Animals
Myeloid-specific Hes1-KO C57BL/6J mice were generated by mating Hes1fl/fl mice (kindly provided by Young-Yun Kong, Seoul National University, Seoul, Korea) with LysM-Cre mice (kindly provided by Heung Kyu Lee, Korea Advanced Institute of Science & Technology, Daejeon, Korea). MMTV-PyMT mice (kindly provided by Han-Woong Lee, Yonsei University, Seoul, Korea) were crossed with LysM-Hes1fl/fl. Age- and sex-matched cohorts of female (8–12-week-old) and male (6–12-week-old) littermates were used in subcutaneous tumor graft experiments. Appropriate littermate controls were used in all experiments. All animal experiments were approved by the Institutional Review Board of the Yonsei University College of Medicine. The mice were housed in pathogen-free facilities, in a 12-hour light/dark cycle in ventilated cages, with ad libitum access to chow and water in accordance with the guidelines for the Care and Use of Laboratory Animals (2018–0289).
Cell culture, transfection, and reagents
The TC-1 lung cancer cell line (from C57BL/6 mice) was kindly provided by Sang-Jun Ha (Yonsei University). The EO771 breast cancer cell line (from female C57BL/6 mice) was provided by Kyu Lim (Chungnam National University, Daejeon, Korea). The MC-38 adenocarcinoma cell line (from female C57BL/6 mice), B16-F10 melanoma cell line (from male C57BL/6 mice), 4T-1 mammary carcinoma cell line (from BALB/c mice), CT-26 colon carcinoma cell line (from BALB/c mice), L-929 fibroblast line (from C3H/An mice), HEK-293, and RAW264.7 cell line (from male BALB/C mice) were purchased from the American Type Culture Collection (Manassas, VA, USA). Cells were maintained in RPMI 1640 or DMEM (Welgene, Kyungsan, Korea) supplemented with 10% FBS and 1% penicillin-streptomycin (Invitrogen, Waltham, MA, USA). All cell lines used in the tumor studies were confirmed to be mycoplasma-negative using a MycoAlert Mycoplasma Detection kit (Lonza, Basel, Switzerland). Cells were cultivated at 37 ℃ under 5% CO2. Transfection with the HES1 expression vector as well as with siRNAs against Stat6, Rbpj, and Hes1 was performed using Lipofectamine 2000 and Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Two siRNAs were used to confirm the results. Stat6 siRNA #1 (5′-GUGAAAGCCUGGUGGAAAU-3′), Stat6 siRNA #2 (5′-GAAACAGGCCAGAGAACUA-3′), Rbpj siRNA #1 (5′-GCACAGAAGUCUUACGGAA-3′), Rbpj siRNA #2 (5′-GCAUGUAGAAGGAGGGAAU-3′), Hes1 siRNA #1 (5′-UGAAAGUCUAAGCCAACUGAATT-3′) and Hes1 siRNA #2 (5′-CCAGCCAGUGUCAACACGACATT-3′) were purchased from GenePharma (Shanghai, China). Mus musculus Hes1 ORF sequences were amplified using high-fidelity PCR from full-length cDNA using primers mHes1-Fw (SalI) 5′-AAATTGTCGACGCCGCCACCATGGATTACAAGGATGACGACGATAAGAGCGC-3′ and mHes1-Rev (NotI) 5′-AATTTGCGGCCGCTCAGTTCCGCCACGGTCTCCAC-3′ and ligated into the pCMV-Sport6 vector (Addgene). DH5α (NEB) cells were used to expand the plasmid, and colonies were selected using 100 µg/mL ampicillin. Plasmid DNA was purified and sequenced to verify the construct. Recombinant murine IL-4 (#214 − 14), IL-10 (#210 − 10), IL-13 (#210 − 13), IFN-γ (#315-05), and macrophage colony-stimulating factor (M-CSF) (#315-02) were purchased from Peprotech (Rocky Hill, NJ, USA). LPS (L3024) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Transwell plates (#3422) were obtained from Corning (Corning, NY, USA).
