Transgenic mouse strains and tumor monitoring
All animal experiments were performed in strict accordance with the protocols approved by the Animal Welfare and Ethics Committee of China Pharmaceutical University (Approval No. 2021-10-017). The construction of transgenic mice with targeted expression of CYP4Z1 transgene in the breast tissue and the MMTV-PyMT (stock no. 022974) transgenic mice were obtained from Shanghai Nanfang Model Biotechnology Co., Ltd. All mice were on a mixed C57BL/6 background with littermate controls (females) used in all experiments. For spontaneous tumorigenesis studies, weaned females were examined by palpation twice weekly for breast tumor development. After 3 weeks of initial detection of palpable tumors, lungs were collected to determine metastatic nodules. Investigators were not blinded to assignments during the experiment and outcome assessment. No randomization method was used, in which mice were segregated into groups based on genotype only. No statistical methods were used to predetermine sample size. All animals were used following the procedures approved by the Shanghai Nanfang Model Biotechnology Co., Ltd and Use Committee (Licence No. 2019-0026). The mice were kept as follows: n = 3 per cage for antitumor study in tumor xenograft mice. They were housed under conventional laboratory conditions at a room temperature maintained at 25 ± 1 °C with a relative humidity range of 40–75% and a regular 12 h light/12 h dark cycle. The mice were fed a standard animal pellet diet and allowed free access to water. At the end of the experiment, the mice were killed by an overdose injection of pentobarbitone (200 mg/kg).
Isolation of Lin-NECs (neoplastic MECs (mammary epithelial cells)) and pNECs (pre-neoplastic MECs)
The pre-neoplastic glands or primary tumors were dissected into pieces, in which fat was removed. The organoids were enzymatically dissociated at 37 °C for 1 h in lysis solution (DMEM/F12 (Keygen BioTECH, Nanjing, China)) containing 5% fetal bovine serum (OmnimAbs, Shanghai, China), 10 ng/ml epidermal growth factor (Sigma-Aldrich, St Louis, MO, USA), 500 ng/ml hydrocortisone (Sigma-Aldrich), 5 μg/ml insulin (Sigma-Aldrich), and 1% penicillin/streptomycin (Sangon Biotech, Shanghai, China)) supplemented with collagenase/hyaluronidase (Sigma-Aldrich). In addition, the sediment was harvested and incubated with trypsin-EDTA (0.25%) for 2 min, 5 mg/ml Dispase (Thermo Fisher Scientific, Waltham, MA, USA) plus 0.1 mg/ml DNase (Sigma-Aldrich) for 5 min, and 0.64% NH4Cl for 5 min at 37 °C. Cells were filtered with a 40-µm cell strainer and re-suspended in Hank’s balanced salt solution buffer containing 0.5% bovine serum albumin. Finally, NEC and pNECs were enriched using the EasySep™ Mouse Epithelial Cell Enrichment Kit II (Catalog no. 19868, StemCell Technologies, Vancouver, British Columbia, Canada).
Cell culture, transfection, and lentiviral-based gene transduction
Human breast cancer cell lines, including luminal A-type (MCF-7, T47D), basal-like type (MDA-MB-231), human embryonic kidney cells HEK-293T, and mice breast cancer cells 4T1 were purchased from ATCC (Manassas, VA, USA) and archived in our laboratory repository. These cell lines were maintained in DMEM (Keygen BioTECH) supplemented with 10% fetal bovine serum at 37 °C in 5% CO2 (v/v). Jet-PRIME (Polyplus Transfection, Illkirch, France) was used for transfection. The lentivirus of CYP4Z1-wt (wild-type) and CYP4Z1K259R/K279R/K502R overexpression was purchased from Corues Biotechnology (Nanjing, China). The final viral titer was 1 × 109 TU/ml. Cells were infected with the lentivirus and selected by puromycin (Sigma-Aldrich, 2 μg/ml).
