Breast-tissue specimens and clinical assessments

Patients at the Harbin Medical University Cancer Center (HMUCC) with a histological diagnosis of breast cancer who had not received chemotherapy or radiotherapy before surgical resection were considered eligible for recruitment to this study. RNA was extracted from breast cancer and normal control tissues, which was stored at − 80 °C immediately after resection. Paraffin-embedded tissue sections were produced from tissue samples stored in 4% formaldehyde at 4 °C immediately after resection. This study was approved by and conformed to the clinical research guidelines of the Research Ethics Committee of Harbin Medical University Cancer Hospital (KY2022-43). Written informed consent was obtained from all patients.

Analysis of NAT10 expression in TCGA and GEO database

Breast cancer mRNA NAT10 expression levels were retrieved from the TCGA database (https://portal.gdc.cancer.gov/), and TNBC patients were screened according to Fudan typing (FUTURE). Scatter plots of expression levels were produced for each group. TNBC immunotherapy-related gene expression data were extracted from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds/). A dataset (GSE124821) was used for our study, and the experiment type was microarray expression profiling. The expression of the NAT10 gene was extracted, and grouped violin plots were produced. All gene expression data were log2-transformed. Results were visualized using the R software package ggplot.

Cell culturing and transfection

Breast cancer cell lines (4T1, BT549, MDA-MB-231, and MDA-MB-468), Jurkat, and THP-1 cells were obtained from the Chinese Academy of Sciences Cell Bank and Cellbio. All cell lines were cultured under standard conditions, as specified by the suppliers, using a culture medium supplemented with 100 U/mL penicillin, 100 mg/mL streptomycin, and 10% fetal bovine serum (FBS) at 37 °C with 5% CO2. Cells were seeded in six-well culture plates, at a density of approximately 70%, and were transfected using JetPrime for 48 h (#114–15, Polyplus, Germany) (2 ml culture medium + 4 μl jet + 0.2 nM siRNA). The concentration of remodelin was 50 μM.

Wound-healing assays

4T1 cells, BT549, and MDA-MB-468 cells (transfected for 36 h or 70 µM remodelin treated for 24h) were seeded in six-well culture plates on RPMI-1640 or DMEM medium containing 5% FBS and were cultivated to a sub-confluent state. A wound was scratched at the bottom of the plate using a 10 µL pipette tip. Cell migration was observed and calculated at the indicated times (0h, 12h, 24h, 36h, 48h), and the size of the remaining wound was measured using an inverted light microscope.

Invasion assays

0.8–1 × 105 4T1 cells, BT 549, and MDA-MB-468 cells (transfected for 36 h or 70 µM remodelin treated for 24h) in serum-free conditioned medium were seeded into a BRAND Insert with Matrigel (#BR782806, Sigma-Aldrich, USA). A complete medium containing 20% FBS was added to the lower chamber. After incubation at 37 °C 48 h, cells that did not migrate to the lower chamber were removed, and the invaded cells were fixed with 4% methanol for 30 min and were stained using crystal violet for 30 min. Cells were imaged and counted under a light microscope.

Cell-viability assays

3–5 × 103 4T1 cells, BT 549, and MDA-MB-468 cells (transfected for 36 h or 70 µM remodelin treated for 24h) were seeded in 96-well culture plates with 200 μl of the medium. Six wells were plated on the same cells as the replicates. Cell proliferation was observed and calculated at the indicated times (0h, 24h, 48h, 72h). Cell Counting Kit-8 (CCK-8) (#MA0218, Kumamoto, Japan) was added to 10% at 37 °C for 1 h before any measurements. The absorbance of each cell suspension was measured at 450 nm wavelength using a microplate reader.

THP-1 monocytic cell cultures

We selected the human monocytic leukemia cell line THP-1 (Chinese Academy of Sciences, China), which showed similar characteristics to human macrophage after differentiation. Then, we employed a differentiation and functional polarization protocol that was shown to be effective for THP-1 cells [33, 34]. THP-1 cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) at 37°C for 48 h. Then, differentiated THP-1 cells were treated with either lipopolysaccharide (LPS, 100 ng/ml) and IFN-γ (20 ng/ml) for 48 h or IL-4 (20 ng/ml) and IL-13 (20 ng/ml) for 48 h to obtain M1-like and M2-like phenotypes, respectively. Cells were treated with remodelin for 24–48 h after which culture supernatants were analyzed for cytokines. Thereafter, cells were collected and analyzed by flow cytometry.

