Cell preparation and culture

Mouse Lewis lung carcinoma cell (LLC), melanoma cell (B16F10), fibroblast cell (3T3), human melanoma cell (SK-MEL-2), lung adenocarcinoma cell (A549), human normal epithelial cell (BEAS-2B) and umbilical vein endothelial cell (HUVEC) lines were purchased from the American Type Culture Collection (ATCC, USA). NCM460 and PC-9 cell line was obtained from Cell Resource Center of East China Normal University. LLC was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1 × penicillin–streptomycin. LLC stably expressing firefly luciferase (LLC-luci) was generated by our lab as described [17, 18] and cultured in complete DMEM medium with 200 ng/mL G418 (Gibco). B16F10, HUVEC, PC-9, A549 and 3T3 were cultured in RPMI 1640 Medium supplemented with 10% fetal bovine serum (FBS), 1 × penicillin–streptomycin. SK-MEL-2 was cultured in Modified Eagle’s Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1 × penicillin–streptomycin. All cell lines cultured maintained at 37 °C and 5% CO2, and were regularly tested for mycoplasma-free.

Chemicals, reagents, and antibodies

RPMI 1640, Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, and fetal bovine serum (FBS) was purchased from Gibco. TRIzol reagent and PrimeScript RT Master Mix were acquired from Takara. SYBR Green PCR Master Mix was purchased from Yeasen. Mouse TNF-α ELISA kit was purchased from Biolegend and Mouse IL-12 p70 ELISA kit was purchased from Invitrogen. GM-CSF and IL-6 were purchased from Proteintech. InVivoMab anti-mouse Ly6G/Ly6C (Gr-1; clone RB6-8C5), InVivoMab IgG2a isotype antibody (clone LTF-2) and InVivoMab anti-mouse PD-1 (clone RMP1-14) were purchased from BioXCell. Mouse Myeloid-Derived Suppressor Cell Isolation Kit, mouse CD4+ T Cell Isolation Kit, mouse CD8+ T Cell Isolation Kit, LS Separation columns, MS Separation columns, MACS BSA Stock Solution, autoMACS Rinsing Solution, autoMACS Running Buffer and mouse T Cell Activation/Expansion Kit were purchased from Miltenyi. FFAR2 Agonist (#371725), FFAR2 inhibitor (GLPG0974, #SML2443), Sodium acetate (#S2889) and Urethane (#U2500) were purchased from Sigma-Aldrich. Gαq inhibitor (YM-254890) and FFAR2 inhibitor (CATPB) were purchased from MCE. Ca2+ inhibitor (2-APB) was purchased from Tocris and PPAR-γ-inhibitor (GW9662) was purchased from Selleck. Live/dead dye and antibodies used for flow cytometry were purchased from Biolegend unless indicated otherwise: Fixable Viability Dye (BD, Horizon™ Fixable Viability Stain 780), FITC-conjugated anti-mouse CD45 (clone 30-F11), APC-conjugated anti-mouse CD3 (clone 17A2), Brilliant Violet 421™-conjugated anti-mouse CD4 (clone RM4-4), PE-conjugated anti-mouse CD8a (clone 53-6.7), PE/Cyanine7-conjugated anti-mouse F4/80 (clone BM8), PE-conjugated anti-mouse CD11c (clone N418), TruStain fcX™ anti-mouse CD16/32 (clone 93), Brilliant Violet 605™-conjugated anti-mouse I-A/I-E (clone M5/114.15.2), PE-conjugated anti-mouse CD8a (BD Pharmingen, clone 53-6.7), BV421-conjugated anti-mouse LY-6G (BD Pharmingen, clone 1A8), APC-conjugated anti-mouse LY-6C (BD Pharmingen, clone AL-21), FITC-conjugated anti-mouse CD11b (BD Pharmingen, clone M1/70), FITC-conjugated anti-mouse CD8a (clone 53-6.7), PE-conjugated anti-human/mouse Arginase 1 (eBioscience™, clone A1exF5), Brilliant Violet 421™-conjugated anti-mouse/human CD11b (clone M1/70), PE-conjugated anti-mouse IFNγ (clone XMG1.2), Brilliant Violet 421™-conjugated anti-mouse/human CD11b (clone M1/70), PE-conjugated anti-mouse IFNγ (clone XMG1.2), FITC-conjugated anti-mouse CD8a (clone 53-6.7), PE-conjugated anti-mouse iNOS (clone W16030C), PE/Cyanine7-conjugated anti-mouse IL-10 (clone JES5-16E3), Alexa Fluor® 700 -conjugated anti-mouse NK-1.1 (clone PK136) and Brilliant Violet 421™-conjugated anti-mouse CD25 (clone A18246A). Antibodies used for Western blotting are as follows: PPAR-γ (#2443), Arginase-1 (#93668), STAT1 (#9172), p-STAT1 (#9171), STAT3 (#4904), p-STAT3 (#9145) and AlexaFluor® 488/555 mouse, rabbit secondary antibodies were purchased from Cell Signaling Technology (CST). C/EBPβ (H7, sc-7962) and p-C/EBPβ (Thr217, sc-16993-R) were purchased from Santa Cruz Biotechnology. Antibodies used for immunofluorescence are as follows: Anti-human FFAR2 (ab124272) and anti-human CD15 (ab17080) were purchased from abcam. Anti-mouse Gr-1 (clone RB6-8C5) was purchased from Biolegend. Anti-CD8 (GB13429), anti-CD4 (GB13064-2), anti-human ARG1 (GB11285) and anti-PPAR-γ (GB112205) were purchased from Servicebio.