Data retrieving
Single-cell dataset GSE225600 was downloaded from the Gene Expression Omnibus database21. The dataset encompassed gene expression profiles from a total of 81,683 cells across 4 primary breast tumor samples and 4 paired lymph metastasis samples. Bulk-seq data of patients with breast cancer were acquired from The Cancer Genome Atlas Genomic Data Commons (TCGA GDC) via UCSC Xena (https://xenabrowser.net/, GDC TCGA Breast Cancer)22. This gene expression dataset included count data for 60,488 genes across 1,217 samples. Additionally, survival data for 1,260 breast cancer samples and clinical data for 1,248 breast cancer samples were obtained from the same source. The spatial transcriptomic dataset GSE213688, which contains three putative Claudin-low tumors, four genomically unstable triple-negative breast cancer samples, and four metaplastic breast cancer samples, was adopted for spatial transcriptomic analysis23.
Cell clustering and annotation
Single-cell RNA (scRNA) data were processed by “Seurat” package (version 4.3.0)24. Briefly, the quality control was performed with parameters set as: 400 < nFeature_RNA < 4,000 and pctMT ≤ 10. Normalization was conducted by the LogNormalize function with default settings, followed by an algorithm named Harmony to integrate all samples and minimize the batch effect. Principal component analysis (PCA) was then carried out for dimensional reduction. Then, the annotation was manually completed based on the conventional markers as reported previously25 (Supplementary Table 1).
Epithelial extraction and re-clustering
Epithelial cells were extracted from annotated scRNA data for dimensional reduction and unsupervised clustering. According to the expression of CYP4Z1 in different epithelial clusters, the CYP4Z1+ Epi and CYP4Z1− Epi cell clusters were annotated manually. Subsequently, via the FindMarkers function, the differential expressed genes between CYP4Z1+ Epi and CYP4Z1− Epi cell clusters were screened, and the top 20 significant differential expressed genes were selected as the characteristic genes of CYP4Z1+ Epi.
Bulk-seq analysis
Patients with breast cancer were separated into high and low groups based on the median value of CYP4Z1 expression. Then, the Gene Set Variation Analysis (GSVA, version 1.46.0) was conducted to evaluate the enrichment of CYP4Z1+ Epi-related genes in patients with breast cancer26. Alternatively, to assess the tumor purity of patients in high and low groups, the “estimate” package was used27. Through R package “corrplot” (version 0.92), the Pearson correlations among StromalScore, ImmuneScore, ESTIMATEScore, tumor purity, and GSVA score were calculated.
Cell stemness analysis
After merging the scRNA data without epithelial cell clusters and new annotated epithelial clusters, 5000 random cells were sampled for further single-cell-level analysis, including cell stemness, pseudotime, and cell–cell communication analysis. To explore the cell stemness of different cell clusters, the “CytoTRACE” package (version 0.3.3) was adopted28. CytoTRACE provides an approach to infer the developmental potential of cell clusters independently and robustly. CytoTRACE score reflects the stemness of each selected cell, respectively, which ranged from 0 to 1. The higher the score, the higher the stemness of the cell.
Pseudo-time analysis and intercellular communication evaluation
The pseudotime analysis was performed via the “monocle” package (version 2.26.0)29. Quality control criteria were set as expressions > 0.1 and dispersion empirical > 1 * dispersion fit cells, and then the cell differentiation states of cell clusters were identified. The expression of CYP4Z1+ Epi cell characteristic genes in different cell states was also visualized. Besides, we used the “Cellchat” package (version 1.6.1) to infer the probability of cell–cell communications between specific types of cells in samples, based on prior knowledge of communications between signaling ligand–receptor pairs and cofactors30.
Spatial transcriptomic analysis
Samples in spatial transcriptomic dataset GSE213688 were used to perform spatial transcriptomic analysis23. In Seurat package, the quality control was conducted by removing mitochondrial genes and genes expressed in less than 10 spots. Subsequently, the normalization was performed via “SCTransform” function. Further dimensionality reduction and clustering were performed with PCA and uniform manifold approximation and projection methods. On the basis of canonical markers of epithelial cells (EPCAM, CDH1, KRT7, and KRT19) and characteristic genes of CYP4Z1+ Epi cells, the “AddModuleScore” function was applied to evaluate the enrichment of epithelial cell genes and CYP4Z1+ Epi characteristic genes, for estimation of fractions of these two types of cells in spatial spots.