Human primary T cell cultures

The peripheral venous blood was collected from healthy volunteers in a heparin anticoagulant tube. Ficoll lymphocyte separation solution (#LTS1077, tbdscience, China) was added to dilute the blood. The PBMC isolation from blood was done using gradient density centrifugation and then counted. The 10 mm dishes were pre-coated with 1 μg/mL anti-CD3 (#16–0037-81, clone OKT3, eBioscience, USA) in PBS at 37°C for 2 h. Next, primary T cells were added in 106 cells/mL suspended in RPMI-1640 which contained 10% FBS, 2 μg/mL anti-CD28(#16–0289-81, clone CD28.2, eBioscience, USA), 200U/mL IL-2, 1% L-glutamine,0.1% β-mercaptoethanol. After three or four days, cells were harvested for experiments.

In vitro CTLA-4 cycling

Human primary T cell was cultured in the presence of anti-CD3 anti-CD28 activation. Then T cell was treated with remodelin for 24–36 h. Staining was carried out at 4 °C to label surface CTLA-4; at 37 °C for 2 h to identify cycling CTLA-4; or after cell fixation and permeabilization to stain the total CTLA-4 pool.

Qrt-PCR

Total RNA was extracted from cells and tissue samples using TRIzol reagent (#269,201, Invitrogen, USA) according to the manufacturer’s instructions, and RNA was reverse-transcribed into cDNA using a High-Capacity cDNA Reverse Transcription Kit (#4,368,814, Applied Biosystems, USA). mRNA expression was quantified by real-time PCR using an SYBR Green PCR Master Mix Kit (#4,913,914,001, Applied Biosystems) with gene-specific primers. β-actin was used as internal control, and qRT-PCR was performed on a 7500 FAST Real-time PCR System (Applied Biosystems). The results were normalized to β-actin expression levels using the 2–ΔΔCt method. The primer sequences of interest are shown in Supplementary Data S6.

RNA immunoprecipitation (RIP)

The RNA immunoprecipitation (RIP) assay was performed according to the protocol of the Magna RIP Kit (Millipore, USA). Briefly, 5 μg NAT10 or FLAG antibodies and 50μl magnetic beads were well mixed and incubated with cell lysates. Then, RNAs were extracted after the removal of proteins. Followed by qPCR, the expression of genes was normalized to input.

RNA pulldown

The cells were transfected with JunB overexpression plasmid (36 h, 500ng/ml). Using biotin labeling to design and synthesize probes based on JunB mRNA. The cell lysate was incubated with beads at 4 °C overnight, which were coated with biotin-tagged JunB. After washing with wash buffer, add elution buffer to separate the RNA–protein complex from the beads. Loading buffer was used to separate the protein and subjected to western blot.

Seahorse xf96 respirometry

3 × 104 per well BT549 (NAT10 knockdown or JunB overexpression) cells were seeded in the XF96 plate and stabilized overnight. The extracellular acidification rate (ECAR) was measured by the XF96 extracellular flux analyzer with glucose stress fuel flex test kits (Agilent). Measurements of ECAR were performed according to the manufacturer’s instructions. The results were analyzed using Wave software (Seahorse/Agilent).

Acrip-qpcr assay

The ac4C immunoprecipitation (acRIP) procedure was performed according to instructions issued by the manufacturer using a GenSeq ac4C RIP Kit (#GS-ET-005, CloudSeq Biotech, China). In brief, 100 μg total RNA was isolated from pretreated cells and was randomly fragmented to a size of 200 nucleotides. RNA samples were then immunoprecipitated using magnetic beads pre-coated with anti-ac4C antibody. The ac4C-modified RNA fragments were eluted with nuclease-free water. Enriched ac4C-modified mRNA was then detected through qRT-PCR.

Chip-qpcr assay

Chromatin-immunoprecipitation (ChIP) assays were performed using a ChIP Assay Kit (#P2078, Beyotime, Shanghai, China), following the manufacturer’s instructions. cells were cross-linked with formaldehyde and sonicated to an average length of 200–1000 bp. The sheared chromatin was immunoprecipitated at 4 ℃ overnight using an anti-JunB antibody (#GTX116011, GeneTex); IgG (BD Biosciences, San Diego, CA) served as the negative control. The precipitated DNA was amplified by RT-PCR.

RNA decay assay

TNBC cells were treated with vehicle (DMSO) or remodelin and were transfected with siNC or siNAT10. Then TNBC cells were seeded in 12-well plates. Actinomycin D was added into each well at a final concentration of 2 µg/mL. The cells were collected after 0, 30, 60, and 90 min, respectively. Total RNA was isolated and subjected to qRT-PCR to quantify the relative expression of JunB. β-actin was used as an internal control.

