Cell lines
For macrophages, the RAW 264.7 cell line was purchased from Shanghai Yu Chun Biotechnology. The THP-1 and Gal9-KO THP-1 cell lines were provided by Prof. Haipeng Liu at Shanghai Pulmonary Hospital.56 Murine BMDMs were generated from bone marrow cells with DMEM (Biosharp, BL304A) complete medium plus 20 ng/mL M-CSF (Novoprotein, CB34) for 7 days. To obtain different subtypes of macrophages, RAW 264.7, THP-1, or BMDMs were incubated with 10 pg/mL LPS (Beyotime Biotech, ST1470) and 20 ng/mL IFN-γ (Peprotech, 210-13-10) to polarize them into M1 subtypes or incubated with 20 ng/mL IL-4 (Peprotech, 214-14-20) and 20 ng/mL IL-13 (Peprotech, 315-05-20) for 48 h to polarize them into M2 subtypes. For tumor cells, the LLC cell line (mouse, male) was provided by Prof. Haipeng Liu. OVA-overexpressing LLC cells (OVA-LLC) were provided by Prof. Weiwei Yang. All cells were cultured in a 5% CO2 incubator at 37 °C with complete medium (glucose-supplemented DMEM with 10% FBS, L-glutamine, and penicillin/streptomycin) and tested annually for mycoplasma contamination.
Mice
C57BL/6 mice (male, 18–20 g) were purchased from Slake Experimental Animal Company (Shanghai, China). Gal9-KO C57BL/6 mice were provided by Prof. Haipeng Liu.56 All mice were bred in the specific pathogen-free (SPF) animal facility of the Laboratory Animal Center of Tongji University in individually ventilated cages (room temperature, 21 ± 1 °C; relative humidity, 40%–70%, and a 12-h light–dark cycle) and had access to food and water ad libitum. Tumor-bearing mice were constructed by subcutaneous injection of tumor cells (1 × 106 cells per mouse) into the flanks. All mice were selected and grouped randomly and observed within ethical limits. All animal experiments were approved and performed under the guidance of the Institutional Animal Care and Use Committee at Tongji University (Shanghai, China).
Magnetic field device
The RMF device was independently developed by our team. The RMF was generated by two rotating NdFeB magnets (the device was fabricated by Chuanshanjia Co., Ltd., Shanghai, China), which were controlled by a motor to adjust the rotation at different frequencies. In addition, the MFG-100 magnetic field system (MagnebotiX AG), controlled by electromagnetic coils and integrated with an inverted fluorescence microscope (Olympus), was used for in vitro video recording of MNM rotational motion. Finally, a custom-built electromagnetic coil system with a field strength of 20 mT, combined with a high-resolution microscope (Nikon), was used for real-time imaging of MNM rotational motions inside cells.
Preparation of MNMs and fluorescent molecule-labeled MNMs
Synthesis of 25-nm zinc-doped iron oxide MNPs
MNPs were synthesized in the organic solution phase by the thermal decomposition method based on previous work.57 Iron (III) acetylacetonate (Fe(acac)3, 97%, 282.5 mg), zinc acetylacetonate hydrate (Zn(acac)2, 97%, 316.3 mg), and 4-phenylbenzoic acid (99%, 400 mg) were dissolved in a mixture of dibenzyl ether (> 98%, 10.4 mL) and oleic acid (AR, 1.2 mL). The solution was sonicated for 10 min for dispersal, followed by argon aeration for 30 min with stirring at 400 rpm. The solution was then heated to 290 °C and held for 30 min with aeration and stirring. After the solution had cooled to room temperature, 10 mL of ethanol was added to promote the precipitation of MNPs. The products were washed sequentially with ethanol and toluene three times each. Finally, the MNPs were dispersed in ethanol.
Synthesis of MNMs (MNPs coated with PLL)
MNMs were synthesized by modifying the surfaces of MNPs with PLL to transform them from hydrophobic to hydrophilic, thus improving biocompatibility and cell internalization. In brief, 100 mg of PLL was dissolved in 4 mL of ddH2O and mixed with 8 mL of ethanol containing 10 mg of MNPs. The mixture was sonicated using an ultrasonic probe (model 120, Fisher Scientific, USA) with 40 W of power (working time 5 s, interval time 2 s) for 120 min to promote coating of PLL onto the MNP surfaces. The MNMs were then washed with ddH2O three times and finally dispersed in ddH2O.
Synthesis of MNM-FITC and MNM-Cy5
MNMs were labeled by chemically coupling the fluorescent molecule fluorescein isothiocyanate (FITC) onto their surfaces to facilitate detection of their intracellular localization. First, 2 mg of MNMs were dispersed in 2 mL ddH2O, and 2 mL of ethanol containing 0.02 mg of FITC was added. After ultrasonic sonication for 30 min, the mixture was stirred overnight. The resulting MNM-FITC particles were collected by magnetic separation and washed with ethanol and ddH2O three times each to remove free FITC.
The fluorescent molecule Cy5 carrying a carboxyl group was also chemically coupled to the surfaces of MNMs. In brief, 10 mg of EDC and 10 mg of NHS were dissolved in 5 mL PBS (pH 5.5), followed by the addition of 100 μg Cy5 and sonication by ultrasound for 2 h. Two milligrams of MNMs were then added, and the mixture was magnetically stirred for 12 h. The resulting MNM-Cy5 particles were collected by magnetic separation and washed with ddH2O three times.
Characterization of MNMs
The morphology and elemental composition of MNMs were characterized by transmission electron microscopy (TEM, JEM-1230, JEOL Ltd.). The hydrodynamic size and zeta potential of MNMs were measured by dynamic and electrophoretic light scattering (Zetasizer Nano ZS90, Malvern Ltd.) dispersed in ddH2O with a nanoparticle concentration of 10 μg/mL. The magnetic properties of dry MNMs were characterized by magnetization vs applied magnetic field (M vs H) curves at room temperature as measured with a vibrating sample magnetometer (VSM, Lakeshore 7407, USA).