Mice

C57BL/6 mice (6–8 weeks old) were purchased from Gempharmatech Co., Ltd. Lyz2-cre mice were purchased from Jackson Laboratory. Global knockout mice Ffar2−/− and conditional knockout mice Ffar2fl/fl were constructed using the CRISPR–Cas9 genome-editing system. Ffar2fl/fl mice were crossed with Lyz2-cre mice to obtain mice with targeted Ffar2 deletion. All mouse breeding and mouse animal experiments are done at the specific-pathogen-free conditions Experimental Animal Center of East China Normal University. All animal experiments were approved by the Institutional Animal Ethics Committee of East China Normal University. The protocol was approved by the East China Normal University Center for Animal Research (m20230201).

Short chain fatty acids (SCFAs) measured by GC–MS

Sample preparation of cell supernatants: 3T3, LLC, B16F10, BEAS-2B, NCM460, HUVEC, PC-9, A549 and SK-MEL-2 cells were cultured in recommended complete medium for a few days, and seeded then in 6-well plates (1 × 106 cells per well), and cultured in RPMI 1640 supplemented with 10% FBS and 1 × penicillin–streptomycin (3 ml complete medium per well) for 16 h. Cell culture supernatants were then collected and kept in −80 °C. Preparation of urethane-induced lung cancer tissue: Urethane-induced mice lung cancer tissue and normal mice lung tissue was dissected, washed twice with precooled PBS, and kept then in −80 °C. All samples were sent to Suzhou Meixin Bioscience Co., Ltd and analyzed and quantified by GC–MS.

Development of mouse MDSCs from bone marrow (BM) precursors

Bone marrow cells were harvested from tibias and femurs of C57BL/6 mice (6–8 weeks old), after red cell lysis, cell suspension was cultured RPMI 1640 supplemented with 1 × Penicillin and Streptomycin, 10% FBS, GM-CSF (40 ng/ml) and IL6 (40 ng/ml) medium for 4 days.