Total RNA isolation, reverse transcription, and qRT-PCR
Total RNA was extracted using TRIzol Reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesized using RT-PCR Master Mix (TOYOBO, Osaka, Japan) from 3–5 µg RNA. qRT-PCR was performed using SYBR premix Ex Taq (Takara, Kusatsu, Shiga, Japan) on an QuantStudio 3 (Applied Biosystems, Waltham, MA, USA). β-actin was used for normalization. RNA quality assessment prior to sequencing was performed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Western blotting. Cells were lysed using 20 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 1.5% MgCl2, 1 mM EDTA, 1 mM Na2VO4, 1 mM phenylmethylsulfonyl fluoride and protease inhibitor cocktail (Xpert Protease Inhibitor Cocktail Solution, GenDEPOT, Katy, TX, USA), pH 7.5. Lysates were briefly vortexed and cleared by centrifugation at 13,200 rpm for 20 min at 4 ℃. Supernatants were transferred to fresh microcentrifuge tubes and protein concentrations were measured using Bradford assay (Thermo Fisher Scientific, Waltham, MA, USA). Proteins (20–50 µg) were separated using SDS-PAGE, electroblotted onto nitrocellulose membranes (Amersham Biosciences, Woburn, MA, USA), and then blocked in 5% skim milk powder and 0.05% Tween-20 in PBS (PBST). Primary antibodies were incubated with blots at dilutions of 1 in 1000–3000 in minimal volumes of 5% BSA in PBST for 1 h at room temperature or overnight at 4°C. Membranes were incubated with horseradish peroxidase–conjugated anti-mouse, anti-rabbit, or anti-goat secondary antibodies (Bethyl Laboratories, Montgomery, TX, USA) at 1:10,000 dilution in 5% skim milk powder in PBST for 1 h at room temperature. Anti-HES1, anti-HEY1, anti-PPARG, anti-STAT3, anti-p-STAT3 (Y705), anti-CD206, anti-GAPDH, anti-p65, anti-p-p65 (S536), anti-CD80, anti-CD86, anti-RBPJ, anti-β-actin, anti-C/EBPA, anti-C/EBPB, anti-AKT, anti-p-AKT (Th308), anti-p-AKT (S473), anti-IKBA, anti-p-IKBA (S32), anti-JNK, anti-p-JNK (Thr183/Tyr185), anti-p38, and anti-p-p38 (Thr180/Tyr182) antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p70S6K, anti-p-S6K (Th389), anti-p-S6K (Th421), anti-STAT1, anti-p-STAT1 (Y701), anti-IRF4, anti-IRF9, anti-SHP2, anti-HIF1A, anti-STAT6, anti-p-STAT6 (Y641), anti-iNOS, anti-ERK1/2, and anti-p-ERK1/2 (Thr202/Tyr204) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-NICD, anti-PDL1, and anti-ARG1 antibodies were obtained from Abcam (Cambridge, UK). The membranes were washed 3 times for 10 min each. A FUSION SOLO imager (Vilber, Marne-la-Vallée, France) was used for image detection according to the manufacturer’s instructions. β-Actin or GAPDH were used as loading controls.
Syngeneic tumor models
TC-1 cells (1.0 × 105), B16F10 cells (2.5 × 105), EO771 cells (5.0 × 105), MC-38 cells (5.0 × 105), 4T-1 cells (1.0 × 106), or CT-26 cells (1.0 × 106) were injected subcutaneously into the middle lower back of mice. In accordance with the ethics committee’s guidelines, two subcutaneous injections were performed, and the tumor was kept from growing so that the sum of the major axes did not exceed 20 mm. Experimenters were blinded to the genotype. Growth was monitored by measuring tumor size using a digital caliper. Volume was estimated using the formula: length × width2 × 0.5. Tumor-infiltrating immune cells were isolated by harvesting tumors 14–18 days after implantation following euthanasia.
Myeloid cell depletion
To achieve in vivo depletion of CSF1R+ cells, cohorts of mice were administered purified anti-CSF1R rat mAb (clone AFS98; BioXCell, Lebanon, NH, USA) or control isotype immunoglobulin (IgG2a) via intraperitoneal injection (400 µg per mouse). The treatment regimen consisted of an initial dose 1 week prior to tumor cell injection, followed by treatments at intervals of 6 d. To specifically deplete Ly-6G+ cells in vivo, purified anti-Ly-6G rat mAb (clone 1A8, BioXCell) or control isotype (clone 2A3, BioXCell) was administered to separate cohorts of mice. Antibodies were administered via intraperitoneal injection (300 µg per mouse) 5 d prior to tumor cell injection and at 48-h intervals thereafter.