Biocompatibility of MNMs and RMF in vitro
Before RMF treatment, the biocompatibility of MNMs was evaluated by a CCK-8 assay (Beyotime Biotech, P0012). MNMs were first sterilized under UV-light irradiation for 30 min, then dispersed into DMEM at concentrations of 10 μg/mL, 20 μg/mL, and 40 μg/mL. Next, 5 × 103 cells were seeded into a 96-well plate and incubated at 37 °C overnight. Fresh medium containing different concentrations of MNMs was added to replace the culture medium in each well. Medium without MNMs served as the control group. After incubation for 24 h, the CCK-8 assay was performed, and absorbance was measured at 450 nm using a microtiter plate reader (ELx808, BioTek).
For RMF treatment, cell viability was again measured by the CCK-8 assay. After incubation with 20 μg/mL MNMs for 24 h, the cells were washed with PBS three times to remove MNMs that had not been taken up into cells. The cells were then exposed to RMF at different frequencies and for different time intervals. After an additional incubation for 4 h, the CCK-8 assay was performed. To assess the effect of dynamic RMF stimulation on cell viability, cells were exposed to RMF (1 Hz, 15 min) once daily for 7 consecutive days. CCK-8 assays were performed on days 1, 3, 5, and 7, and absorbance was measured at 450 nm using a microtiter plate reader.
To observe the content of MNMs in macrophages, the iron concentration was detected. Macrophages were seeded onto dishes (35 mm Φ) at a concentration of 1 × 105 cells per dish and co-cultured with MNMs at concentrations of 10 μg/mL, 20 μg/mL, and 40 μg/mL for 24 h. The cells were homogenized and lysed in aqua regia after digestion and collection. The amount of Fe in different groups was determined by ICP-MS.
Theoretical calculation and finite element simulation within lysosomes
The assembled number of MNMs within lysosomes and the torque generated under RMF stimulation were first determined through theoretical calculations.58,59,60,61 A deep learning system was then used to quantify the number of assembled nanomotors in Bio-TEM images, thus providing experimental validation for the theoretical predictions. In parallel, finite-element simulations were performed to assess the changes in lysosomal membrane pressure induced by MagLMP at different magnetic field frequencies. To obtain more accurate lysosomal mechanical simulation parameters, lysosomal biophysical characteristics in macrophages were measured experimentally. Detailed procedures are described in Supplementary information, Materials and Methods.
Preparation of SUVs and SUV-MNMs and sulforhodamine B (SRB) release assay under RMF stimulation
On the basis of previous work,38 fluorophore-filled SUVs and fluorophore-filled small magnetic lipid vesicles (SUV-MNMs) were prepared by the thin-film rehydration method. For SUV preparation, DOPC (10 mM, 700 µL) and DOPE (10 mM, 300 µL) were mixed and dissolved in chloroform, then added to a 10-mL round-bottom flask. The solution was rotated in a rotary evaporator (RV 10 Digital, IKA, Germany) at 40 °C for 20 min to remove the chloroform and form a lipid film, then dried overnight at room temperature. After 30 min of ultrasound, the solution was frozen and thawed 5 times using liquid nitrogen and a 50 °C water bath. The SUVs were extruded 25 times through a 200-nm polycarbonate membrane (Avanti Polar Lipids, USA) using an extrusion kit (Avanti Polar Lipids, USA), then purified using a PBS-filled NAP-5 column (GE Healthcare, UK). The purified SUVs were equilibrated at 4 °C overnight. SUV-MNMs were prepared as described for SUVs, except that the buffer of the lipid film was replaced with PBS containing 50 mM SRB (Rhawn, 3520-42-1) and 200 µg/mL MNMs. Both the SUVs and SUV-MNMs were stored at 4 °C in the dark and used within 48 h. The diameters of SUVs and SUV-MNMs were determined by DLS with a nanoparticle potentiometer (Zeta SIZER NANO ZS90, Malvern) and then negatively stained with 2% sodium phosphotungstate for characterization by TEM (JEM-1230, JEOL).
To observe the proportion of SRB released under different frequencies of RMF stimulation, 100-µL aliquots of SUVs and SUV-MNMs were added to a 96-well plate, and the fluorescence intensity was measured at 586 nm (after excitation at 565 nm) using a multimode microplate reader (Tecan, Germany). This measurement provided the baseline fluorescence intensity, owing to the fact that the fluorescence of high SRB concentrations inside SUVs is self-quenching. Next, we collected and stimulated the 100 µL SUVs and SUV-MNMs with RMF at frequencies of 0.2 Hz, 0.8 Hz, 1 Hz, 2 Hz, and 5 Hz for 15 min to induce the release of SRB by mechanically mediated vesicle damage and then measured fluorescence intensity again. Finally, the solutions were collected and freeze-thawed twice in liquid nitrogen and a 50 °C water bath to enable maximum SRB release, and the maximum fluorescence intensity was measured.
Cell transfection
Two plasmids, EGFP-Gal3 (provided by Dr. Bin Liu at Novo Nordisk in Denmark) and YFP-Gal9 (provided by Prof. Haipeng Liu), were used to characterize LMP and the localization of Gal9. RAW 264.7 cells and BMDMs were first seeded onto 35-mm confocal dishes (1 × 105 cells per dish), cultured for 24 h, and transfected with 2.5 μg plasmid using 4 μL jetPRIME transfection reagent (Polyplus, 1010000027) per dish for 24 h to 48 h. Two milliliters of 20 µg/mL MNMs-Cy5 were incubated with the cells for 24 h, and the cells were then stimulated under 1 Hz RMF for 15 min and washed three times with PBS. Lysosomes were stained with LysoTracker Red reagent (Beyotime Biotech, C1046) in DMEM at a dilution of 1:1000 for 30 min at 37 °C, and nuclei were stained with Hoechst (blue) in DMEM at a dilution of 1:500 for 15 min at 37 °C. Confocal laser scanning microscopy (TCS SP8, Leica) was used to capture fluorescence images after cells had been fixed in 4% PFA for 15 min. The Opera Phenix High-Content Screening System (PerkinElmer, USA) was used to continuously observe and analyze changes in the intensity of Gal3 fluorescent spots.