Ac4c dot blot

Total RNA was heated to 95 ℃ for 3 min and was placed on ice, and loaded onto Hybond-N + membranes. Membranes were crosslinked at 150 mJ/cm2 in a UV254 nm Stratalinker 2400 (Stratagene, USA), and were blocked with 5% nonfat milk in 0.1% Tween 20 PBS (PBST) for 1 h at room temperature (RT) followed by incubation with an anti-ac4C antibody (#ab252215, Abcam, USA) in PBST (1:250) at 4 ℃ overnight. The membranes were then washed three times using PBST, incubated with an HRP-conjugated secondary anti-rabbit IgG in PBST (1:1000) at RT for 1 h, and washed three times with PBST. The ac4C modification was visualized using an enhanced chemiluminescence detection system (Western Lightning, Perkin Elmer, Norwalk, CT, USA) and the total RNA was visualized by methylene blue.

Western blot

Cells were lysed with 10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM NaF, 10 mM DTT, 5% glycerol, 5000 U/ml proteinase inhibitors, and 2% SDS. Protein concentrations were measured using a protein assay kit (#5,000,001, Bio-Rad, Richmond, USA); equal amounts of protein were separated using SDS-PAGE and were then transferred onto polyvinylidene fluoride membranes blocked with 5% skim milk in TBST for 1 h at RT. Then, the membranes were incubated overnight at 4 ℃ with the primary antibody diluted in TBST. After washing three times using TBST, the membranes were incubated with HRP-labeled secondary antibodies for 1 h. After washing with TBST, protein bands were visualized using an enhanced chemiluminescence detection system (Western Lightning, Perkin Elmer).

Enzyme-linked immunosorbent assay (ELISA)

Cells were seeded in a six-well plate. Cells were treated with vehicle (DMSO) or remodelin and were transfected with siNC or siNAT10. Then supernatants were collected to measure the total levels of several cytokines using respective human ELISA kits according to the manufacturer’s instructions. The plates were read using a microplate reader at 450 nm wavelength.

Immunohistochemistry and multiplex immunofluorescence staining

Tumor tissues were fixed, embedded, and sectioned (4 μm thickness), followed by backing for 2 h at 60 °C. The slides were then deparaffinized in xylene and rehydrated in gradient ethanol. First, antigen retrieval of slides was performed with citrate buffer (pH 6.0) or EDTA (pH 8.0), using a steamer (95–100 ℃) for 10 min. The slides were incubated in 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity followed by 10% BSA incubation for 1 h at RT. The first primary antibody was applied for 4 ℃ overnight followed by washing three times using PBS. The slides were incubated with appropriate secondary universal immuno-peroxidase polymer, anti-rabbit, or anti-mouse antibody for 30 min, followed by washing thrice. Detection was performed using liquid DAB + (#9018, Golden Bridge, China) and counterstaining with Carazzi’s hematoxylin (#BL702A, Biosharp, China). The stained sections were independently analyzed by two pathologists.

Multiplex immunofluorescence staining was performed by using the four-color multiple fluorescent immunohistochemical staining kit (#abs50012, Absin, China) based on the tyramide signal amplification (TSA) technique, according to the manufacturer’s manual. As described above, after performing antigen retrieval and blocking, we sequentially incubated primary antibodies for 30 to 60 min at 37 ℃, followed by incubation of HRP-conjugated secondary antibody and TSA with Opal. Lastly, sections were counterstained using DAPI and were mounted in glycerol and gelatin mounting medium. Antibody information is listed in Supplementary Data S1. Tissue sections were imaged using a Nikon A1 scanning confocal microscope. Confocal images were captured and the image data were collected using NIS Elements (Nikon, V4.50.00).

Animal experiment

The animal experiment was approved by the Ethics Committee of the National Center for Nanoscience and Technology (NCNST21-2203–0610). Six-to-eight-week-old female Balb/c mice were obtained from the Beijing Vital River Laboratory Animal Technology Company. Approximately 5 × 104 4T1 cells in 50 μl of serum-free RPMI1640 medium and 50 μl Matrigel matrix were injected directly into the right mammary fat pad. For in vivo 4T1 tumor-growth experiments, remodelin was administered at a dosage of 3 mg/kg by intraperitoneal injection every four days. Anti-CTLA-4 antibody (#BP0164, BioXCell, USA) was injected intraperitoand and nearly administered at a dosage of 250 µg. Tumor growth was measured once every 2 days using calipers, and tumor volume was calculated as 1/2 (length × width2).

