Reagent

HN-1, a 21 mer peptide (FALGAVTKLLPSLLCMITRKC), was synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China) using reverse-phase high-performance liquid chromatography and electronic spray ionization mass spectrometry to attain ~ 95% purity (Supplementary Fig. 1). BI-6C9 was obtained from MedChemexpress (MCE, New Jersey, USA). Both the anti-CD8α antibody and the isotype IgG1 antibody were purchased from BioXcell (New Hampshire, USA).

Cell culture

The human breast cancer cell lines MDA-MB-453 and MDA-MB-231 and the mouse breast cancer cell lines 4T1 and EMT6 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). MDA-MB-453 and MDA-MB-231 were maintained in L-15 medium (HyClone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The 4T1 and EMT6 cells were cultured in RPMI-1640 supplemented with 10% fetal bovine serum.

Immunofluorescence staining

The MDA-MB-231 and MDA-MB-453 cells were treated with 20 µM HN-1, then fixed with 95% ethanol, and permeated with PBS containing 1% Triton X-100. Next, the cells were incubated with an anti-calreticulin monoclonal antibody (1:75; ab2907; Abcam), a dsDNA monoclonal antibody (1:50; 15,635; Cayman Chemical), or Photoshop Histone H2A.X (1:200; 9718; Cell Signaling Technology) overnight at 4 °C. The cells were washed three times with PBS and then incubated with the secondary antibody goat anti-rabbit Alexa Fluor 488 (1:200; ab150077; Abcam) or goat anti-mouse Alexa Fluor 488 (1:200; ab150113; Abcam) for an additional 30 min, after which Hoechst 33,342 was used to stain the cell nucleus. Finally, the cells were observed by confocal fluorescence microscopy (Olympus, FV1000, Tokyo, Japan), and the ImageJ software (version 1.51j8; National Institutes of Health and University of Wisconsin, Bethesda, MD, USA) was used for quantification.

DC culture

We recruited two female volunteers, aged 30 and 38 years, who provided informed consent. Isolated peripheral blood mononuclear cells (PBMCs) were stained with FITC-conjugated HLA-A2 (343,304; Biolegend, San Diego, CA, USA), and human leukocyte antigen (HLA) subtypes were detected via flow cytometry. The PBMCs were cultured in 75 cm2 cell culture flasks for 1 h. After removing the suspended cells, the adherent cells were cultured in fresh X-VIVO 15 medium (Lonza, Alpharetta, GA, USA) supplemented with 5% plasma, 500 U/ml IL-4, and 1000 U/ml GM-CSF (Promega, Madison, WI, USA). After 5 days of stimulation, the DCs were collected for further investigation.

Enzyme-linked immunosorbent assays (ELISAs)

The MDA-MB-231 and MDA-MB-453 cells were treated with 20 µM HN-1, and the culture supernatant was collected to detect the secretion of ATP (Promega, Madison, WI, USA) and HMGB1 (Signalway Antibody, MD, USA). The MDA-MB-231 cells were pretreated with HN-1 and co-cultured with DCs for 48 h, after which the IL-2, IL-10, IL-12, and IFN-γ levels were detected. The quantification of cytokines in the supernatant was performed using a human ELISA detection kit (IL-2, D2050; IL-10, D1000B, IFN-γ, DIF50C; R&D Systems, MN, USA) in accordance with the instructions provided by the manufacturer.

Mouse tumor model

All the procedures were performed according to protocols approved by China Medical University’s Institutional Committee for the Use and Care of Laboratory Animals. Five-week-old female BALB/c mice or BALB/c nude mice were obtained from Vital River Laboratories (Beijing, China). For the immunocompetent mice study, 2 × 105 4T1 or EMT6 mouse breast cancer cells were inoculated into mammary fat pads. For the nude mice study, 3 × 106 MDA-MB-231 or MDA-MB-453 cells were implanted into mammary fat pads. After the long and short diameters of the tumors on both sides reached 5 mm, the mice were randomly divided into a control group and an experimental group. The control group of mice was intraperitoneally injected with PBS, while the HN-1 group was administered 4 mg/kg HN-1 every other day for 14 days. The tumor volume was measured daily with a quantitative value of [length × width2]/2. Finally, we euthanized the mice, weighed the tumors, and collected primary tumor cells for a flow cytometry analysis.

CD8+ T cell depletion

When the long and short diameters of the tumors on both sides reached 5 mm, 200 µg anti-CD8α antibody started to be intraperitoneally administered every 4 days to deplete CD8+ T cells, as previously reported [22].

Flow cytometry analysis

Briefly, 1 × 106 cells were suspended in 100 µL of PBS and incubated at room temperature for 30 min with CRT-AF488 (92,516, Abcam), HLA-A2-FITC (343,304, Biolegend), CD11c-APC (301,614, Biolegend), HLA-DR-PerCP-Cy5.5 (307,630, Biolegend), CD80-FITC (305,206, Biolegend), CD86-PE (305,406, Biolegend), and CD83-APC (305,312, Biolegend) antibodies.