For precise evaluation of the cell proportion of spatial spots in samples, the “SPOTlight” package was used31. The deconvolution process was used to match the annotated breast cancer tissue scRNA dataset GSE225600 and spatial transcriptomic data GSE213688. The proportions of CYP4Z1+ Epi cells and CYP4Z1− Epi cells in each spatial spot were estimated and visualized thereafter.
Bayes deconvolution of single-cell data
To further evaluate the cell composition of TCGA-BRCA bulk-seq samples, BayesPrism (https://github.com/Danko-Lab/BayesPrism) package was used. BayesPrism was modeled a prior from cell-type-specific expression profiles from scRNA-seq to jointly estimate the posterior distribution of cell-type composition and cell-type-specific gene expression from bulk RNA-seq expression of tumor or non-tumor samples. After obtaining the cell proportions of each sample, the overall survival of patients with different CYP4Z1+ epithelial cell proportions was analyzed using the “survival” package. Specifically, the CYP4Z1 ratio was adopted for survival analysis: \({CYP}4Z1\mathrm{ratio}=\mathrm{prop}({CYP}4Z1+\mathrm{epithelial}\,\mathrm{cell})\ast \exp ({CYP}4Z1)\).
Plasmids and small interfering RNAs (siRNAs)
Plasmids, chemicals, and siRNAs used in this study were listed in Supplementary Tables 2–4. Plasmids expressing mutant proteins were generated using Mut Express II Fast Mutagenesis Kit V2 (Vazyme, Nanjing, China), following the manufacturer’s instructions. The detailed primer sequences for vector construction were indicated in Supplementary Table 5. Mutations were confirmed by Sanger sequencing. siRNAs were purchased from Invitrogen (Carlsbad, CA, USA).
Whole-mount staining of mammary gland and histology
The fourth inguinal mammary glands were isolated and fixed in Carnoy’s solution (60% ethanol, 30% CHCl3, and 10% glacial acetic acid) for 2–4 h, followed by hydration and carmine red staining overnight. Stained glands were mounted after flattening and dehydration. For histology, 4% paraformaldehyde-fixed tissues were embedded in paraffin and sectioned, followed by hematoxylin and eosin (HE) staining.
Flow cytometry
Single cells derived from cell lines or purified mouse primary cells were re-suspended and incubated for 30 min on ice with CD44-APC (BioLegend, Catalog no. 338806, Clone no. BJ18, San Diego, CA, USA), CD24-PE (BioLegend, Catalog no. 311106, Clone no. ML5), CD24-APC (BD Biosciences, Catalog no. 562349, Clone no. M1/69), CD29-PE (BD Biosciences, Catalog no. 562801, Clone no. HM β1-1). The aldehyde dehydrogenase (ALDH) activity of purified cells was measured using ALDEFLUOR™ Kit (StemCell Technologies, Catalog no. 01700), following manufacturer’s instructions. Spontaneous single cells were prepared from bone marrow for the hematopoietic stem cell analysis. All antibodies were purchased from BioLegend. The antibodies such as anti-Gr-1-FITC (108405), Ter119-FITC (116205), B220-FITC (103205), CD19-FITC (115505), IgM-FITC (406505), CD127-FITC (121105), and CD3e-FITC (100305) were used for lineage markers. Anti-Sca1-PE (108107), c-Kit-APC (105811), CD34-PE/Cyanine7 (119325) and CD16, and 32-Brilliant Violet 510™ (101333) antibodies were used for hematopoietic progenitor cell population analysis; anti-CD150-Brilliant Violet 421™ (115925) and CD48-PerCP/Cyanine5.5 (103421) antibodies were used for hematopoietic stem cell (HSC) population analysis. All tests were detected by flow cytometer (BD, USA), and FACS analysis was performed using FACS Canto, Diva software, and FlowJo.
Mammosphere assay
Single cells were seeded into ultralow-attachment plates (Corning, NY, USA) with DMEM/F12 (Keygen BioTECH) with epidermal growth factor (200 ng/ml, MedChem Express, Monmouth Junction, NJ, USA), basic fibroblast growth factor (200 ng/ml, Sigma-Aldrich), and B27 (50×, abs9120, Absin, Shanghai, China). Mammospheres were photographed and counted 8 days later. Spheroids with a diameter greater than 50 μm are considered to be spheroids.