Cytokine panel

Blood was collected after the animal experiments. The samples were centrifuged at 3,000 × g at 4 ℃ for 20 min; serum was collected and stored at − 80℃ until use. Serum samples were processed by Univ Bioscience using the Millipore MILLIPLEX MAP Mouse Cytokine/Chemokine 32-Plex panel (#MCYTMAG 70K PX32). Samples were examined according to the manufacturer’s protocol, as follows: serum was thawed and diluted to one-part serum to one-part assay butter on the day of the assay. Fifty microliters sample or standards was plated with 50 µl assay buffer, and 50 µl beads in each well, which were then sealed and agitated for 30 min at RT. The plate was washed three times, then 25 µl of detection antibodies were added to each well and the plate was sealed, covered with foil, and agitated for 30 min at RT. Fifty microliters of streptavidin–phycoerythrin were added to each well. The plate was sealed, covered with foil, and agitated for 10 min at RT. The plate was washed three times, then 125 µl of assay buffer was added to each well, and beads were resuspended on a plate shaker for 30 s. Plates were read on a Luminex 200.

RNA-sequencing (RNA-seq)

BT549 was transfected with siNC or siNAT10 for 48 h, and total RNA was sent to Majorbio Bio-pharm Biotechnology Co. Ltd. (Shanghai, China) for the construction of libraries. Small RNA libraries were constructed using the Illumina TruSeq Small RNA Kit (Illumina, USA), and strand-specific libraries were constructed using the Illumina TruseqTM RNA sample prep Kit method. RNA-sequencing was performed on an Illumina Novaseq 6000/HiSeq X ten platform (Illumina), according to the manufacturer’s instructions.

Flow cytometry analysis

Cell surface markers of CD8+T, CD4+T, macrophage, and cytokines (IL-2, TNF-α, and IFN-γ) assessment was performed by immunofluorescence staining followed by flow cytometry analysis. In brief, cells were collected at 800 × g for 5 min and were washed with, and suspended in FACS buffer (#00–4222-26, Thermo Fisher Scientific, USA) (1.0 × 106 cells/100 μl), and all steps were performed in the dark unless stated otherwise. Then cells were incubated with fluorochrome-conjugated anti-CD3, anti-CD4, anti-CD8, anti-CD86, anti- CX3CR1, and anti- MHCII antibodies at 4 °C for 30 min.

For measurement of intracellular CD206, IL-2, TNF-α, and IFN-γ, cells were stimulated with PMA (20 ng/mL), ionomycin (1 ug/mL), and brefeldin A (10 ug/mL) for 4–6 h. And then the cells were washed using FACS buffer, fixed, and permeabilized with intracellular staining fixation buffer (#420,801, BioLegend, USA) at RT for 20 min and intracellular staining permeabilization wash buffer (10X) (#421,002, BioLegend) at 4 ℃ for 40 min followed by staining with fluorochrome-conjugated anti-IL-2, anti-TNF-α, anti-IFN-γ antibodies at RT for 30 min. After incubation, cells were washed with intracellular staining permeabilization wash buffer and FACS buffer and were then suspended in FACS buffer. The specificity of the antibodies was verified by staining with respective isotype control antibodies and FMOs. The samples were analyzed immediately or within 24 h using BD canton II (Thermo Fisher Scientific). Data evaluation was performed using the FlowJo software.

Time-of-flight mass cytometry (cytof)

Mass cytometry was performed by PLTTech Inc. (Hangzhou, China). Cells isolated from mouse breast cancer tissues (control group and remodelin treated group) were incubated with a surface antibody mix panel and were stained overnight with the DNA Intercalator-Ir (FLUIDIGM, South San Francisco, CA) to differentiate live cells from debris. After fixation and permeabilization, cells were stained using intracellular/nuclear antibodies. The immuno-labeled samples were then barcoded using a unique barcode isotope combination for 30 min, re-suspended in deionized water, and subjected to a CyTOF instrument (Helios, FLUIDIGM). The antibodies used in the assay are listed in the Supplementary Data S1. Following density clustering, the immune cells were identified via t-distributed stochastic neighbor embedding (t-SNE).

NAD + /NADH assay and ATP assay

TNBC cells were treated with vehicle (DMSO) or remodelin, and transfected with siNC or siNAT10 for 48 h. Then the lysate provided with the kits was used to collect the intracellular fluid for subsequent assays. NAD + , NADH, and total NAD (NADt) (NAD +  + NADH = NADt) were measured using a NAD + /NADH assay kit (#S0179, Beyotime, China) according to the manufacturer’s instructions. ATP was detected using a kit from the Nanjing Jiancheng Bioengineering Institute (#A095-1–1, Nanjing Jiancheng, China).

Statistical analyses

Statistical analyses were conducted using GraphPad Prism (GraphPad Software, USA) using the Student’s t-test and ANOVA test. Spearman’s correlation coefficients were calculated for correlation analyses. All experiments were conducted using three replicates. Statistical significance was defined as P < 0.05.