T-cell depletion
Mice were treated with 200 µg rat-anti-CD4 (clone GK1.5, BioXCell), rat-anti-CD8 (clone 2.43, BioXCell), or rat IgG2b anti-KLH isotype control (clone LTF2, BioXCell) antibodies diluted in InVivo Pure pH 7.0 Dilution Buffer (IP0070, BioXCell) via intraperitoneal injection, with a treatment schedule of every three days commencing one day before the tumor cell injection.
Tumor digestion
Tumors were harvested, minced, and further dissociated using a gentleMACS Dissociator (#130-093-235, Miltenyi Biotec, Bergisch Gladbach, Germany), then digested with a MACS Miltenyi Tumor Dissociation Kit for mice (#130-096-730, Miltenyi Biotec) according to the manufacturer’s instructions. The resulting tumor cells were washed with DMEM and dissociated by gently pushing and pulling a syringe plunger and pipetting. The cell suspension was passed through a 70-µm cell strainer (SPL Life Science, Pocheon-si, Korea) and red blood cells were lysed using RBC Lysis Solution (Biosesang, Seong-nam, Korea). The tumor cells were washed again and collected from the 40 − 80% interface of a Percoll gradient.
Flow cytometry
Single-cell suspensions were prepared from spleen, bone marrow, and tumor tissues of normal and tumor-bearing mice. Spleens and bone marrow cells were labeled using fluorochrome-conjugated antibodies against CD3 (clone 17A2), CD4 (clone GK1.5), CD8 (clone 53 − 6.7), CD45R/B220 (clone RA3-6B2), Ly-6G/Ly-6C (Gr1) (clone RB6-8C5), Ly-6A/E (Sca-1) (clone D7), and CD117 (c-kit) (clone 2B8), as well as isotype-matched IgG controls (BioLegend, San Diego, CA, USA). Tumor cells were prepared as described above before being resuspended in PBS containing 2% FBS and 2 mM EDTA for flow cytometry. A LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (L-10119, Invitrogen) was used with cells in combination with anti-mouse CD16/CD32 Fc blocker antibody (#14-0161-81, Invitrogen) for 15 min on ice in the dark. Cells were washed and incubated with the fluorochrome-conjugated antibodies (BioLegend) against CD45 (clone 30-F11), CD4 (clone GK1.5), CD8 (clone 53 − 6.7), CD45R/B220 (clone RA3-6B2), NK-1.1 (clone PK136), F4/80 (clone BM8), CD11b (clone M1/70), Ly-6C (clone HK1.4), Ly-6G (clone 1A8), CD11c (clone N418), I-A/I-E (clone M5/114.15.2), CD86 (clone GL-1), CD80 (clone 16-10A1), and CD206 (clone C068C2) at the manufacturer’s recommended dilution for 30 min on ice in the dark. For samples requiring intracellular staining, cells were fixed with Fixation/Permeabilization Diluent (#00-5223-56, Invitrogen) for 30 min at RT, washed twice with Permeabilization Buffer (#00-8333-56, Invitrogen), and incubated with Alexa Fluor-488 − conjugated antibodies against HES1 (clone EPR4226), FOXP3 (clone FJK-16s), and ARG1 (clone A1exF5) in permeabilization buffer for 30 min at room temperature. For assessment of cytokine production by tumor-infiltrating lymphocytes, antibodies against IFN-γ (clone XMG1.2), granzyme B (clone GB11), IL-12/p40 (clone C15.6), and TNF-α (clone MP6-XT22) and purified cells were incubated with cell activation cocktail containing brefeldin A (#423303, Biolegend, San Diego, CA, USA) for 4 h at 37 ℃ under 5% CO2. Surface staining was carried out as described above; cells were fixed and permeabilized using Cyto-Fast Fix/Perm Buffer set (#426803, Biolegend) and intracellular staining was carried out according to the manufacturer’s instructions. Following staining, cells were washed again with permeabilization buffer, then with PBS, and resuspended in FACS Buffer for flow cytometric analysis on the BD LSRFortessa cell Analyzer (BD Biosciences, San Jose, CA, USA) at the Flow Cytometry Core of the Avison Biomedical Research Center in Yonsei College of Medicine. A total of 10,000–1,000,000 cells were analyzed per sample per mouse using BD FACS Diva Software. Data were analyzed using the FlowJo software. For TAM sorting, single-cell suspensions of tumor tissues were stained with fluorescently-labeled antibodies and sorted using a FACSAria flow cytometer (BD Biosciences). Live/Dead−CD45+CD11b+F4/80+ cells were collected for gene expression analysis.