Western blotting and immunohistochemistry (IHC) analysis
For western blotting analysis, lysates prepared using RIPA buffer (Beyotime, Beijing, China) were subjected to SDS–PAGE and transferred onto polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 2 h. After overnight incubation with primary antibodies at 4 °C, the membranes were incubated with secondary antibodies for 40 min and then developed using the Tanon™ High-sig ECL Western Blotting Substrate (Tanon, Catalog no. 180-5001, Shanghai, China). Gray scale analysis of western blot bands was performed using ImageJ software. For IHC analysis, tissues were fixed in 4% paraformaldehyde for 48 h at room temperature and then embedded in paraffin. Paraffin-embedded sections were deparaffinized and rehydrated, followed by antigen retrieval. After incubation with primary and secondary antibodies, the slides were treated with diaminobenzidine (Dako, USA). Details of the primary antibodies used were listed in Supplementary Table 6. IHC image analysis was conducted using Image-Pro Plus software.
Co-IP and proteomics assays
Cells were homogenized in RIPA buffer supplemented with phenylmethylsulfonyl fluoride (Beyotime), Phosphatase Inhibitor Cocktail (Beyotime), and E3876 (Sigma-Aldrich). Lysates were cleared by centrifugation at 13,000 rpm for 15 min at 4 °C, and the supernatant was collected for protein quantification using a BCA assay kit. To remove nonspecific proteins and reduce background, 100 μl of Protein A agarose beads was added to 1 ml of total protein and gently shaken at 4 °C for 10 min. After centrifugation at 4 °C, 14,000×g for 15 min, the supernatant was transferred to a new tube, and the Protein A beads were discarded. Next, a specific volume of primary antibody was added to 500 μl of total protein, and the antigen–antibody mixture was gently shaken overnight at 4 °C. The antigen–antibody complex was captured by adding 100 μl of Protein A agarose beads, followed by gentle shaking at room temperature for 1 h. The agarose bead–antigen–antibody complex was collected after centrifugation at 14,000 rpm for 5 s. The complex was re-suspended in 2× sample buffer, gently mixed, and then boiled for 5 min. Subsequently, SDS–PAGE was performed. After electrophoresis, the proteins were transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% skimmed milk at room temperature for 1.5 h, incubated with the primary antibody overnight at 4 °C, and washed thrice with TBST. After being incubated with the secondary antibody at room temperature for 1 h, the membranes were washed three times with TBST. Finally, protein expression changes were detected using an enhanced chemiluminescence detection system. Alternatively, after electrophoresis, the gel was stained with Coomassie Brilliant blue, which was sent to Beijing Novogene Science and Technology Co., Ltd (Beijing, China) for proteomic analysis of interacting proteins.
ISGylation assay and proteomics analysis on ISGylation sites
MCF-7 or MDA-MB-231 cells were treated with 20 μM MG132 for 8 h before harvesting. Cells were lysed by boiling in buffer (50 mM Tris–HCl, 150 mM NaCl, 1% SDS, 0.5% deoxycholate, 10 mM dithiothreitol) for 10 min. The cell lysates were then diluted tenfold with lysis buffer containing 1% NP-40 (Beyotime), phenylmethylsulfonyl fluoride (Beyotime), Phosphatase Inhibitor Cocktail (Beyotime), and E3876 (Sigma-Aldrich). These lysates were subjected to IP analysis using a CYP4Z1 antibody (DF10128, Affinity, Cincinnati, OH, USA). The ISGylation of endogenous CYP4Z1 was detected by western blotting. To analyze the exogenous CYP4Z1 ISGylation, HEK-293T cells were transfected with plasmids encoding Flag-tagged CYP4Z1 and either His-tagged or HA-tagged ISG15. Co-IP assays were performed to assess the ISGylation of CYP4Z1. For the identification of ISGylation sites, the ISG15 coding sequences were inserted into the pcDNA4/HisMax plasmid (pc-ISG15). Site-directed mutagenesis was used to mutate the LRGG amino acid sequence at the C terminus of the ISG15 protein to LKGG, resulting in pc-ISG15-mut. HEK293T cells were co-transfected with pc-ISG15-mut and pc-Z1-wt, followed by a co-IP experiment using an anti-Flag antibody. The Coomassie Brilliant blue-stained gel strips from the co-IP electrophoresis were sent to Beijing Novogene Co., Ltd for qualitative proteomic analysis of PTMs. Endoproteinase Lys-C (Sequencing grade, Catalog no. 11047825001, Sigma-Aldrich), which specifically recognizes the LKGG sequence, was used for enzymatic digestion.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from cells using the TransZol Up reagent (ET111-01-V2, TransGen Biotech, Beijing, China) according to the manufacturer’s instructions, followed by cDNA preparation using Hiscript III Reverse Transcriptase (Vazyme). The real-time PCR assays were performed in triplicate using SYBR Green Supermix (Vazyme) with the Applied Biosystems QuantStudio 5 (A28139, Thermo Fisher Scientific). The sequences of the primers used for qRT-PCR were described in Supplementary Table 7.
