Ethical approval
All the mice were maintained under a specific pathogen-free (SPF) condition in the animal facility of Zhejiang University. Carbon dioxide was used for euthanasia. All the animal experiments were strictly conducted in accordance with the protocols approved by the the Animal Research Committee guidelines of Zhejiang University.
Cell Lines
HEK293T (ATCC CRL-3216), THP-1 (ATCC TIB-202), K562 (ATCC CCL-243), Nalm6 (ATCC CRL-3273) and AsPC-1 (ATCC CRL-1682) were obtained from the National Collection of Authenticated Cell Cultures and cultured according to standard protocols. HO-8910 (MZ-0089) was purchased from NingboMingZhoubioCO.,Ltd (Zhejiang, China). Human iPSCs were obtained from the reprogramming of peripheral blood mononuclear cells from a volunteer donor, as described before10. The experiment was approved by the Human Subjects Committee of Jinling Hospital, Nanjing University. Approval number: 2020DZSKTZX-007. Human iPSCs were cultured in mTeSR medium (85852, STEMCELL Technologies) with Matrigel Matrix (354277, Corning) coated plates.
Plasmid construction and single guide RNA cloning
All the Cas9-expressing THP-1 cells or iPSC lines in this study were derived by lentiviral transduction with a Cas9 expression vector containing an optimized sgRNA backbone (LentiCRISPR v2; Addgene, 52961). All of the sgRNAs were cloned into the LentiCRISPR v2 vector following the protocol described before41. And the sgRNAs used in this study were shown in Supplementary Table 1. The annealed sgRNA oligonucleotides were ligated with T4 DNA ligase (M0569S, NEB) to the BsmB1-digested LentiCRISPR v2 vector.
Lentivirus production
We produced lentivirus using HEK293T cells cultured in DMEM supplemented with 1% penicillin-streptomycin and 10% FBS. The CRISPR library vectors (Human CRISPR Metabolic Gene Knockout Library; Addgene, Pooled Library #110066)12 or the single sgRNA vectors, envelop vector pMD2.G, and packaging vector psPAX2 were mixed in a 4:3:1 ratio in OPTI-MEM (Thermo Fisher Scientific, 31985070) and Polyethylenimine (PEI) (Polysciences, 9002-98-6), and transfected into HEK293T cells at 80% to 90% confluence in 10-cm tissue culture plates. The supernatant was collected at 24, 48, and 72 h post-transfection, filtered via a 0.45 μm filtration unit (Millipore, Cat# SLHVR33RB), and mixed overnight at 4 °C with one-third volume of 30% PEG8000. The medium was concentrated at 3200 × g for 30 min at 4 °C. The pellet was resuspended in PBS and stored at -80 °C.
Transduction of lentivirus containing sgRNAs
For transfection of THP-1 cells and iPSCs, we infected cells with lentivirus and 5 μg/mL polybrene overnight, and the medium was changed the following day. After puromycin (1 μg/mL for THP-1 cells and 250 ng/mL for iPSC) selection for seven days, >95% of the population was transfected, and the cells were ready to be used for the subsequent experiments.
Pooled CRISPR screen
1.5 × 107 THP-1 cells were transduced with a viral library for 24 h (MOI = 0.3). After puromycin (1 μg/mL) selection for seven days, 1.5 × 107 transduced cells were collected as input samples. The other transduced cells were treated with PMA (50 ng/mL) for 48 h, then stimulated by LPS (50 ng/mL) plus IFN-γ (50 ng/mL) for 24 h. The stimulated cells were harvested and stained with PE anti-human CD80 (Biolegend, Cat:305208, Clone: 2D10, Lot: B330518) for 15 min at room temperature. The CD80high and CD80low cells were separated by flow cytometry sorting. The genomic DNA of cells was isolated, and the sgRNA library was barcoded and amplified for two rounds of PCR. PCR products were purified for sequencing on an Illumina HiSeq 2500. The sequencing data was analyzed by MAGeCK42.