Preparation of LLC tumor explant supernatants

Mice were subcutaneously injected with LLC (1 × 106 cells/mouse), and LLC tumors were excised at day 21. LLC tumors were minced into small pieces (less than 3 mm in diameter) and re-suspended into T75 culture flask with RPMI 1640 medium (without FBS) and incubated for 16–18 h. After incubation, supernatants were collected. After centrifugation and filtration (0.22 μm filters), supernatants were directly used or kept in −80 °C.

CD4+ T cells, CD8+ T cells and MDSCs isolation from mouse spleen

Disrupt spleen in recommended buffer and pass through 70 μm nylon cell strainer (Corning) and followed red blood cell lysis. Prepared single cell suspensions were determined cell number, and used for following cell isolation. CD4+ T cells and CD8+ T cells were isolated from naïve C57BL/6 mouse spleen via Mouse CD4+ T Cell Isolation Kit (Miltenyi) or Mouse CD8+ T Cell Isolation Kit (Miltenyi), following the manufacturer’s instructions. MDSCs were isolated from LLC tumor-bearing C57BL/6 mouse spleen via Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi), following the manufacturer’s instructions.

CD8+ T cells isolation from LLC tumors of Ffar2fl/fl and Ffar2fl/flLyz2-cre mouse

LLC cells were subcutaneously injected into Ffar2fl/fl and Ffar2fl/flLyz2-cre mice (1 × 106 cells/mouse), and LLC tumors were excised at day 21. LLC tumors were minced into small pieces (less than 3 mm in diameter) and resuspended with RPMI 1640, 400 U/mL Collagenase IV (Gibco) and 30 U/mL DNase I (Gibco). Tumor small pieces were incubated at 37 °C for half an hour. Stopping tumor samples digestion used complete medium (RPMI 1640 + 10% FBS), and tumor samples were filtered through 70 μm nylon cell strainer (Corning). After red cell lysis, and CD8+ T cells isolated from generated single-cell suspensions via Mouse CD8a Positive Selection Kit II (STEMCELL Technologies) following the manufacturer's instructions.

Flow cytometry analysis

For tumor infiltrating immune leukocytes, tumor single-cell suspensions created as described (CD8+ T cells isolation from LLC tumors). For spleen infiltrating immune leukocytes, disrupt spleen in PBS supplemented with 2% FBS and pass through a 70 μm nylon cell strainer (Corning) and followed red blood cell lysis. All samples were blocked FcγII/III with anti-CD16/32 (BD Pharmingen) at 4 °C for 30 min, and surface marker was stained at 4 °C for 30 min. Samples were then stained with indicated fluorescence-conjugated antibodies. Fixable Viability Stain 780 (BD Pharmingen) was used to gate out non-viable cells. For intracellular Arg1 and IFNγ staining, Cytofix/Cytoperm Soln Kit (BD Pharmingen) was used to fix and permeabilize cells, following the manufacturer’s instructions. All samples were run on LSRFortessa (BD Pharmingen) and analyzed by FlowJo software (Tree Star).

T cell suppression assays

CD4+ T cells, CD8+ T cells isolation from naïve mice spleen and MDSCs isolation from tumor-bearing mice spleen described as above. MDSCs were plated in the 48-well plates and cocultured with 1 μM carboxyfluorescein succinimidyl ester (CFSE) labeled CD4+ or CD8+ T cells at different ratios in the complete medium (RPMI 1640 supplemented with 10% FBS). For T cells activation, T Cell Activation/Expansion beads (Miltenyi) was added to coculture of T cells and MDSCs. After 72 h, T cells proliferation and IFNγ expression were measured by flow cytometry.