Immunofluorescence (IF) assay
For IF assays, cells were cultured in chamber slides overnight (if a probe (refer to Supplementary Table 8) was used, it was applied before the cells are fixed, and the use of the probe should follow the instructions provided, incubating at 37 °C for 30 min) and fixed with 3.7% formaldehyde in PBS for 10 min at 4 °C, followed by permeabilization with 0.5% Triton X-100 in PBS for 10 min. Cells were then blocked for nonspecific binding with 10% goat serum in PBS and 0.1% Tween-20 (PBST) for 1 h at room temperature and incubated with the indicated antibody overnight at 4 °C, followed by incubation with Alexa Fluor-labeled or FITC-labeled secondary antibody for 50 min at room temperature. Coverslips were mounted on slides using the anti-fade mounting medium with 4′,6-diamidino-2-phenylindole (Beyotime). IF images were acquired on a Zeiss LSM 800 Confocal Laser Scanning Microscopy (Jena, Germany). For each channel, all images were acquired with the same settings. The relevant probes used in the experiments were listed in Supplementary Table 8.
Enzyme activity assay for CYP4Z1
Lytic P450-dependent bioluminescence assays were conducted according to the manufacturer’s instructions (Promega, Madison, WI, USA). To detect CYP4Z1 activity, 12.5 μl of an inhibitor mixture was added to each well at the indicated concentrations before the CYP reactions. Then, 12 μl of HEK293T cell lysate, with or without CYP4Z1 overexpression, and 0.5 μl of Luciferin-CEE (Catalog no. V8752, Promega) were mixed with the inhibitor mixture in 96-well plates. The plates were preincubated at 37 °C for 10 min, after which 25 μl of a 2× NADPH regeneration system (Catalog no. V9510, Promega) was added to each well. Once biotransformation was completed, an equal volume of Luciferin detection reagent (Promega) was added to each well, and the plates were gently swirled to facilitate the formation of cell lysates. The plates were then incubated at room temperature for 20 min, and luminescence was measured using an Infinite M200 PRO Multimode Microplate Reader (Tecan; Infinite).
Wound healing analysis
When cell confluence reached 90% in six-well plates, a wound was created on the cell surface, and cell debris was washed away with PBS twice. To exclude the effects of cell proliferation on cell migration ability, cells were then cultured in a serum-starved medium (≤2%) containing the compounds or solvent. The migration rate was calculated as follows: Migration rate = (Dt − D0)/D0, in which Dt represents the distance between the wound edges at different time points and D0 represents the initial wound distance at 0 h.