Generation of CRISPR/Cas9 knockout cells
LentiCRISPR v2 vectors targeting KEAP1 and ACOD1 were constructed as described before41. The THP-1 cells and iPSC were infected with lentivirus expressing Cas9 and sgRNAs targeting KEAP1 and ACOD1. After puromycin selection for seven days, the THP-1 cells were expanded, and knockout efficiency was verified using qPCR and western blotting. After puromycin selection for three days, iPSCs were passaged, and the clones grown from single cells were picked up and expanded. The knockout efficiency of iPSC was verified by sequencing, qPCR, and western blot analyses.
Derivation of iMACs from iPSCs
The derivation of iMAC from iPSCs has been previously described10. Briefly, 8000 iPSCs were seeded in 96-well round-bottom plates with APEL2 medium (05271, STEMCELL Technologies) containing 100 ng/mL human Stem Cell Factor (SCF), 50 ng/mL human Vascular Endothelial Growth Factor (VEGF), 10 ng/mL recombinant human Bone Morphogenetic Protein 4 (BMP-4), 5 ng/mL human FGF-basic (154 a.a.), and 10 mM Rho kinase inhibitor (ROCK inhibitor, Y27632, Sigma). After eight days of hematopoietic differentiation, spin embryoid bodies (EB) were transferred into Matrigel-coated 6-well plates under macrophage differentiation conditions. Macrophage differentiation medium is StemSpan-XF (100-0073, STEMCELL Technologies) containing 10 ng/mL human FGF-basic (154 a.a.), 50 ng/mL human Vascular Endothelial Growth Factor (VEGF), 50 ng/mL human Stem Cell Factor (SCF), 10 ng/mL recombinant human Insulin-like Growth Factor-1 (IGF1), 20 ng/mL IL-3, 50 ng/mL recombinant human M-CSF, and 50 ng/mL recombinant human GM-CSF. The floating cells were collected from the supernatant and directly transferred into uncoated 6-well plates in macrophage culture medium. The macrophage culture medium is StemSpan-XF containing 50 ng/mL recombinant human M-CSF and 50 ng/mL recombinant human GM-CSF.
Flow cytometry
The tMACs or iMACs were stimulated with LPS and IFN-γ for the indicated time. The single-cell suspensions were then prepared and incubated with an antibody or antibody cocktails for 15 min at room temperature for cell surface staining. Antibodies used in this study were PE Mouse IgG1, κ isotype (Biolegend, Cat: 400113, Clone: MOPC-21, Lot: B245984), APC Mouse IgG1, κ isotype (Biolegend, Cat: 400119, Clone: MOPC-21, Lot: B243042), FITC Mouse IgG1, κ isotype (Biolegend, Cat: 400107, Clone: MOPC-21, Lot: B199152), APC anti-human CD206 (Biolegend, Cat: 321109, Clone: 15-2, Lot: B348965), APC anti-human CD86 (Biolegend, Cat: 305411, Clone: IT2.2, Lot: B351349), PE anti-human CD80 (Biolegend, Cat:305208, Clone: 2D10, Lot: B330518), PE anti-human CD163 (Biolegend, Cat: 333606, Clone: GHI/61, Lot: B347256), FITC anti-human CD14 (Biolegend, Cat: 325604, Clone: HCD14, Lot: B268830) and APC anti-humanCD11B (Biolegend, Cat: 301309, Clone: ICRF44, Lot: B278346). These antibodies were used at a 1:100 dilution. Data were recorded on Beckman DxFLEX and analyzed with the FlowJo V10 software.
In vitro tumor killing assay
The functionality of CAR-iMACs was assessed by co-culturing them with luciferase-expressing tumor cells in a 96-well plate (WHB-96-2). CAR-iMACs and 2 × 103 tumor cells were mixed in a total volume of 100 μL RPMI 1640 complete medium, and the number of cells was determined based on the indicated E:T ratio. After co-culturing for 24 or 48 h, 25 μL of 33 mg/mL D-Luciferin (GoldBio, 115114-35-9) was added to each well, and luminescent signals were analyzed using a microplate reader (TECAN, SPARK).
For the 4-OI supplement assay, iMACs were pre-treated with 4-OI (MCE, HY-112675, 250 μM) or DMSO (Sigma-Aldrich, 41639) control for 3 h before challenging them with LPS (InvivoGen, tlrl-eblps) plus human IFN-γ (PeproTech, 300-02-100UG) (50 ng/mL each). The iMACs were then co-cultured with luciferase-expressing tumor cells.