Mouse tumor models

For urethane-induced lung cancer, Ffar2+/+, Ffar2−/−, Ffar2fl/fl and Ffar2fl/flLyz2-cre mice were intraperitoneally injected with urethane (1 g/kg body weight in 200 μl PBS) once per week for 10 weeks, lung tissues were excised and collected at 28 weeks. Then, the lungs were soaked in 4% paraformaldehyde in a fixed shape for 2 weeks. After that, lung nodules were quantified and photographed. For mice subcutaneous tumor model, LLC, LLC-luci or B16F10 cells were injected subcutaneously into Ffar2+/+, Ffar2−/−, Ffar2fl/fl and Ffar2fl/flLyz2-cre mice (1 × 106 cells/mouse), and tumor volume assessed using calipers and calculated using the formula [(small diameter)2 × (large diameter) × 0.5]. For MDSCs deletion in vivo, Ffar2+/+ or Ffar2−/− mice were injected subcutaneously with LLC cells (1 × 106 cells/mouse) and injected intraperitoneally with isotype or anti-Gr-1 antibody (200 μg/mouse, every 4 days) from day 4 to day 23. For bone marrow chimeras, bone-marrow cells after red blood cell lysis were collected from Ffar2+/+ or Ffar2−/− mice. Prepared Ffar2+/+ or Ffar2−/− mice were lethally irradiated with 8.5 Gy, and lethally irradiated mice received bone marrow transplants from Ffar2+/+ or Ffar2−/− mice. Ten weeks after transplantation, chimeric mice were subcutaneously injected with LLC (1 × 106 cells/mouse), and tumor growth was recorded. For the combined treatment with FFAR2 inhibitor and anti-PD-1 antibody, WT mice were injected subcutaneously with LLC (1 × 106 cells/mouse). LLC-tumor bearing mice treated with FFAR2 inhibitor (5 mg/kg per day, single esophageal gavage), anti-PD1 antibody (200 μg/mouse every 4 days), FFAR2 inhibitor + anti-PD1 antibody or control (PBS containing 0.5% DMSO). Tumor-bearing mice were treated starting at day 4 post-tumor injection. Tumor growth and survival curve were recorded in two independent experiments. For survival analysis, mice were euthanized when total tumor burden approached IACUC guidelines with a tumor burden exceeding 1500 mm3 in volume.

Coinjection of MDSCs and mouse tumor cells

MDSCs were isolated from tumor-bearing mice spleen via Myeloid-Derived Suppressor Cell Isolation Kit (Miltenyi) as described. WT mice were then injected with tumor cells (LLC or B16F10; 5 × 105 cells/mouse) or co-injected with tumor cells and Ffar2+/+ MDSCs (5 × 105:5 × 105) or tumor cells and Ffar2−/− MDSCs (5 × 105:5 × 105). Tumor volumes were recorded.

Histology and immunofluorescence assay

Urethane-induced lung cancer tissues, LLC and B16F10 tumors were dissected, and washed twice by precooled PBS. Samples were fixed then in 4% paraformaldehyde for overnight, and embedded into paraffin. All paraffin embedded samples were sent to Servicebio. Hematoxylin and eosin (H&E) and immunofluorescence assay were stained and analyzed by Servicebio.

Western blotting analysis

Generated bone marrow-derived MDSCs were first resting overnight, and then stimulated by GM-CSF (40 ng/ml) + IL6 (40 ng/ml) for the indicated time. After stimulation, BM-MDSCs were harvested and lysed with radio immunoprecipitation assay (RIPA) buffer (CoWin Biosciences, China, catalog# CW2333) supplemented with complete Mini Protease and Phosphatase inhibitor Cocktail (Roche, catalog# 4693159001 and 4906837001). Cell lysates were separated by standard SDS-PAGE and analyzed by immunoblotting.

Enzyme-linked immunosorbent assay (ELISA)

Sample preparation of cell supernatants: generated bone marrow-derived MDSCs were first resting overnight, and then stimulated by GM-CSF (40 ng/ml) + IL6 (40 ng/ml) for 48 h. Remove particulates by centrifugation and assay immediately or store samples at -80℃. Preparation of LLC tumor tissue extracts: Mice were subcutaneously injected with LLC (1 × 106 cells/mouse), and LLC tumors were excised at day 21. Add appropriate amount of PBS to the tissue and mash it. Centrifuge at 3000 rpm for 10 min to take the supernatants and kept then in -80℃. The concentration of L-Arginine from cell supernatants and LLC tumor tissue extracts were quantified using mouse L-Arginine (L-Arg) ELISA Kit (Shanghai Coibo Bio Technology Co.Ltd). Consumption of L-Arginine by MDSCs was calculated according to the formula: (concentration of L-Arginine in medium-concentration of L-Arginine in culture supernatants) × volume. TNF-α and IL-12 p70 in the culture supernatants and lysates of MDSCs were determined by ELISA following the manufacturer's instructions.

