and 10 μL of transduction enhancer. Incubate the plate at 37°C with 5% CO₂ for 6 hours
• After 6 hours, remove the supernatant from each well and replace it with complete growth medium. Culture for an additional 24 hours. After 24 hours, transfer the cells from the 12-well plate into larger culture dishes for further expansion
• Once the cells reach approximately 80% confluency, add the appropriate antibiotic for selection. After successful selection, the desired cell line—designated Syn-M-INPUT Test 1 or 2—is obtained

Fluorescence Imaging and Flow Cytometry

• Co-culture the constructed iBMDM (designated Syn-M-INPUT Test 1 or 2) with AML12 expressing mCherry red fluorescence. After cell counting, seed cells into 6-well plates at a 1:2 ratio of macrophages to hepatocytes, with a total cell count of 4.5 × 10^5 cells per well. Add 2 mL of complete medium and incubate
• After 24 hours, observe cell changes under a fluorescence microscope and take photographs
• Simultaneously, collect cells from another 6-well plate into a flow cytometry tube. Centrifuge at 350g for 4 minutes, discard the supernatant, resuspend cells in 1 mL of flow cytometer buffer, and centrifuge again at 350g for 4 minutes
• Add 10 μL rat serum for 10 min blocking. After 10 min, add 40 μL BV421-F480 antibody for 15 min staining (dilution ratio 1:100). After staining, resuspend cells in 1 mL flow cytometer buffer and centrifuge to remove excess antibody
• Aspirate the supernatant, resuspend cells in 300 μL flow cytometer buffer, and analyze using a flow cytometer
• Flow cytometer buffer composition DPBS(48mL) FBS(1mL) EDFA(1mL)

Cell Transfection

• Seed iBMDM cells into a 12-well plate and allow them to grow until they reach 70–80% confluency per well
• Aspirate the culture medium, then add 500 μL per well of viral supernatant prepared by Xi’an Tsingke Biotechnology Co., Ltd. on behalf of our team. Add an additional 500 μL of serum-free DMEM and 10 μL of transduction enhancer. Incubate the plate at 37°C with 5% CO₂ for 6 hours
• After 6 hours, remove the supernatant from each well and replace it with complete growth medium. Culture for an additional 24 hours. After 24 hours, transfer the cells from the 12-well plate into larger culture dishes for further expansion
• Once the cells reach approximately 80% confluency, add the appropriate antibiotic for selection. After successful selection, the desired cell line—designated Syn-M—is obtained

Transcriptome Sequencing

Co-culture and Magnetic Bead Sorting

• Prepare 6-well plates and co-culture Syn-M and AML12 at a ratio of 1:2 for 24 hours
• Aspirate and discard the supernatant of the cells, add 1 mL of magnetic bead buffer, resuspend the cells and transfer them to a flow cytometry tube, then centrifuge at 350g for 4 minutes
• Discard the supernatant, add 40 μL of Biotin CD11b antibody, vortex to mix well, and incubate at 4°C for 15 minutes in the dark
• Resuspend the cells again with 1 mL of magnetic bead buffer, then centrifuge. Discard the supernatant, add 10 μL of BD magnetic beads, vortex to mix well, and incubate at 4°C for 30 minutes in the dark
• After adding magnetic bead buffer, centrifuge to wash away excess beads and antibodies
• Add 1.5 mL buffer to resuspend cells. Place the flow cytometry tube on a magnetic stand for 8 minutes
• Aspirate the negative cells away from the magnetic rack, then repeat the previous step
• Collect positive cells in a 1.5 mL Eppendorf tube

Transcriptome Sequencing

After magnetic bead sorting, Syn-M cells that were either co-cultured with or without AML12 treated with Trizol reagent for preservation, and submitted for analysis. Three groups each were prepared for the control and experimental groups, which were subsequently sent to the Beijing Biomarker Biotechnology Co., Ltd. for transcriptome sequencing

Immunofluorescence for P65 and SIRPα

Cell Mounting

• Add 10 μL DPBS to each well of the prepared 12-well plate. Using forceps, hold a round coverslip and briefly heat it over an alcohol lamp before placing it onto the bottom of the well, ensuring it adheres to the well base
• Digest cultured Syn-M and AML12 cells. Aspirate supernatant, add 2 mL trypsin for 2 min digestion, then terminate digestion with complete medium (DMEM containing 10% serum). Centrifuge at 350g for 4 min
• After cell counting, seed the cells into a 12-well plate at a Syn-M to AML12 ratio of 1:2, add 2 mL of complete medium (containing serum and dual antibiotics), and incubate in a cell culture incubator
• After 24 hours, remove the 12-well plate. Aspirate the supernatant, wash three times with PBS, and add 100 μL 4% paraformaldehyde to fix cells for 15 minutes
• Wash three times with PBS, add 100μL fluorescent blocking solution and block for 30 min
• Remove blocking solution, wash three times with PBS, add P65 or SIRPα antibody diluted in primary antibody dilution buffer, incubate overnight at 4°C
• Recover primary antibody and store at -20°C. Wash three times with PBS (5 min each). Add corresponding secondary antibody diluted in secondary antibody buffer and incubate at 37°C for 1 hour
• Aspirate secondary antibody. Wash three times with PBS for 5 min each. Add Hoechst stain diluted in secondary antibody diluent at 1:5000 dilution. Incubate at room temperature for 15 min
• Wash three times with PBS. Remove PBS, lift the slide using a needle, add 10 μL of 50% glycerol, mount the slide, and observe results under a fluorescence microscope