To test the function of NRF2, SFN (MCE, HY-13755, 10 μM) was added to the co-culture system.
To test the function of cytokines, the supernatant was collected after iMACs were co-cultured with HO-8910 cells for 24 h (E:T = 10:1). Then human IgG1 isotype control (BioXcell, BE0297), neutralizing antibody (10 μg/mL) of IFN-γ (BioXcell, BE0235), or TNF-α (BioXcell, SIM0006) was added to the supernatant, and the cytotoxicity activity was measured by luciferase assay.
Enzyme-linked immunosorbent assay
The supernatant of iMAC culture or tumor-iMAC co-culture was collected and centrifuged at 300 × g for 10 min to remove the precipitate. Human IL-6, IL-1β, CXCL-10, IFN-γ and TNF-α were quantified using Elisa kits (MultiSciences, EK106, EK101B, EK168, EK180, EK182) following the manufacturer’s protocols.
In vivo anti-tumor assay
For in vivo experiments, 6–8-week-old NOD/SCID/IL2rγnull (NSG) mice (Stock No: 005557) (The Jackson Laboratory, Bar-Harbor, Maine, USA) were maintained under pathogen-free conditions under the Zhejiang University Institutional Animal Care and followed the committee’s approved protocols. All mice were maintained in suitable temperature (25 °C) and humidity environment (typically 50%), 12 h dark/light cycle, and fed with sufficient water and food. The catalogue number of the diet is 1010085 (Xietong).
Mice were sacrificed when the average tumor diameter exceeds 20 mm in mice or when tumor volumes exceeded 2000 mm3. Mice will also be sacrificed when the tumor metastasizes or rapidly grows to the point of ulceration, causing infection or necrosis. For survival studies, mouse were monitored carefully and tumor burdens were measured once a week after initial inoculation.
In the first ovarian cancer mouse model, 6–8-week-old female NSG mice were used, 2 × 105 luciferase gene expressing HO-8910 cells were inoculated IP before treatment (day -4). After tumor cell inoculation, mice were randomly assigned to experimental groups (n = 5 per group). Four days later, 4 × 106 MSLN-CAR-iMACs or ACOD1-/- MSLN-CAR-iMACs were inoculated IP (day 0) for therapy. The tumor burden was determined by BLI using an In Vivo Imaging system (IVIS) Imaging System (BiospaceLab photonimager).
In the ovarian cancer orthotopic injection mouse model with CAR-iMAC and anti-CD47 antibody combined therapy, 6–8-week-old female NSG mice were used, 1 × 105 luciferase gene expressing HO-8910 cells were inoculated directly into ovary before treatment (day -4). After tumor cell inoculation, mice were randomly assigned to experimental groups (n = 5 per group). Four days later, mice received a single in situ injection of 6 × 106 MSLN-CAR-iMAC or ACOD1-/- MSLN-CAR-iMACs (day 0) combined with a low-dose CD47 antibody (50 μg/mouse, twice a week) for therapy. Tumor burden was determined by BLI.
In ovarian cancer mouse model with CAR-iMAC and the anti-PD1 antibody (Sintilimab) (Chemstan, Cat: CSD00572) combined therapy, 6–8-week-old female NSG mice were used, 2 × 105 luciferase gene expressing HO-8910 cells were inoculated IP before treatment (day -4). After tumor cell inoculation, mice were randomly assigned to experimental groups (n = 5 per group). Four days later, mice received a single injection of 4 × 106 MSLN-CAR-iMAC or ACOD1-/- MSLN-CAR-iMACs IP (day 0) combined with a low-dose anti-PD1 antibody (100 μg/mouse, twice a week) for therapy. The tumor burden was determined by BLI using an IVIS Imaging System (BiospaceLab photonimager).
In the pancreatic cancer mouse model, 6–8-week-old male NSG mice were used. 1 × 105 AsPC−1 cells were inoculated IP before treatment (day -4). After tumor cell inoculation, mice were randomly assigned to experimental groups (n = 5 per group). AsPC-1 cells grow fast in vivo, in order to get a better therapeutic effect, a higher E:T ratio was used. Four days later, 1.5 × 107 MSLN-CAR-iMACs or ACOD1-/- MSLN-CAR-iMACs were IP injected (day 0). The tumor burden was determined by BLI later.