Patients

Human lung adenocarcinoma tissues and adjacent normal tissues were obtained from Huzhou Central Hospital, Affiliated Hospital of Zhejiang University (Huzhou, 313000, Zhejiang, China) and statements that informed written consent of lung adenocarcinoma patients were obtained. All tissues were collected and handled according to the ethical and safety procedures approved by the Clinical Ethics Committee of the Huzhou Central Hospital, Affiliated Hospital of Zhejiang University (reference ethics number 20180701-02). The lung cancer tissue array (HLugA180Su08) was purchased from Shanghai Outdo Biotech.

RNA sequencing and analysis

Ffar2+/+ and Ffar2−/− bone marrow-derived MDSCs (1 × 106 cells/well) were seeded in a 6-well plate and cultured in complete RPMI 1640 medium overnight. After overnight resting, BM-MDSCs were re-stimulated by combination GM-CSF (40 ng/ml) with IL-6 (40 ng/ml) for 24 h. After that, RNA was extracted using the RNA extraction kit (Magen, R4801-02) and sequenced by BGI (Beijing Genomic Institute in ShenZhen). The sequencing data were filtered with SOAPnuke (v1.5.2) by removing reads containing sequencing adapters, removing reads whose low-quality base ratio (base quality less than or equal to 5) was more than 20%, and removing reads whose unknown base (“N” base) ratio was more than 5%. After this, the clean reads were obtained and stored in a FASTQ format. The clean reads were aligned to the reference genome using HISAT2 (v2.0.4). Fusion genes and differential splicing genes (DSGs) were detected through Ericscript (v0.5.5), and rMATS (v3.2.5). Bowtie2 (v2.2.5) was used to align the clean reads to the gene set, a database for this organism was built by BGI (Beijing Genomic Institute in ShenZhen), coding transcripts were included, and the expression levels of genes were calculated using RSEM (v1.2.12). Differential expression analysis was performed using DESeq2 (v1.4.5) with a P value ≤ 0.05 and |log2 (fold change)|≥ 0.5. Volcano map was plotted by using the EnhancedVolcano package in R (v4.5.0). GSEA of KEGG pathway was performed by clusterProfiler package and visualized by ggplot2 package and gseaplot2 package. P value ≤ 0.05 was set as the cut-off criteria.

RNA extraction and quantitative real-time RT-PCR

Total RNA of cells or tissues was isolated with TRIzol (TaKaRa), and RNA concentration was measured by NanoDrop 2000 (Thermo Fisher Scientific). RNA was reverse transcribed into cDNA by PrimeScript RT Master Mix (TaKaRa). Quantitative real-time RT-PCR (RT-qPCR) was performed using the QuantStudio 3 Real Time PCR System (Applied Biosystems). The expression of each gene was normalized to the expression level of GAPDH and reported as relative mRNA expression (2−ΔΔCt) or fold change. The sequence-specific primers are shown in Additional file 2: Table S1.

Statistical analysis

Statistical analyses were analyzed by Prism 6.0 (GraphPad Software). Statistical differences between two groups were analyzed using unpaired two-tailed Student’s t-test. The statistical differences for more than two groups were analyzed using ANOVA. Survival analysis was performed using the Log-rank (Mantel-Cox) test. All data were shown as mean ± SEM. P value ≤ 0.05 was considered to be statistically significant. (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, NS, not significant).