Antibody Dilutions

• NFKB/P65 (1:400) or SIRPα (1:1000)
• F4/80 (1:100)
• 1. Alexa Fluor 488 donkey anti-rabbit IgG (H+L) （1:2000） AF594 donkey anti-rabbit IgG （1:1000）

ELISA for Inflammatory cytokines

• Collect the supernatant from co-culturing Syn-M with AML12 for subsequent testing
• Sample addition: Add 100μL of appropriately diluted test samples to the pre-coated reaction wells. (Concurrently prepare blank wells and serially diluted standard wells. Note: If feasible, include negative control wells and positive control wells as quality control points)
• Incubation: Cover wells with sealing film and incubate at 37°C for 1-2 hours
• Washing: Follow procedure step 2
• Antibody addition: Add 100μL of diluted biotinylated antibody working solution to each well
• Incubation: Cover wells with sealing film and incubate at 37°C for 1 hour
• Wash: Repeat Step 2
• Add Enzyme Conjugate: Add 100 μL of diluted enzyme conjugate working solution to each well
• Incubation: Cover with a sealing membrane and incubate at 37°C in the dark for 30 min
• Wash: Repeat Step 2
• Add colorimetric substrate: Add 100 μL of TMB substrate solution to each well. Incubate at 37°C in the dark for 10–30 min until a distinct color gradient appears in the wells containing the serially diluted standards
• Stop reaction: Add 100 μL of 2 M sulfuric acid to each reaction well. The color changes from blue to yellow

Read the results: Within 10 minutes, measure the OD value of each well at 450 nm on an enzyme-linked immunosorbent assay (ELISA) reader after zeroing with the blank control well.

Quantitative real-time polymerase chain reaction（qRT-PCR ）for Pro-inflammatory and anti-inflammatory cytokines

Co-culture and Magnetic bead sorting

• Prepare 6-well plates and co-culture Syn-M and AML12 at a ratio of 1:2 for 24 hours
• Aspirate and discard the supernatant of the cells, add 1 mL of magnetic bead buffer, resuspend the cells and transfer them to a flow cytometry tube, then centrifuge at 350g for 4 minutes
• Discard the supernatant, add 40 μL of Biotin CD11b antibody, vortex to mix well, and incubate at 4°C for 15 minutes in the dark
• Resuspend the cells again with 1 mL of magnetic bead buffer, then centrifuge. Discard the supernatant, add 10 μL of BD magnetic beads, vortex to mix well, and incubate at 4°C for 30 minutes in the dark
• After adding magnetic bead buffer, centrifuge to wash away excess beads and antibodies
• Add 1.5 mL buffer to resuspend cells. Place the flow cytometry tube on a magnetic stand for 8 minutes
• Aspirate the negative cells away from the magnetic rack, then repeat the previous step
• Collect positive cells in a 1.5 mL Eppendorf tube

Extract cellular RNA

• Using a 6-well plate as an example, add 1 mL Trizol to each well and lyse on ice for 5 min. Note: Cells have been transferred to EP tubes
• Add 200 μL chloroform at a Trizol:chloroform ratio of 5:1. Gently invert to mix. Let stand on ice for 5 min until liquid phases separate. Centrifuge at 12,000 rpm, 4°C for 20 min
• Using a new EP tube, slowly aspirate 200 μL of the upper clear layer and transfer to the EP tube. Add an equal volume of isopropanol, gently invert to mix, let stand for 5 min, then centrifuge at 12,000 rpm, 4°C for 15 min
• Discard supernatant. Add 1 mL anhydrous ethanol. Centrifuge at 12,000 rpm, 4°C for 10 min
• Discard supernatant. Invert EP tube onto paper towel and let stand for 5 min to air-dry. Add appropriate volume of DEPC-treated water to dissolve RNA
• Quantify RNA using a nucleic acid/protein analyzer