Real-time observation of MNM motion
To observe the assembly and rotation of MNMs in vitro, MNMs dispersed in ddH2O were continuously imaged using the MFG-100 magnetic field system. To visualize the fluid vortices induced by MNM rotation, 500 nm polypropylene nanospheres were added as tracer particles to monitor flow behavior around the rotating MNM assemblies. To visualize the rotational motion of MNMs within lysosomes of macrophages, RAW 264.7 cells were transfected with the EGFP-Gal3 plasmid and incubated with 20 µg/mL MNMs for 24 h. Lysosomes and nuclei were stained using LysoTracker Red and Hoechst, respectively, as described above. A custom-built high-resolution fluorescence microscope integrated with a magnetic control system was used to observe the self-assembly and rotational motion of MNMs within lysosomes under applied RMF at 4, 8, 12, and 20 mT.
To evaluate magnetically guided enrichment of MNMs under flow conditions, a peristaltic pump (Henghe Fluid Electronics Technology) and artificial blood were used to simulate blood circulation in vitro. Flow rates were set to 0.1 mL/min, 0.3 mL/min, and 0.5 mL/min. One milliliter of artificial blood solution containing MNMs (50 µg/mL) was flowed through a silicone tube, and a 1-cm round magnet was placed adjacent to the tube. After each flow cycle, ICP-MS was performed to quantify the Fe content.
Fluorescence imaging
Both macrophages and tumor cells were incubated with 20 µg/mL MNMs or MNMs tagged with fluorescent molecules (MNM-FITCs and MNM-Cy5s) for 24 h to characterize their uptake ability. Macrophages were first induced with 20 µg/mL IL-4 and 20 µg/mL IL-13 for 48 h to polarize them into the M2 subtype. MNMs were incubated with M2 macrophages for 4 h, 8 h, 12 h, and 24 h, and lysosomes and nuclei were stained with LysoTracker Red (red) and Hoechst (blue) as described above. Confocal laser scanning microscopy (TCS SP8, Leica) was used to photograph the co-localization of MNMs and lysosomes in macrophages. For 3D fluorescence confocal imaging, macrophages were co-incubated with MNMs for 2 h and 24 h. After staining with LysoTracker Red and Hoechst, z-stack imaging was performed to visualize the spatial co-localization of MNMs and lysosomes.
To detect changes in lysosome pH of macrophages stimulated by RMF, macrophages were seeded on 35-mm confocal dishes at a concentration of 1 × 105 cells per dish and cultured for 24 h. As a positive control, 10 µg/mL CQ was used to stimulate cells for 1 h before stimulation with 1 Hz RMF for 15 min, then cultured for 0 h and 24 h. For the RMF-twice group, cells were stimulated again with 1 Hz RMF 24 h after the first RMF stimulation. All cells were washed three times with PBS. The LysoSensor probe (Yisheng Biotechnology) was used to measure lysosome pH at a 1:1000 dilution in DMEM for 30 min at 37 °C. Lysosomes and nuclei were stained as described above. After the cells were washed with PBS, confocal laser scanning microscopy (TCS SP8, Leica) was used to capture fluorescence images of the cells, and the images were analyzed using ImageJ.
To observe the effects of RMF stimulation on mitochondrial and nuclear membranes, fluorescent staining was performed using corresponding dyes. Macrophages were seeded on 35-mm confocal dishes, and MNMs were added and co-cultured for 24 h. After stimulation with an RMF of 1 Hz, the cells were washed three times with PBS, stained with mitochondrial staining probes (JC-1, C2006; TMRE, C2001S; Beyotime Biotech) according to the manufacturer’s instructions, and fixed in 4% PFA for 15 min. To stain the nuclear membranes of macrophages, the cells were directly fixed in 4% PFA for 15 min after RMF stimulation, then stained for immunofluorescence with anti-mouse Lamin B1 (Proteintech, 12987-1-AP). Cells were observed by confocal laser scanning microscopy (TCS SP8, Leica).
To directly observe the rotational motion of the assembled magnetic nanomotors within lysosomes, macrophages were seeded on 35-mm confocal dishes, and MNMs were added for 24 h. Lysosomes and nuclei were stained with LysoTracker Red and Hoechst (blue) as described above. The independently developed magnetic control devices and high-resolution fluorescence microscope (Nikon, ECLIPSE Ti2) were successfully assembled. Continuous images of the rotating magnetic nanomotors within lysosomes were directly observed under RMF stimulation.
RT-qPCR detection
Macrophages were seeded on 35-mm dishes at a density of 1 × 105 cells per dish and cultured for 24 h. After exposure to different treatment conditions, RNA was extracted from cells using 1 mL TRIzol reagent (Ambion, 15596018). Then, 200 μL trichloromethane was added, and the mixture was shaken for 15 s. After centrifugation at 15,000 rpm for 10 min at 4 °C, the upper layer of clear solution was collected. Isopropyl alcohol (500 μL) was used to precipitate the RNA, and the mixed solution was allowed to stand for 10 min at 4 °C. After centrifugation under the same conditions, the pellet was retained and washed with 1 mL 75% ethanol. Total RNA (1 μg) was reverse transcribed into complementary DNA using the ReverTra Ace qPCR RT Master Mix kit (TOYOBO, FSQ-201) containing a mixture of oligo(dT) and random primers. The cDNA was used for qPCR with the 2× SG Fast qPCR Master Mix kit (Sangon Biotech, B639272) in duplicate wells on a QuantStudio 7 Flex Real-Time PCR System (Tongji University Advanced Institute of Translational Medicine) with the following cycle conditions: activation at 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 60 s; and a melting curve for assessment of amplicons. For tumor tissues, tumor pieces were mechanically disrupted with surgical scissors in PBS, then homogenized using a tissue grinder, and RNA was extracted as described above. All primer sequences used in this study are provided in Supplementary information, Materials and Methods.