Preparation of BMDMs
Cohorts of male mice aged 6–12 weeks were euthanized and both femur and tibia were dissected free of adherent tissue. Bone marrow cells from the femur and tibia were subjected to red blood cell lysis using red blood cell lysis buffer, and surviving cells were cultured for 6 d in differentiation medium comprising Dulbecco’s minimum essential (DME) medium supplemented with 30% L929 cell culture supernatant, 20% heat-inactivated FBS, and 1% penicillin/streptomycin [21]. On day 3, 5 mL medium was added. Cells were harvested using cold PBS, washed, resuspended in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin, and used at a density of 2 − 10 × 105/mL in subsequent experiments. To confirm the development of BMDMs, antibodies (BioLegend) against CD11b (clone M1/70), F4/80 (clone BM8), and MHC class II (I-A/I-E; clone M5/114.115.2) were used. Cells were analyzed using a BD LSRFortessa Cell Analyzer or GUAVA easyCyte 5HT (Cytek Biosciences, Fremont, CA, USA) flow cytometer. Appropriate gates were set for forward and side scatter in analyzing each population. To polarize the BMDMs, classical or alternative activation was induced by culturing the cells for 1 − 2 d. Classical activation was induced with 20 ng/mL of recombinant mouse IFN-γ and 100 ng/ml LPS, while alternative activation was induced with 20 ng/mL recombinant mouse IL-4. To rule out unintended effects of L929 cell culture supernatant on BMDMs, BMDMs were differentiated using recombinant M-CSF for comparison.
L929- and tumor-CM
L929 cells were maintained in DMEM supplemented with 10% FBS as the biological source of CSF-1 for BMDM culture. L929 cells were plated at a density of 4.7 × 105 cells/well in a 75-cm2 flask containing 55 mL 10% FBS/DMEM. Cells were placed in a humidified incubator with 5% CO2 at 37 ℃ for 7 d. The supernatant was collected and filtered (0.45 µm). Supernatants were prepared using B16-F10, EO771, MC-38, and TC-1 cells. Each cell line was grown in DMEM-complete medium or RPMI complete medium in a 75-cm2 flask. Supernatants were collected on days 1, 2, 3, and 4 and filtered using a sterile 33-mm filter (#PR03691, Millex-GV, Merck, Darmstadt, Germany) using a 10-mL syringe. Supernatants were stored at − 20 ℃ until use.
Transwell coculture
Bone marrow cells were collected from the femur and tibia of male C57BL/6 mice and differentiated into BMDMs using a previously described protocol [21]. On day 7, cells were harvested from a 90 × 15 mm Petri dishes (#10090, SPL) in cold PBS, washed, and resuspended in medium (DMEM with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 1% HEPES, and 1% sodium pyruvate). Cells were centrifuged at 1,500 rpm for 5 min and the pellet was resuspended in DMEM. BMDMs (1.5 × 105 per well) were plated in the lower compartment of 6.5-mm polycarbonate transwell inserts with a pore size of 0.4 µm (#3413, Corning) in maintenance medium one day prior to seeding of other cells. The next day, the medium was replaced with fresh medium 1 h before adding B16F10, EO771, MC-38, TC-1 or HEK-293 cells (1.5 × 105 per well), into the transwell inserts. Cells were co-cultured for 1 − 3 d in a humidified chamber at 37°C. Control wells contained only BMDMs in the lower compartment with medium.