Extract cellular RNA

• 1. RNA reverse transcription system: Reverse Transcription Reagent 4x Hifair III SuperMix Plus (5 µl) RNA (1000 ng) DEPC-treated water (Up to 20 µl)
• 2. Reverse transcription procedure: 37℃ （30min） 85℃ （5min） 4℃ （forever）

qRT-PCR Reaction System

Cell Phagocytosis Assay of Syn-M

Co-culture and Magnetic bead sorting

• Prepare 6-well plates and co-culture Syn-M and AML12 at a ratio of 1:2 for 24 hours
• Aspirate and discard the supernatant of the cells, add 1 mL of magnetic bead buffer, resuspend the cells and transfer them to a flow cytometry tube, then centrifuge at 350g for 4 minutes
• Discard the supernatant, add 40 μL of Biotin CD11b antibody, vortex to mix well, and incubate at 4°C for 15 minutes in the dark
• Resuspend the cells again with 1 mL of magnetic bead buffer, then centrifuge. Discard the supernatant, add 10 μL of BD magnetic beads, vortex to mix well, and incubate at 4°C for 30 minutes in the dark
• After adding magnetic bead buffer, centrifuge to wash away excess beads and antibodies
• Add 1.5 mL buffer to resuspend cells. Place the flow cytometry tube on a magnetic stand for 8 minutes
• Aspirate the negative cells away from the magnetic rack, then repeat the previous step
• Collect positive cells in a 1.5 mL Eppendorf tube

CFSE Staining

• Aspirate the supernatant from cultured Hepa1-6 hepatocellular carcinoma cells or LLC lung carcinoma cells, wash with DPBS, then add 2 mL trypsin and digest for 2 min
• Upon completion of digestion, add complete medium to terminate digestion. Transfer to a 15 mL centrifuge tube and centrifuge at 350g for 4 min at 4°C
• Remove supernatant, resuspend cells in DPBS, and perform cell counting
• Centrifuge again, resuspend cells in PBS containing 0.1% serum at a concentration of 1×10⁶ cells/mL
• Add CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester) at a final concentration of 5 nM. Incubate at room temperature in the dark for 7 min
• After staining, add an equal volume of serum to terminate the reaction. Incubate for 8 min
• After incubation, centrifuge at 350g for 4 minutes. Discard the supernatant, resuspend cells in complete medium for later use. Simultaneously, take a small sample for flow cytometric analysis of staining

Cell Phagocytosis

• Count the magnetic bead-sorted positive macrophages and co-incubate with LLC cells at a ratio of Hepa1-6 or LLC:Syn-M = 2:1 for 2 hours
• After 2 hours, digest cells into suspension, centrifuge at 350g for 4 minutes, discard supernatant, add 50μL BV421-F4/80 antibody (1:100 dilution), and incubate at 4°C in the dark for 15 minutes
• Resuspend cells in 1 mL flow cytometer buffer, centrifuge at 350g for 4 min to wash off excess antibody, discard supernatant, resuspend in 300 μL flow cytometer buffer, and mix thoroughly. Analyze phagocytosis by flow cytometry
• Isolate the FITC-positive population from the F4/80-positive cell population, which represents tumor-phagocytosing Syn-M

Preparation and Characterization of Lipid Nanoparticle (LNP)

Composition ratio of LNP

DLin-MC3-DMA/Distearoylphosphatidylcholine (DSPC)/Cholesterol/DSPE-PEG-M2pep = 40/10/40/10 mol/mol.

Preparation process of LNP

• DLin-MC3-DMA, DSPC, cholesterol and DSPE-PEG-M2pep were solubilized in ethanol at a molar ratio of 40/10/40/10, respectively. The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol and lipid concentration of 30% (vol/vol) and 6.1 mg/mL respectively and allowed to equilibrate at room temperature for 2 min before extrusion
• The hydrated lipids were extruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22℃ using a Lipex Extruder (Northern Lipids, Vancouver, Canada) until a vesicle diameter of 70-90 nm, as determined by dynamic light scattering analysis, was obtained.This generally required 1-3 passes
• The Syn-M related plasmids (solubilized in a 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) was added to the vesicles (pre-equilibrated to 35℃) at a rate of ~5 mL/min with mixing. After a final target plasmid/lipid ratio of 0.06 (wt/wt) was achieved, the mixture was incubated for a further 30 min at 35℃ to allow vesicle re-organization and encapsulation of the plasmid
• The ethanol was then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by dialysis

Properties characterization of LNP

• Using a dynamic light scattering (DLS) instrument, appropriately dilute the LNP sample and measure its hydrodynamic diameter (nm), polydispersity index (PDI), and zeta potential at 25°C, with each sample measured in triplicate
• Place a drop of the diluted LNP onto a carbon-coated copper grid, negatively stain (with 2% phosphotungstic acid), air-dry, and then observe particle morphology, size, and dispersion status by transmission electron microscopy (TEM), followed by image acquisition

All experimental data were statistically analyzed using GraphPad Prism 8 software. Comparisons between the control group and experimental groups were performed using unpaired t-tests or one-way analysis of variance (ANOVA). Results were considered statistically significant at P < 0.05.