Flow cytometry analysis in vitro
To assess the effect of macrophage polarization or changes in MHC I expression on LLC cells induced by MagLMP, RAW 264.7 or LLC cells were seeded as described previously. Cells were incubated with 20 μg/mL MNMs and stimulated by 1 Hz RMF for 15 min. All groups were washed with PBS three times and then collected into 1.5-mL centrifuge tubes. Blocked Purified Rat Anti-Mouse CD16/CD32 (BD Biosciences, 553141) was used at a 1:400 dilution in flow buffer (2.5% FBS mixed into 97.5% PBS) to block non-specific sites on cells for 20 min at 4 °C. The reaction was terminated with 1 mL flow buffer, and cells were collected after centrifugation at 1400 rpm for 4 min. Cells were incubated with BV421 Rat Anti-Mouse CD86 (1:100, BD Biosciences, 565388) and Alexa Fluor 647 Rat Anti-Mouse CD206 (1:100, BD Biosciences, 565250) for 30 min to label M1 and M2 subtypes. LLC cells were incubated with PE Rat Anti-Mouse MHC I (1:100, BioLegend, 116507) for 30 min. After the reaction was terminated and the cells were centrifuged, flow buffer was used to dilute the cells, and flow cytometry was performed.
Enzyme-linked immunosorbent assay (ELISA)
All macrophages (RAW 264.7 and BMDMs) were seeded and polarized into M1 or M2 subtypes with the corresponding inducing factors as described previously. M2 macrophages were incubated with 20 μg/mL MNMs and stimulated by 1 Hz RMF for 15 min. After culturing for 24 h, the supernatants of the different groups were collected, and the contents of IL-1β, IL-4, IL-6, and IL-10 were detected using corresponding ELISA kits (Wuhan Biofavor Biotech Service, China).
Western blot analysis
All macrophages (RAW 264.7 and THP-1 wild-type cells or Gal9-KO cells) were seeded and induced to obtain M1 and M2 subtypes. After incubation with 20 μg/mL MNMs, M2 macrophages were stimulated by 1 Hz RMF for 15 min. After treatment, the M2 and M2-RMF groups were cultured for 6 h and 12 h. Compound C (10 μM; Selleck, 866405-64-3) or 1 mM metformin (Selleck, 657-24-9) was also used to stimulate the related groups for 1 h. All cells were collected using sodium dodecyl sulfate (SDS) loading buffer, lysed with RIPA buffer, separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to PVDF membranes. After blocking with 5% milk, the membranes were incubated with the related antibodies overnight at 4 °C. The mixture was purified, and horseradish peroxidase-linked rabbit anti-goat secondary antibody was added. The signal was detected using a gel and blot imaging system, and band densities were analyzed using ImageJ.
For co-immunoprecipitation, cells from different groups were lysed after stimulation, and whole-cell lysate extracts were incubated with 3 μg AMPK (CST, 5832 T) antibody or IgG control overnight at 4 °C. Extracts were then incubated with protein A/G Sepharose beads for 2 h at 4 °C, washed five times with cell lysis buffer, and eluted in 2× loading buffer at 95 °C for 10 min. Eluates and cell lysate extracts (input samples) were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore, ISEQ00010), and immunoblotted.
Transwell migration assay
To observe the migration of tumor cells after RMF stimulation, a Transwell assay was performed. LLC cells were seeded in the upper compartment of the Transwell (8.0-µm pore polycarbonate membrane, Costar, 3428) at a density of 1 × 104 cells per chamber, and 20 μg/mL MNMs were added and co-cultured for 24 h. After 1 Hz RMF stimulation for 15 min and culture for 24 h, the cells were fixed in 4% PFA. The upper layer of LLC cells was removed using a cotton swab, and cells in the lower layer were stained with 0.1% crystal violet for 20 min. After three washes with PBS, 33% glacial acetic acid was used to elute the crystal violet for 10 min. The absorbance of the solution was measured at 570 nm using a microtiter plate reader (ELx808, BioTek).
To examine the ability of MNMs to penetrate the endothelial barrier, a Transwell-based endothelial penetration assay was performed. Endothelial cells were seeded in the upper chamber at a density of 1 × 105 cells per well to form a confluent monolayer. Formation of a tight monolayer was confirmed by measuring transendothelial electrical resistance (TEER) using a voltohmmeter (Millicell ERS-2, Merck Millipore), and experiments were initiated when TEER values reached approximately 200 Ω·cm2. Fifty micrograms of MNMs were added to the upper chamber, with or without placing a magnet beneath the culture plate to provide magnetic guidance. At 30 min, 1 h, 2 h, and 4 h, the medium from the lower chamber was collected, and the Fe content was quantified by ICP-MS.
Seahorse extracellular flux analysis
Respiration and glycolytic rates were measured in RAW 264.7 cells using a 24-well Seahorse XF24 Extracellular Flux Analyzer. Macrophages in Control, MNM, and RMF groups were plated in XF24 microplates (2 × 105 cells per well) and incubated overnight.
For mitochondrial respiration measurement, the Agilent XF Cell Mito Stress Test Kit (#103015) was used. Before the assay, cells were incubated with Seahorse XF DMEM containing 10 mM glucose, 5 mM sodium pyruvate, and 2 mM glutamine (pH 7.4) in a CO2-free incubator for 1 h. The oxygen consumption rate (OCR) was measured at baseline and after sequential addition of oligomycin (1 μM, ATP synthase inhibitor), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, 1.6 μM, uncoupler of oxidative phosphorylation), and a combination of rotenone (0.5 μM, complex I inhibitor) and antimycin A (0.5 μM, complex III inhibitor).
For measurement of glycolytic function, the Agilent XF Glycolysis Stress Test Kit (#103020) was used. Cells were incubated with Seahorse XF Base Medium supplemented with 2 mM glutamine (pH 7.4) in a CO2-free incubator for 1 h. Extracellular acidification rate (ECAR) was measured at baseline and after sequential addition of glucose (10 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (2-DG, 50 mM, a glycolysis inhibitor).
