Journal: Advanced Science

Article Title: GPX4 Inhibitor Resistance and Metastatic Features in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202523198

Figure Lengend Snippet: Generation and in vitro characterization of GPX4‐inhibitor‐resistant TNBC cells. (A) Experimental scheme of the generation of 4T1 and M231 cell lines resistant to small molecule GPX4 inhibitors ( n = 3 independent generations) (B) Parental, RSL3 R , and RSL3 R DH cell lines were incubated with indicated doses of RSL3, ML210, and Erastin2 for 48 h. Cell viability was assessed using an absorbance‐based assay (MTT), and results are expressed as a ratio normalized to the untreated condition ( n = 3–8 independent experiments). (C–F) Immunoblot analyses of protein extracts from 4T1 (C) and M231 (D) parental, RSL3 R , and RSL3 R DH collected after 24 h. Protein level quantifications relative to the parental cells are represented in (E,F) ( n = 4–6 independent protein extracts). (G,H) Parental, RSL3 R, and RSL3 R DH cell lines were incubated with 1 µ m Liprox or vehicle for 24 h. Lipid peroxidation was evaluated by C11‐BODIPY 581/591 staining. Representative plots (G) and BODIPYox/BODIPYred, ratio of oxidized to reduced BODIPY relative to parental control group (H) are shown ( n = 4–8 independent experiments). (I) GSH/GSSG ratio in 4T1 and M231 RSL3 R and RSL3 R DH relative to the parental control group collected after 24‐hours ( n = 4 independent experiments). (J) NADP + /NADPH ratio in 4T1 and M231 RSL3 R and RSL3 R DH relative to the parental control group collected after 24‐hours ( n = 3 independent experiments). Data in (B) are displayed as mean. Data in (E,F) are displayed as mean ± s.d. and were analyzed by one‐way ANOVA and Dunnett's multiple comparison. Data in (H,I,J) are displayed as mean ± s.d. and were analyzed by one‐sample t ‐test.

Article Snippet: The human MDA‐MB‐231 (M231, Cat#HTB‐26, RRID: CVCL_0062) and murine 4T1 (Cat#CRL‐2539, RRID: CVCL_0125) cell lines were purchased from the American Type Culture Collection in July 2022 and November 2022, respectively.

Techniques: In Vitro, Incubation, Western Blot, Staining, Control, Comparison

Journal: Advanced Science

Article Title: GPX4 Inhibitor Resistance and Metastatic Features in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202523198

Figure Lengend Snippet: GPX4‐inhibitor resistant tumors form fewer distant metastases. (A,B) Experimental scheme of the 4T1‐Balb/c (A) and M231‐NSG (B) model of spontaneous metastasis from a surgically removed primary tumor ( n = 2 independent experiments per model). (C,D) Tumor growth curves of 4T1‐Balb/c (C) and M231‐NSG (C) models ( n = 7–18 mice per group). (E–H) Representative images of lung sections stained with hematoxylin and eosin (H&E) from 4T1‐Balb/c (E) and M231‐NSG (F) models. Quantification of the percentage of the tumor burden area covering the lung is shown in (G,H) ( n = 6–11 mice per group). (I) Percentage of mice with distant metastases visually quantified in lungs, liver, kidneys (kid.), pancreas (panc.), GI tract, heart, and spleen in M231‐NSG model ( n = 5–6 mice per group). (J) Percentage of mice with cancer cells isolated from tdLNs in 4T1‐Balb/c and M231‐NSG models ( n = 4–10 mice per group). (K,L) Survival curves of 4T1‐Balb/c (K) and M231‐NSG (L) models ( n = 5–10 mice per group). (M,N) Lipid peroxidation levels quantified by C11‐BODIPY 581/591 staining in cancer cells isolated from the resected primary tumor in the 4T1‐Balb/c model (M) and in the M231‐NSG model (N) ( n = 7–10 mice per group). (O) Cancer cells were isolated from the resected primary tumor in 4T1‐Balb/c and M231‐NSG models and incubated with dose‐range RSL3 and Erastin2 for 48 h. Cell viability was assessed using an absorbance‐based assay (MTT), and results are expressed as a ratio to the untreated condition ( n = 6–8 mice per group). (P–S) Immunoblot analysis of protein extracts from cancer cells isolated from the resected primary tumor in 4T1‐Balb/c (P) and M231‐NSG (Q) using the indicated antibodies. Protein level quantification relative to the average of parental control group are represented in (R,S) ( n = 4–11 mice per group). Data in (C,D) are displayed as mean ± s.d. and were analyzed by two‐way ANOVA and Tukey's multiple comparation. Data in (G,H,M,N,R,S) are displayed as mean ± s.d. and were analyzed by two‐sided Mann–Whitney test. Kaplan–Meyer plots in (K,L) were analyzed by the Mantel–Cox log‐rank test. Data in (O) are displayed as the mean.