Transwell invasion analysis
Millicell® hanging cell culture inserts (Catalog no. MCEP24H48, Merck) precoated with Matrigel matrix (Catalog no. 356234, Corning) were placed into 24-well plates. A total of 2 × 105 MCF-7 or MDA-MB-231 cells in 200 μl of serum-free medium containing different compounds or solvents were added to the upper chamber, and 800 μl of medium containing 20% serum was used as a chemoattractant in the lower chamber. After 24 h for MDA-MB-231 cells and 48 h for MCF-7 cells, the cells were fixed with 70% ethanol for 20 min at room temperature. The invaded cells were then stained with crystal violet staining solution (Catalog no. C0121, Beyotime) and observed under a microscope. Finally, the chambers were washed with 33% glacial acetic acid, and the absorbance was measured at 570 nm using a Bio-Rad iMark (Hercules, CA, USA). The measured values represent the number of invaded cells.
Flow cytometry analysis of lipid droplet formation
Cells were cultured under the relevant research conditions. At the time point of interest, a 10 μM BODIPY™ 493⁄503 (Catalog no. D2191, Thermo Fisher Scientific) staining solution was prepared in PBS. The cells were washed twice with PBS, followed by adding the BODIPY staining solution and incubating at 37 °C for 15 min. After two additional PBS washes, the cells were trypsinized to obtain a single-cell suspension, which was subsequently analyzed by flow cytometry.
Lipidomic identification and analysis
Lipidomics analysis was conducted by Beijing Novogene Science and Technology Co., Ltd. The experimental process included sample collection, lipid extraction, and liquid chromatography–mass spectrometry (MS)/MS detection. The raw data obtained from MS were processed using CompoundDiscoverer 3.1 software for spectral analysis and database searching to achieve qualitative and quantitative results of lipid compounds. Quality control measures ensured data accuracy and reliability. Multivariate statistical analyses were performed to identify differences in metabolic patterns between groups, including PCA and partial least-squares discriminant analysis. Hierarchical clustering analysis and correlation analysis were subsequently used to elucidate relationships between samples and lipid compounds.
Cellular thermal shift assay (CETSA)
Cells were collected and proteins were extracted. The samples were divided into two equal parts and incubated with an equal volume of DMSO or BM-51 (500 μM) at room temperature for 30 min. Subsequently, each group of samples was further divided into 12 portions and incubated at temperatures ranging from 54 °C to 76 °C, with each 2 °C interval, for 5 min. After incubation, the samples were cooled to room temperature, centrifuged, and the supernatant was collected. Loading buffer (5×) was added and the mixture was boiled. The resulting samples were subjected to 10% SDS–PAGE for western blotting.
Drug Affinity Responsive Target Stability (DARTS)
Cells were collected, and proteins were extracted. The protein concentration was adjusted to 4–6 μg/μl using 10× TNC (Tris–NaCl–Casein) buffer, and the samples were divided into two equal parts. Each portion was incubated with either an equal volume of DMSO or BM-51 (500 μM) at room temperature for 30 min. Subsequently, each group of samples was divided into six parts and incubated with Pronase solution (Catalog no. 10165921001, Roche, Basel, Switzerland) at varying ratios (1 μg Pronase per 100 μg protein and so on) at room temperature for 30 min. The reaction was terminated by adding 5× loading buffer and boiling the mixture. The resulting samples were subjected to 10% SDS–PAGE for western blotting.
Molecular docking studies
For docking purposes, the homology model of CYP4Z1 was used as a protein receptor, which was generated according to our previous study32. The protein and ligand preparation module in Schrodinger was used to deal with the protein and ligand. Grid generation module was used to generate grid box centered around the heme iron atom with 8 × 8 × 8 Å. The dockings were performed using the Glide standard precision (GlideSP) mode with the ligands as flexible, reject poses with Coulomb–van der Waals energy greater than 5 kcal/mol, and all other options were left at their default values. Then the poses were visualized with PyMOL.
Molecular simulation dynamics
To investigate the interaction dynamics between BM-51 and CYP4Z1, molecular dynamics (MD) simulations were conducted. The initial structure for MD simulations was based on the predicted binding mode of BM-51 to the active site of CYP4Z1 obtained from molecular docking. First, the complexes were prepared using the ProteinPrepWizard module of MAESTRO. The prepared complexes were solvated in a cubic box using the TIP3P water model, with a 15 Å buffering distance between the protein and the box edges. To neutralize the system charge, an appropriate amount of sodium or chloride ions was added, and an additional 0.15 M NaCl was included to mimic physiological conditions. After setting up the solvated system, energy minimization and relaxation were performed using the default protocol in the Desmond module. MD simulations were carried out in the NPT ensemble with periodic boundary conditions. Temperature and pressure were maintained at 300 K and 1 atm, respectively, using the Nose−Hoover thermostat and isotropic scaling. Subsequently, 200 ns MD simulations were executed using the DESMOND simulation engine and the OPLS-AA force field. Trajectories of the complexes were saved every 100 ps for analysis. Finally, the last conformation of the complex was extracted for binding mode analysis, and root-mean-square deviation and root-mean-square fluctuation analyses were conducted using DESMOND.