Each condition was performed in three biological replicates. After the assay, cells were fixed in 4% paraformaldehyde (PFA), and OCR/ECAR values were normalized to cell numbers (quantified by Hoechst staining) and to the baseline value at the initial time point.
RNA sequencing
RNA was extracted and purified from all groups following the standard procedures described for RT-qPCR, and the purity and concentration of total RNA were measured using the NanoDrop 2000 system. For RNA sequencing, mRNA was purified by polyT selection. PCR was used to enrich the template and obtain the cDNA library. A Qubit fluorometer was used to measure the concentration of the cDNA library, and qPCR was then performed to obtain accurate quantification. The samples were sequenced on the Illumina NovaSeq 6000 system at Berry Genomics Corporation to obtain 150-bp paired-end reads. Differentially expressed genes (DEGs) between the M2-RMF and M2 groups were identified using DESeq2.62 GSEA was performed using clusterProfiler, and the results were plotted with enrichplot.63 The P values were corrected using the Benjamini–Hochberg approach, and genes with a P value < 0.05 were considered significant.
Quantification of metabolites by liquid chromatography-mass spectrometry (LC-MS)
RAW 264.7 cells were washed with PBS and lysed with 200 μL pre-cooled extraction buffer (40:40:20 methanol: acetonitrile: water). Cells were then scraped from plates using cell scrapers and transferred into Eppendorf tubes. After 30 s of vortexing, the samples were centrifuged at 16,000× g for 20 min at 4 °C. The supernatants were transferred to new 1.5-mL tubes and centrifuged at 16,000× g for 30 min at 4 °C, and the resulting supernatants were transferred to LC-MS vials for analysis.
LC separation was performed on a Vanquish UHPLC system (Thermo Fisher Scientific) with a HILIC column (2.1 × 150 mm, 5 μm, HILICON) and a column temperature of 25 °C. Solvent A was 95% water and 5% acetonitrile with 20 mM ammonium acetate and 20 mM ammonium hydroxide (pH 9.4), and solvent B was acetonitrile. The gradient was 0–2 min, 95% B; 3–7 min, 75% B; 8–9 min, 70% B; 10–12 min, 50% B; 13–14 min, 25% B; 16–20.5 min, 0% B; and 21–28 min, 90% B, with an injection volume of 5 μL. The Q Exactive Plus mass spectrometer was operated in switching negative/positive ion mode, scanning m/z 60–900 with a resolution of 140,000 at m/z 200 (AGC target 3e6, maximum IT 200 ms). Raw data were converted to mzXML format using MSconvert and processed using EI-MAVEN for peak detection, extraction, alignment, and integration.
scRNA-seq
scRNA-seq of tumor tissues was performed at Berry Genomics Corporation. CellRanger (Version 7.0.0) was used to align scRNA-seq reads to the mm10 reference genome and to filter and count barcodes and UMIs. Data normalization, cell clustering, and dimensional reduction were performed using the Python package Scanpy (Version 1.7.2).64
For quality control, genes expressed in fewer than three cells were excluded from the merged cell-gene count matrix. Cell filtering thresholds were determined on the basis of data distribution and expression patterns. Cells that expressed fewer than 200 genes and cells with greater than 10% mitochondrial gene counts were excluded to ensure cell quality. Cells that expressed more than 8000 genes were excluded to avoid potential doublets. After clustering, cell clusters that overexpressed markers of more than two cell types were also removed from the analysis.
The resulting matrix included 94,218 cells and 25,964 genes. Data were normalized using the Scanpy function pp.normalize_per_cell, and pp.log1p was used for log transformation. Highly variable genes were identified using the pp.highly_variable_genes function with default parameters, followed by data scaling with the pp.scale function. Principal component analysis was carried out using the tl.pca function for linear dimensionality reduction on highly variable genes. A neighborhood graph was computed with pp.neighbors using the top 40 PCs, and Uniform Manifold Approximation and Projection (UMAP) was performed using the tl.umap function. Leiden clustering was performed using the tl.leiden function with a resolution of 0.4. The resulting clusters were annotated on the basis of their expression of canonical markers. Macrophages, T cells, and neutrophils were extracted separately from the integrated single-cell dataset on the basis of initial cluster annotations. For each cell type, a second round of dimensionality reduction and clustering was performed. Subtype annotations were based on the expression of canonical marker genes.
Differential expression analysis of macrophage cells was performed using the GSEA Java tool.65 The log2 mean expression fold-change between treatment and control was calculated for each gene, and the log2 fold change rank data were used as input for GSEA preranked analysis. The hallmark gene set (mh.all.v2023.2.Mm.symbols.gmt) was used as the analysis database.
Subcutaneous tumor implantation and antitumor therapy in vivo
Two implantation methods for mouse tumor models were used to assess whether MagLMP could inhibit tumor growth in vivo. First, 1 × 106 LLC cells were directly implanted subcutaneously into the thighs of male C57BL/6 mice. Tumor volume was calculated as V = (π × L × W2)/6, where L is the maximum tumor diameter, and W is the minimum tumor diameter. When the tumor volume of LLC-tumor-bearing mice reached ~50 mm3, MagLMP antitumor therapy was performed for 14 days. Tumor volumes and body weights of all mice were measured every day. For macrophage depletion in tumor tissues, mice were treated with CL using two dosing regimens: (1) intraperitoneal injection of 200 μL (5 mg/mL, YEASEN) once daily for three consecutive days before RMF treatment, and (2) intravenous injection of 200 μL on day 1, followed by 100 μL every three days during the treatment period until completion. To ensure consistency in CL administration, except for the validation experiments comparing the efficacy of the two dosing regimens (Supplementary information, Fig. S9a–d), all other experiments used the pre-treatment intraperitoneal injection protocol.
After 14 days of observation, mice were deeply anesthetized by intraperitoneal injection of 1% sodium pentobarbital and then cardiac-perfused with 10 mL PBS followed by 10 mL 4% PFA. After tumor tissues from all groups were photographed, all tissues were stored in 4% PFA for further experiments. Four groups of mouse tumor models were established through direct implantation of LLC cells.