Western blotting
Pellets from 1 × 106 cells were collected and resuspended with 100 μL RIPA Buffer (Beyotime, Cat: #P0013J). The samples were incubated on ice for 30 min and centrifuged at 15000×g for 15 min at 4 °C. The supernatant was collected, and the protein concentration was measured by BCA analysis (Thermo Scientific, Cat: #23225). Approximately 50 μg of total protein was loaded for western blotting. Antibodies used in this study were HRP AffiniPure Goat anti-Rabbit IgG (H + L) secondary antibody (EARTHOX, Cat: 620822, 1:2000), β-Actin (13E5) Rabbit mAb (Cell Signaling Technology, Cat: #4970, 1:1000), NRF2 (D1Z9C) Rabbit mAb (Cell Signaling Technology, Cat: #12721, 1:1000), Anti-Keap1 antibody (abcam, Cat: ab227828, 1:1000), Anti-IRG1 antibody (abcam, Cat: ab222411, 1:1000). Representative images were obtained using the ChemiDoc Touch Imaging System (Bio-Rad).
Real-time reverse transcription-PCR
RNA was extracted from macrophages or tumor cells using Total RNA Isolation Kit V2 (Vazyme, Cat# RC112-01). Reverse transcription from RNA to cDNA use Hiscript Reverse Transcriptase (Vazyme, Cat# R302-01). PCR reactions were performed on a CFX96 Real-Time PCR System (Bio-Rad Laboratories) using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Cat# Q711-02). All the primers for qRT-PCR used in this study were shown in Supplementary Table 2.
Metabolic studies
OCR was measured by Seahorse XFe96 Analyzer (Agilent) using a Seahorse XF Cell Mito Stress Test Kit (Agilent, 103015-100). iMACs were resuspended in an RPMI1640 medium containing LPS (50 ng/mL) plus IFN-γ (50 ng/mL) and then seeded at 5 × 104 cells/well in an XF96 plate. Eight hours later, the RPMI 1640 medium was changed to XF RPMI medium. The OCR was measured (pmol/min) real-time in an XFe96 Extracellular Flux Analyzer. iMACs were stimulated with LPS and IFN-γ for 24 h, and the OCR was in response to 1.5 μM oligomycin, 2 μM fluorcarbonylcyanide phenylhydrazone (FCCP) and 500 nM rotenone and antimycin A. Basal OCR, MRC, ATP linked respiration, and mitochondrial spare respiratory capacity (SRC) was calculated by WAVE V2.6 software.
RNA-seq
Total RNA was isolated and purified using FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01) from 2 × 106 tMACs according to the manufacturer’s protocol. RNA qualification was performed using Nanodrop to check RNA purity (OD260/0D280) and Agilent 2100 to check RNA integrity. A total amount of 2 μg RNA per sample was used for RNA-seq libraries preparation. RNA-seq libraries were prepared using VAHTS Stranded mRNA-seq Library Prep Kit for Illumina V2 (Vazyme, NR612-02) according to the manufacturer’s protocol and sequenced on an Illumina Hiseq 2500. The threshold of differentially expressed genes is p-adj <0.05. The color descending from red to blue in the heatmaps of differentially expressed genes indicated log10 (FPKM + 1) from large to small.
Gene set enrichment analysis
To identify biological signatures depleted or enriched following CD80-based sorting or in the KEAP1 knockout macrophages, we used DAVID Bioinformatics Resources (https://david.ncifcrf.gov/). We focused on the biological oncology of the GO gene sets to obtain the indicated enrichment score.
Statistical analysis
All data are presented as mean ± SD. Comparisons between different groups were analyzed by the one-way analysis of variance (ANOVA), two-way analysis of variance (ANOVA), and unpaired two-tailed Student’s t-test. Kaplan-Meier survival curves were compared with the log-rank test. Statistical analyses were performed in GraphPad Prism 9.0.0 software using the statistical tests indicated for each experiment. All tests were considered significant at p < 0.05.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.