ChIP
ChIP was performed on 5 × 106 cells using a Pierce Agarose ChIP kit (#26156, Thermo Fisher Scientific) according to the manufacturer’s instructions. Nuclear lysates were precipitated with anti-Hes1 (NBP1-47791, Novus Biologicals, Littleton, CO, USA), anti-STAT6 (#9362, Cell Signaling Technology), or normal rabbit IgG (#sc-2027, Santa Cruz Biotechnology) antibodies overnight at 4°C. The recovered DNA was amplified using qRT-PCR. HES1 binding sites were predicted by the JASPAR website (https://jaspar.genereg.net/).
Luciferase assay
The murine Arg1 promoter sequence was cloned into the pGL3-Basic reporter plasmid (pGL3-Basic-Arg-1) spanning from position − 3,200 to + 100 bp and validated by sequencing. Using Lipofectamine 2000, cells were co-transfected with the Arg1 reporter plasmid and either Hes1 expression plasmid (100 ng) or Hes1 siRNA together with pCMV-β-Gal plasmid. After 18 h, cells were stimulated with IL-4 (20 ng/mL) or EO771 CM for the times indicated. The murine Hes1 reporter plasmids pHes1 (467)-luc (Addgene plasmid #41723; http://n2t.net/addgene:41723; RRID: Addgene_41723) and pHes1 (467 RBPj (-))-luc (Addgene plasmid #43805; http://n2t.net/addgene:43805; RRID: Addgene_43805) were gifts from Ryoichiro Kageyama (RIKEN Center for Brain Science, Kyoto University). pGL2 basic, pHes1 (467)-luc, or pHes1 (467 RBPj (-))-luc, together with pCMV-β-Gal, were co-transfected into BMDMs using Lipofectamine 2000. At 18 h after transfection, cells were stimulated with IL-4 or tumor supernatants for 6 h. Cells were washed with PBS and lysed with Cell Culture Lysis Reagent (Promega, Madison, WI, USA), and luciferase activity was normalized to β-galactosidase activity in the cell lysate.
PD-1 checkpoint blockade
Once tumors became palpable, mice were randomly assigned to treatment groups and administered with 200 µg antibodies. The treatment, either an isotype control (clone 2A3, cat. #BE0089, BioXCell) or anti-PD-1 antibody (clone RMP1-14, cat. #BE0146, BioXCell), was administered intraperitoneally every three days, starting as soon as the tumors began to form.
QuantSeq 3′ mRNA sequencing
Reads were aligned to the genome assembly sequence or the representative transcript sequences using Bowtie2 (version 2.3.4.3). The resulting alignment file was used for transcript assembly, estimation of abundance, and differential gene expression analyses. Differentially expressed genes were determined based on counts from unique and multiple alignments using coverage in Bedtools (version 2.26). The read count data were normalized using the quantile normalization method with EdgeR within R version 4.0.3 (R development Core Team, 2016) using Bioconductor. Gene sets were obtained from the Molecular Signature Database (version 7.0). Gene classification was performed by DAVID (http://david.abcc.ncifcrg.gov/).
Immunohistochemistry
Staining was performed on 5-µm-thick formalin-fixed paraffin-embedded tissue sections. Sections were deparaffinized using xylene and dehydrated using a graded ethanol series. Endogenous peroxidase activity was quenched using 3% H2O2 for 20 min at room temperature. The sections were subjected to antigen retrieval and incubated with antibodies against CD45, CD11b, CD3e, F4/80, ARG1, CD163, and HES1. Antibody-labeled sites were visualized using 3,3′-diaminobenzidine, lightly counterstained with hematoxylin, dehydrated in ethanol, and cleared in xylene. Negative controls (rabbit IgG and omission of primary antibody) were prepared concurrently. Slides were scanned using an Aperio AT2 digital scanner with a 40× objective (Leica Biosystems, Wetzlar, Germany). Twenty representative fields were captured from virtual microscopic images, and numbers of positive cells were counted using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Mean numbers of positive cells per unit area (0.71 mm2) were calculated from 20 areas for each case.
Statistical analysis
Data were analyzed using Prism 7.0 (GraphPad Software Inc., San Diego, CA, USA) and are presented as means ± SD or SEM as indicated. Statistical comparisons among multiple treatment groups were conducted using a one-way ANOVA for comparison of the means among groups. Two-tailed unpaired t-tests were used for comparisons between two groups. Statistical significance was set at p < 0.05.