Group 1: observing the antitumor effect of MagLMP therapy with or without CL treatment in a subcutaneous tumor model. When the tumor volume reached the requirements described above, MNMs were injected into tumor tissues at a dosage of 50 μg MNMs/25 μL PBS per mouse for 4 consecutive days. For the control group, PBS (25 μL) was intratumorally injected for 4 consecutive days. For the RMF group, tumors were stimulated with 1 Hz RMF for 30 min on 14 consecutive days, beginning on the second day after MNM injection. For the CL/RMF group, CL was injected intraperitoneally at 200 µL once daily for three consecutive days, followed by MNM injection and RMF stimulation as described above.
To further assess the role of macrophages in MagLMP-mediated antitumor effects, BMDMs were isolated from wild-type mice and differentiated by M-CSF treatment (20 ng/mL) in vitro. Mature BMDMs were then incubated with 100 μg/mL MNMs for 24 h. The MNM-loaded BMDMs were adoptively transferred (106 cells per mouse) via intratumoral injection into subcutaneous tumors of macrophage-depleted mice that had been pretreated with CL (200 µL injected intraperitoneally once daily for three consecutive days). Tumors were then treated with or without RMF stimulation (1 Hz, 30 min) for 14 consecutive days.
To evaluate the effects of MagLMP on LLC cells in vivo, LLC cells were co-cultured with MNMs (100 μg per 1 × 106 cells) for 24 h. The MNM-loaded LLC cells were then directly implanted subcutaneously into the thighs of male C57BL/6 mice, followed by daily RMF stimulation (1 Hz, 30 min) for 14 consecutive days.
Group 2: observing the antitumor effect of MagLMP therapy in 4T1 and B16 subcutaneous tumor models. 4T1 and B16 cells (1 × 106) were directly implanted subcutaneously in the thighs of male C57BL/6 mice. When the tumor volume reached the requirements described above, MNMs were injected into tumor tissues at a dosage of 50 μg MNMs/25 μL PBS per mouse for 4 consecutive days. For the control group, PBS (25 μL) was intratumorally injected for 4 consecutive days. For the RMF group, the tumors were stimulated with 1 Hz RMF for 30 min on 14 consecutive days, beginning on the second day after PBS (RMF (MNM–) group) or MNM (RMF (MNM + ) group) injection.
Group 3: observing the antitumor effect of MagLMP therapy by tail vein injection of MNMs in a subcutaneous tumor model. For the MNM group, MNMs were injected through the tail vein (100 μg MNMs/25 μL PBS per mouse) once a day for 4 times. For the control group, 100 μL PBS was injected through the tail vein 4 times. For the RMF group, RMF stimulation was performed for 14 consecutive days beginning on the second day. For the RMFMF Guide group, a magnet was fixed to the tumor site within 4 days of MNM injection to guide MNM accumulation in the tumor tissues, and RMF stimulation was performed as described above.
Group 4: observing the antitumor effect of MagLMP by tail vein injection of MNMs in AIS of the lung in a mouse model. After being deeply anesthetized, the mice were fixed in the supine position on the operating table, and the anterior chest wall was disinfected with 75% alcohol. Next, a small incision of ~5 mm was made at ~1.5 cm above the costal arch of the left anterior axillary line. The skin and subcutaneous tissue were separated, and the chest wall was exposed. A total of 100 μL of LLC cells and Matrigel suspension (106 LLC cells dispersed in 50 μL PBS and mixed with 50 μL Matrigel) were injected into the left lung. The injection depth was ~3 mm, and the incisions were closed sequentially. Two batches of in situ lung cancer mouse models were generated in parallel. In one batch, three mice were sacrificed on days 6, 9, 12, 15, and 20 according to the prescribed procedure, and lung tissues were removed to visualize the tumor volumes. Another batch of mice was randomly divided into 4 groups with 10 mice in each group. The PBS, MNM, RMF, and RMFMF Guide groups received the treatments described above. After MagLMP therapy for 14 days, 1 mouse was randomly chosen for the removal of lung tissue as described above, and the tissue was fixed in 4% PFA for subsequent use. The remaining nine mice in each group were observed for 120 days to assess overall survival.
Second, a mouse-derived allograft (MDA) model was used to maintain the tumor microenvironment and tumor heterogeneity. First, 1 × 106 LLC cells were implanted subcutaneously in the right thighs of several male C57BL/6 mice, which were used to produce additional tumor tissue for transplant. After the tumor volume reached 100–500 mm3, the tumor tissue was removed, and a tissue block of ~1 mm in diameter was cut. The small pieces of tissue were replanted subcutaneously in the thighs of C57BL/6 mice with a trocar. When the tumor volume of LLC-tumor-bearing mice reached approximately 50 mm3, MagLMP antitumor therapy was performed for 14 days. Tumors were observed and data recorded after MagLMP treatment as described above. Four groups of mouse tumor models were established on the basis of the MDA mouse model.
Group 1: verifying the antitumor effect of MagLMP combined with anti-PD-1 antibody (BioXcell, BE0146) in a subcutaneous tumor model. The subcutaneous MDA mouse model was created first. Then, 100 μg MNMs/25 μL PBS were injected through the tail vein once daily for 4 consecutive days, with 100 μL PBS injected similarly in the control group. For the RMF group, the tumors were stimulated with 1 Hz RMF for 30 min on 14 consecutive days, beginning on the second day after MNM injection. For the iPD-1 and iPD-1/RMF groups, anti-PD-1 antibody was injected intraperitoneally every two days at a dosage of 100 μg PD-1 inhibitor/100 μL PBS per mouse on the second day after MNM injection for 14 days, accompanied by or without MagLMP treatment for 14 days.