Article Snippet: The human MDA‐MB‐231 (M231, Cat#HTB‐26, RRID: CVCL_0062) and murine 4T1 (Cat#CRL‐2539, RRID: CVCL_0125) cell lines were purchased from the American Type Culture Collection in July 2022 and November 2022, respectively.

Techniques: Staining, Isolation, Incubation, Western Blot, Control, MANN-WHITNEY

Journal: Advanced Science

Article Title: GPX4 Inhibitor Resistance and Metastatic Features in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202523198

Figure Lengend Snippet: GPX4i‐resistant 4T1 tumors form fewer distant metastases in immunocompromised NSG mice. (A) Experimental scheme of the survival surgery model of the 4T1‐NSG model ( n = 2 independent experiments). (B,C) Tumor growth curve of D22 cohort (B) and Survival cohort (C) in 4T1‐NSG model ( n = 5 mice per group). (D,E) Representative images of lung sections stained with hematoxylin and eosin (H&E) from 4T1‐NSG (D) models. Quantification of the percentage of the tumor burden area covering the lung is shown in (E) ( n = 3 mice per group). (F) tdLNs size from 4T1‐NSG model ( n = 6 mice per group). (G) Survival curves of 4T1‐NSG model ( n = 5 mice per group). (H) Cancer cells were isolated from the resected primary tumor from D22 and Survival cohorts in the 4T1‐NSG model and incubated with dose‐range RSL3, ML210, and Erastin2 for 48 h. Cell viability was measured using an absorbance‐based assay (MTT), and results are expressed as a ratio to the untreated condition ( n = 10 mice per group). (I,J) Immunoblot analysis of protein extracts from cancer cells isolated from the resected primary tumor in 4T1‐NSG using the indicated antibodies (I). Protein level quantification relative to the average of the parental control group is represented in (J) ( n = 8 mice per group). (K) Lipid peroxidation quantified by C11‐BODIPY 581/591 staining in cancer cells from the resected primary tumors of the D22 cohort in the 4T1‐NSG model ( n = 10 mice per group). Data in (B,C) are displayed as mean ± s.d. and were analyzed by two‐way ANOVA and Tukey's multiple comparison. Data in (E,J,K) are displayed as mean ± s.d. and were analyzed by two‐sided Mann–Whitney test. Kaplan–Meyer plot in (G) was analyzed by Mantel–Cox log‐rank test.

Article Snippet: The human MDA‐MB‐231 (M231, Cat#HTB‐26, RRID: CVCL_0062) and murine 4T1 (Cat#CRL‐2539, RRID: CVCL_0125) cell lines were purchased from the American Type Culture Collection in July 2022 and November 2022, respectively.

Techniques: Staining, Isolation, Incubation, Western Blot, Control, Comparison, MANN-WHITNEY

Journal: Advanced Science

Article Title: GPX4 Inhibitor Resistance and Metastatic Features in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202523198

Figure Lengend Snippet: GPX4i‐resistant tumors show distinct lipid adaptations in the immunocompetent model. (A–C) Lipid profiling was performed on cancer cells isolated from the resected primary tumor in 4T1‐Balb/c by LC/MS ( n = 7 mice per group). FC of lipid peak intensities and p ‐value for RSL3 R ‐ versus parental‐derived cancer cells comparisons calculated using MetaboAnalyst are shown in (A), and significant changes in lipid peak intensities are highlighted (FC ≥ 1.5 and ≤0.66, p < 0.05). MUFA and PUFA composition of significantly increased phospholipids are represented in (B,C) respectively. (D–G) Spatial lipid profiling was performed on the resected primary tumor in 4T1‐Balb/c by MALDI‐MSI ( n = 2 mice per group). Images of primary tumor sections stained with hematoxylin and eosin (H&E) and the corresponding spatial distribution of representative lipid species from the same section are shown in (D) (scale bar = 250 µm). FC of lipid peak intensities in tumor versus non‐tumor areas is represented in (E). Unsaturation levels are represented in percent for each lipid class in tumor areas (F) and in non‐tumor areas (G). (H–K) Lipid profiling was performed on cancer cells isolated from the resected primary tumor in M231‐NSG by LC/MS ( n = 5–7 mice per group). FC of lipid peak intensities and p ‐value for RSL3 R ‐ versus parental‐derived cancer cells comparisons calculated using MetaboAnalyst are shown in (H), and significant changes in lipid peak intensities are highlighted (FC ≥ 1.5 and ≤0.66, P < 0.05). MUFA and PUFA composition of significantly decreased triglycerides are represented in (I,J) respectively. Fatty acyl chain composition of triglycerides is represented in (K). AcCa: acyl carnitine, Cer: ceramides, ChE: cholesterol ester, CoQ 9 : coenzyme Q9, PC(‐P/‐O): phosphatidylcholine (‐plasmalogen/‐alkyl ether), PE(‐P/‐O): phosphatidylethanolamine (‐plasmalogen/‐alkyl ether); PI: phosphatidylinositol; PS: phosphatidylserine, PUFA: polyunsaturated fatty acids, MUFA: monounsaturated fatty acids, SM: sphingomyelin, TG: triglycerides.