Xenograft animal model
Six-week-old BALB/c male nude mice were purchased from GemPharmatech Co., Ltd (Nanjing, China) and used in the xenograft experiments. MCF-7 cells with or without stably-ectopic expression of transcripts were implanted into the dorsal flanking sites of nude mice at 1 × 105, 1 × 106, and 1 × 107 cells in 200 µl PBS. Lin-NECs were implanted into the dorsal flanking sites of nude mice at 1 × 104, 1 × 105, and 1 × 106 cells in 200 µl PBS. Two weeks after injection, mice-bearing tumors were sacrificed for the assessment of tumor size and weight examination, as well as stem cell frequencies through extreme limiting dilution analysis33.
Tumor metastatic analysis
BALB/C nude mice aged 4–6 weeks were selected. The cell suspension was injected into caudal vein (5 × 105 cells/200 μl) twice on 2 days. After 3 weeks, the lung and liver tissues of mice were removed entirely and photographed. After fixation, HE staining and microscopic examination were performed.
Evaluation of the antitumor effects of BM-51 in vivo
For evaluating the effects of BM-51 on Adriamycin sensitivity of breast cancer cells, all nude mice were inoculated with MCF-7 cells (5 × 106 cells per mouse) mixed 1:1 with Matrigel matrix (BD Biosciences) under the left subcutaneous. When the tumors reached the volume of ~100 mm3, we randomly allocated the mice to groups in which they received Adriamycin (0.5 mg/kg, Shenzhen Main Luck Pharmaceuticals Inc., China), PBS, 10 mg/kg BM-51, Adriamycin + 10 mg/kg BM-51. Tumor growth was monitored by caliper measurements. Tumor volume was calculated by the following formula: Volume (mm3) = L (length) × W (width)2 × 1/2. Mice were euthanized 14 days after the inoculation. The weight of each tumor was measured and mice organs were used for HE staining (Servicebio, Wuhan, China).
For evaluating the effects of BM-51 on the tumor-initiating ability of breast cancer cells, 6-week-old BALB/c male nude mice were used in the xenograft experiments. MCF-7 and MDA-MB-231 cells were pretreated with BM-51 for 72 h and then were implanted into the dorsal flanking sites of nude mice at 1 × 105, 1 × 106, and 1 × 107 cells in 200 µl PBS. Two weeks after injection, mice-bearing tumors were sacrificed for the assessment of tumor size and weight examination, as well as stem cell frequencies through extreme limiting dilution analysis33.
For evaluating the effects of BM-51 on the metastatic ability of breast cancer cells, MDA-MB-231 cells (2 × 106 cells per mouse) were injected (intravenously) into mice, which were randomly allocated to groups with or without BM-51 (10 mg/kg) treatment. After 21 days, mice were euthanized and lung tissues were subjected to HE staining to observe the lung metastatic nodes. Additionally, PyMT-MMTV and PyMT-CYP4Z1 mice were treated with BM-51 from week 9 and lasted for 6 weeks. The process of tumor progression between PyMT-MMTV and PyMT-CYP4Z1 mice with or without BM-51 treatment was compared by observing tumor initiation, growth, metastasis, and whole-mount staining of the mammary gland.
Chemical synthesis
See Supplementary Note.
Statistical analysis
Statistical analysis was performed using Prism 8.0 (GraphPad Software, La Jolla, CA, USA). For two-group comparisons, two-tailed unpaired t tests were performed; for multiple-group comparison, two-way analysis of variance of post hoc test was performed. P-values less than 0.05 were considered significant.