Group 2: verifying the effect of AMPK on the MagLMP strategy for antitumor immunity using an AMPK inhibitor. The subcutaneous MDA mouse model was created, and MNMs were then injected into tumor tissues at a dosage of 50 μg MNMs/25 μL PBS per mouse for 4 consecutive days when tumor volumes reached approximately 50 mm3. The tumors were stimulated with 1 Hz RMF for 30 min on 14 consecutive days, beginning 24 h after MNM injection. For the treatment group that received MagLMP combined with the AMPK inhibitor, the mice were intraperitoneally injected with an AMPK inhibitor (Compound C, 20 mg/kg per mouse) every two days.
Group 3: verifying the effect of Gal9 on the MagLMP strategy for antitumor immunity using Gal9-KO BMDMs. Gal9 gene knockout was performed on C57BL/6 wild-type mice. BMDMs were isolated from both C57BL/6 wild-type and Gal9-KO mice and differentiated using M-CSF (20 ng/mL). Mature BMDMs were incubated with 100 μg/mL MNMs for 24 h. The MNM-loaded BMDMs were then adoptively transferred into subcutaneous tumors (106 cells per mouse) of macrophage-depleted mice (pretreated with CL) via intratumoral injection. Tumors that received wild-type BMDMs were subsequently stimulated with RMF (1 Hz, 30 min) daily for 14 consecutive days.
Group 4: verifying the dependence of the MagLMP strategy for antitumor immunity on Gal9 and AMPK using Gal9-KO BMDMs. BMDMs were isolated from Gal9-KO mice and differentiated using M-CSF (20 ng/mL). Lentiviral particles encoding AMPK-targeting shRNA were used to transduce BMDMs for AMPK knockdown, and particles carrying negative control (NC) shRNA served as the control. Mature BMDMs were incubated with 100 μg/mL MNMs for 24 h. The MNM-loaded BMDMs were subsequently adoptively transferred into subcutaneous tumors (106 cells per mouse) of macrophage-depleted mice (pretreated with CL) via intratumoral injection. Tumors that received these BMDMs were stimulated with RMF (1 Hz, 30 min) daily for 14 consecutive days.
Flow cytometry analysis of in vivo tissues
After completing all treatments, mice were sacrificed by neck amputation after being deeply anesthetized by intraperitoneal injection with 1% sodium pentobarbital, and the entire tumor tissue was quickly extracted. Surgical scissors were used to cut up the tumor tissue as much as possible, and the cut tissue was placed in a mixed enzyme solution (0.05 mg/mL type I collagenase, 0.05 mg/mL type IV collagenase, 0.025 mg/mL hyaluronidase, 0.01 mg/mL deoxyribonuclease, and 0.05 mg/mL trypsin inhibitor). The mixed solution was placed in a shaker at 37 °C for 15 min. After the undigested solid tissue blocks were discarded, red blood cell lysis buffer (Beyotime Biotech, C3702) was added for 5 min, and lysis was terminated with flow buffer (2.5% FBS in 97.5% PBS). Cell blocking and staining were performed in vitro as described above. PE Rat Anti-Mouse F4/80 (BD Biosciences, 565410), PerCP-Cy5.5 Rat Anti-CD11b (BD Biosciences, 550993), BV421 Rat Anti-Mouse CD86, and Alexa Fluor 647 Rat Anti-Mouse CD206 were used to label different macrophage subtypes in order to analyze the ratio of M1 and M2 macrophages in the tumor tissue. Finally, the cells were resuspended in 300 μL flow buffer and passed through 40-μm filters.
Macrophage subtypes in the spleen were also analyzed by flow cytometry. Mouse spleens were completely removed. After mechanical trituration, cells were filtered through 40-μm filters. Red blood cell lysis buffer was added for 5 min, and the reaction was terminated with flow buffer. Cells were blocked and stained separately as described above. The data were analyzed using CytExpert Software (Version 2.4.0.28).
To analyze the ratio of different cell types that internalized MNMs in tumor tissues, LLC cells were implanted subcutaneously in the thighs of C57BL/6 mice. Once the tumors reached the appropriate size, MNMs were directly injected into the tumor once daily, followed by RMF stimulation for 14 consecutive days. In the intravenous injection model, mice were administered MNMs (100 μg per mouse) via tail vein injection. An external magnet was applied to guide the enrichment of MNMs at the tumor site, followed by RMF stimulation for 14 consecutive days. At the end of the treatment period, tumors were excised and enzymatically digested as described previously. After red blood cell lysis, all cells were magnetically separated for 20 min. All the fluid in the tubes was removed, and the remaining cells adsorbed on the tube walls by the magnet were those with internalized MNMs. Fc receptors were blocked with anti-CD16/32 antibody. BV-570 FVS was used to distinguish living cells. APC-Cy7 CD45 (BD Biosciences, 564406) was used to differentiate monocytes and tumor cells. Macrophages were screened by BV785-F4/80 and BV605-CD11b. M1-like macrophages were further distinguished by CD80 expression. Neutrophils were labeled with PE-Cy7 and PerCP-Cy5.5 CD11b. DCs were identified using BB700-conjugated CD11c and BV421-conjugated MHC-II antibodies.
To evaluate the depletion efficiency of CL on tumor-associated macrophages, mice were pretreated with CL intraperitoneally once daily for three consecutive days. To assess the in vivo persistence of intratumorally transferred BMDMs, cells were labeled with DiD dye (1:1000, Beyotime Biotech, C1039) for 20 min prior to injection into the tumor tissues. In both experiments, tumors were collected on days 1, 7, and 14 after treatment and processed as described above, and endogenous macrophages were stained for analysis. DiD labeling was used to track the adoptively transferred BMDMs.