Mouse primary tumor cells were chopped and digested with the EZ enzyme (Nitta Gelatin Company, Osaka, Japan) to obtain single-cell suspensions. Then, the single-cell suspensions were stained with TruStain FcX (anti-mouse CD16/32 101,320; Biolegend) to block nonspecific staining. Next, the cells were stained on ice for 30 min using the following combinations: CD45-PerCP (103,130, Biolegend)/CD11b-FITC (101,205, Biolegend)/CD11c-APC (117,309, Biolegend); CD3-FITC (100,203, Biolegend)/CD4-PE (100,407, Biolegend)/CD8a-APC (100,711, Biolegend)/CD45-PerCP; CD3-FITC/CD4-BV785 (100,551, Biolegend)/CD25-PE (102,007, Biolegend)/Foxp3-AF647 (126,407, Biolegend); CD45-perCP/CD11b-FITC/Ly-6G-APC (127,613, Biolegend)/Ly-6 C-BV421 (128,031, Biolegend); and CD45-perCP/CD11b-FITC/F4/80-APC (123,115, Biolegend)/Gr-1-PE (108,407, Biolegend). Treg cells were stained with surface markers and, intracellularly, with Foxp3-AF647 after fixation and permeabilization with the True-Nuclear Transcription Factor Buffer Set (Biolegend). The specific gating strategy is shown in Supplementary Fig. 2. Finally, the samples were detected with BD FACSCelesta (BD Biosciences, San Jose, CA, USA).

Western blotting

The cells were lysed using the RIPA buffer, and the protein lysates were separated via 10% polyacrylamide gel electrophoresis (SDS–PAGE) and subsequently transferred to a PVDF membrane. Next, the membranes were blocked with 10% skim milk and incubated with primary antibodies overnight at 4 °C. Afterwards, the membrane was washed carefully and incubated with a secondary antibody (goat anti-rabbit IgG-HRP (1:10000, ab6721, Abcam) or goat anti-mouse IgG-HRP (1:15000, ab205719, Abcam)) for one hour at room temperature. A Supersignal West Pico Plus (Thermo Fisher Scientific, Inc.) was used for chemiluminescence, and a Bio-Rad GelDoc XR + system was used for detection (Bio-Rad, Berkeley, CA, USA). The ImageJ (version 5.2.1) software was used to analyze the data.

The primary antibodies used were anti-GAPDH (1:1000, 2118, Cell Signaling Technology), anti-STING (1:1000, 13,647, Cell Signaling Technology), anti-phospho-STING (1:1000, 50,907, Cell Signaling Technology), anti-TBK1 (1:1000, 38,066, Cell Signaling Technology), anti-phospho-TBK1 (1:1000, 5483, Cell Signaling Technology), anti-IRF-3 (1:1000, 4302, Cell Signaling Technology), anti-phospho-IRF-3 (1:1000, 79,945, Cell Signaling Technology) and anti-mouse Phospho-STING (Ser365) (1:1000, 72,971, Cell Signaling Technology).

Transfection

MDA-MB-231 and MDA-MB-453 cells were transfected with 100 pmol of the following STING-specific siRNAs: si-STING-1, 5’-GCCCUUCACUUGGAUGCUUTT-3’; si-STING-2, 5’-GCAUUACAACAACCUGCUATT-3’; and si-STING-3, 5’-GCAUCAAGGAUCGGGUUUATT-3’. The scrambled RNA 5’-ACGUAACACGCCCGGAGUAGT-3’ was transfected as a control. The STING mRNA and protein levels were detected via quantitative real-time PCR and Western blotting, respectively.

RNA sequencing

A total of 2 × 106 MDA-MB-231 and MDA-MB-453 cells were treated with 20 µM HN-1 for 24 h, after which the total RNA was extracted with TRIzol. These samples were sequenced at the Novogene Technology Laboratory (Novogene Technology, Inc., Beijing, China). We analyzed the differential gene expression between the two groups based on the DESeq2 R package (1.16.1). The data were subsequently transformed into Venn diagrams and heatmaps. Cluster Profiler R was used to conduct Gene Ontology (GO) and KEGG enrichment analyses of the DEGs.

Primary breast cancer cell sensitivity test

Drug sensitivity was assessed via a collagen gel droplet-embedded culture drug sensitivity assay (CD-DST, Kurabo, Osaka, Japan), according to the manufacturer’s instructions [23]. Briefly, fresh breast cancer samples were obtained after surgery, minced, and digested. The disassociated cells were cultured in a collagen droplet, which provided a three-dimensional environment for breast cancer cells. Every droplet contained 1-1.5 × 103 cells, with 3 droplets per well, and was treated with 20 µM HN-1 for 24 h in 6-well plate. The culture medium was replaced with prepared culture media-2 (PCM-2, Kurabo) without FBS for an additional 7 days. Viable cells were stained with neutral red and fixed with neutral formalin. After air-drying, cell viability was quantitatively evaluated utilizing ImageJ.

Statistical analysis

The SPSS software version 21 (IBM Corp., Armonk, NY, USA) was used to conduct the data analysis. Unless otherwise specified, three biological replicates were routinely used in the experiment. The standard deviation of the average values of three independent experiments was represented by the mean ± standard deviation. The two groups were compared with t tests or one-way ANOVA. A P value < 0.05 indicated a statistically significant difference (*P < 0.05, **P < 0.01).