To detect the activation of tumor-specific CD8+ T cells in the spleen, LLC cells with OVA-LLC were constructed and implanted subcutaneously into C57BL/6 mice. MNMs were injected into the tumor directly before MagLMP was performed on these mice. Mice were treated with or without CL. After tumors were treated with programmable MagLMP for 14 days, the spleen of each mouse was dissected and prepared into a single-cell suspension. These cells were co-cultured with OVA-LLC cells for 6 h in the presence of APC-Cy7 CD107a (BioLegend, 121616) and brefeldin A (MCE, 20350-15-6). BV650 CD45 (BioLegend, 103151) was used to differentiate monocytes. PerCP-Cy5.5 CD3 (BD Biosciences, 551163) and FITC CD8 (MBL, 553035) were used to label CD8+ T cells. PE OVA tetramer (MBL, TS-5001-1C), BV421 granzyme B (BioLegend, 396414), and APC IFN-γ (BD Biosciences, 554413) were used to identify the activation of tumor-specific CD8+ T cells.
To evaluate changes in MHC I expression on tumor cells after MagLMP treatment, GFP-labeled LLC cells were implanted subcutaneously into C57BL/6 mice. MagLMP treatment was performed for 14 consecutive days as described above. Tumors were harvested on days 1, 7, and 14 post-treatment for flow cytometric analysis. Tumor tissues were dissociated into single-cell suspensions, and PE-conjugated anti-MHC I antibody was used to detect MHC I expression on GFP+ LLC tumor cells.
All the processed cells were resuspended in 300 μL flow buffer and analyzed by flow cytometry. Data were processed using CytExpert software.
Flow cytometric sorting of tumor-associated macrophages
LLC cells were implanted subcutaneously into C57BL/6 mice. MNMs were administered intratumorally as described above, followed by 14 days of MagLMP treatment. For the intravenous injection model, MNMs were injected via the tail vein and guided to accumulate in the tumor tissue using an external magnet. Tumor tissues were then dissociated into single-cell suspensions. Fc receptors were blocked with an anti-CD16/32 antibody, and BV-570 FVS was used to exclude dead cells. Tumor-associated macrophages were sorted and collected using APC-Cy7 CD45, BV785-F4/80, and BV605-CD11b markers. Subsequently, Fe levels in sorted macrophages were quantified by ICP-MS. Total RNA was extracted according to standard protocols for RT-qPCR analysis of macrophage-related genes. In parallel, proteins were extracted to assess AMPK, p-AMPK, and iNOS expression by western blotting.
In the animal experiment involving Gal9-KO BMDMs transduced with AMPK-targeting shRNA and adoptively transferred into tumors, tumor tissues were dissociated into single-cell suspensions after 7 days of MagLMP treatment. The transferred BMDMs were sorted on the basis of GFP positivity (as the AMPK-targeting shRNA construct carried a GFP reporter) by flow cytometry. Proteins were then extracted from the sorted cells to evaluate AMPK expression by western blotting, thus confirming the in vivo knockdown efficiency of AMPK.
In vivo fluorescence imaging
To evaluate the accumulation of MNMs in tumors in the intravenous injection model, Cy5.5-labeled MNMs (100 μg per mouse) were administered via tail vein injection into subcutaneous tumor-bearing mice. Immediately after injection, a magnet was placed over the tumor site to guide MNM enrichment. Fluorescence intensity at the tumor site was monitored at 30 min, 1 h, 2 h, and 4 h post-injection using an in vivo imaging system (IVIS Lumina XRMS, PerkinElmer).
Detection of serum inflammatory factors
Mice were intratumorally injected with 50 μg MNMs/25 μL PBS per mouse for 4 consecutive days, treated with MagLMP for 14 days, and sacrificed at different time points. Retro-orbital blood was collected after the mice were deeply anesthetized by intraperitoneal injection with 1% sodium pentobarbital, and the mice were then immediately sacrificed by neck amputation. After standing at room temperature for 2 h, the blood was centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatants from different groups were collected, and the contents of IL-1β and TNF-α were measured using corresponding ELISA kits (Elabscience Biotechnology, China).
Quantification of Fe in mouse blood and tissues
Mice were intratumorally injected with 50 μg MNMs/25 μL PBS per mouse for 4 consecutive days. After MagLMP therapy for 14 days, heart, liver, spleen, lung, kidney, brain, and tumor tissues were obtained using standard operating procedures after transcardiac perfusion. The isolated tissues were homogenized and lysed in aqua regia, and ICP-MS was performed to quantify the amount of Fe in the different tissues.
To evaluate the biodistribution of MNMs in the intravenous injection model, mice were intravenously administered 100 μg MNMs per mouse with or without magnetic guidance. Blood and tumor tissues were collected at 30 min, 1 h, 2 h, and 4 h after injection under anesthesia. ICP-MS was performed to quantify Fe levels in blood and tumor tissues from the different groups.
Tissue sectioning, staining, and immunofluorescence
Tumor tissues and organs from different groups were cut into thin slices of approximately 5 mm × 5 mm × 2 mm. To observe cell morphology in tumor tissues and organs, hematoxylin and eosin (H&E) staining (Solarbio, G1120) was performed on the slices. To explore the distribution of MNMs in different organs, sections from the different groups were stained with Prussian blue (Solarbio, G1422). To explore the proportions of different macrophage subtypes in tumor tissues after MagLMP treatment, macrophages in the sections were stained by immunofluorescence with anti-mouse CD206 (R&D Systems, AF2535), anti-mouse CD80 (Proteintech, 14292-1-AP), anti-mouse F4/80 (R&D Systems, MAB5580), and DAPI (Beyotime Biotech, C1002). To explore the infiltration of CD4+ and CD8+ T cells into tumor tissues, sections were stained with anti-mouse CD4 antibody (Abcam, ab316866), anti-mouse CD8 antibody (Abcam, ab308264), and DAPI. All stained sections were observed by confocal laser scanning microscopy (Leica TCS SP8).
Quantification and statistical analysis
Statistical details of experiments, including n and P values, can be found in the figures, figure legends, and methods. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software, CA), and data are presented as means ± SD. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. Comparisons among multiple groups were performed using one-way ANOVA or two-way ANOVA, followed by Tukey’s honestly significant difference (HSD) post hoc test or Bonferroni’s multiple comparisons post-test, unless otherwise noted. P < 0.05 was considered statistically significant. For co-localization experiments, percent co-localization was determined using Manders’ co-localization coefficient (MCC).