Article Snippet: The human MDA‐MB‐231 (M231, Cat#HTB‐26, RRID: CVCL_0062) and murine 4T1 (Cat#CRL‐2539, RRID: CVCL_0125) cell lines were purchased from the American Type Culture Collection in July 2022 and November 2022, respectively.

Techniques: Isolation, Liquid Chromatography with Mass Spectroscopy, Derivative Assay, Staining

Journal: Advanced Science

Article Title: GPX4 Inhibitor Resistance and Metastatic Features in Triple‐Negative Breast Cancer

doi: 10.1002/advs.202523198

Figure Lengend Snippet: GPX4i‐resistant TNBC cells can survive in the blood and colonize the lungs. (A) Experimental schematic of breast cancer cell survival and colonization in lungs of NSG mice after intravenous injection with M231 breast cancer cells pre‐treated with vehicle or with Liprox ( n = 2 independent experiments). (B,C) Quantification of breast cancer cell survival and colonization in lungs (B) based on ex vivo bioluminescence imaging (C) in NSG mice intravenously injected with M231 cells treated with vehicle or Liprox. (D) Experimental schematic of breast cancer cell survival and colonization in lungs of NSG mice after intravenous injection with M231 parental or M231 RSL3 R breast cancer cells ( n = 2 independent experiments). (E,F) Quantification of breast cancer cell survival and colonization (E) based on ex vivo bioluminescence imaging (F) in NSG mice intravenously injected with parental or RSL3 R cells derived from the M231 cell line. (G) Experimental schematic of breast cancer cell survival and colonization in lungs of Balb/c mice after intravenous injection with 4T1 parental or 4T1 RSL3 R cells pre‐treated with vehicle or with Liprox ( n = 1 experiment). (H,I) Quantification of breast cancer cell survival and colonization (H) based on ex vivo bioluminescence imaging (I) in Balb/c mice intravenously injected with parental or RSL3 R cells derived from the 4T1 cell line and treated with vehicle or liprox. Data in (B,E,H) are displayed as mean ± s.d. and were analyzed by two‐sided Mann–Whitney test.

Article Snippet: The human MDA‐MB‐231 (M231, Cat#HTB‐26, RRID: CVCL_0062) and murine 4T1 (Cat#CRL‐2539, RRID: CVCL_0125) cell lines were purchased from the American Type Culture Collection in July 2022 and November 2022, respectively.

Techniques: Injection, Ex Vivo, Imaging, Derivative Assay, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: TOE suppresses malignant phenotype of triple negative breast cancer cells. (A) The effect of TOE on cell proliferation in MDA-MB-231 and 4T1 cells. Cells were treated with varying concentrations of TOE for 24 hours, and cell proliferation was assessed using the CCK-8 assay. Data are expressed as the mean ± SEM (n = 3). Statistical significances were calculated via Student’s t-test. **p < 0.01 and ***p < 0.001 and ****p < 0.0001. (B) Transwell migration and invasion assay of MDA-MB-231 and 4T1 cells after treatment with TOE for 24 hours. Representative images of the migrated and invaded cells from randomly selected fields of Transwell inserts are shown on the left, while quantitative data for cell numbers are presented on the right. Cell numbers were calculated and expressed as the mean ± SEM of three independent experiments. Statistical significance was determined by t-test, with ** p < 0.01 and *** p < 0.001 and **** p < 0.0001 indicating significant differences between TOE-treated and DMSO-treated cells. Scale bar = 100 μm. (C) MA plot of DGEs in MDA-MB-231 treated with TOE. (D) Enrichment and scatter map of KEGG pathway of DGEs.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: CCK-8 Assay, Migration, Invasion Assay

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: Dig and AA inhibited tumor growth in breast cancer mouse models. (A) Inhibition of growth by Dig and AA in MDA-MB-231 and 4T1 cells for 24 h. MDA-MB-231 and 4T1 cells were treated with Dig and AA (at various concentration) for 24 hours, and cell proliferation was assessed using the CCK-8 assay. Data are presented as the mean ± SEM from three independent experiments (n = 3). Statistical significance was determined using unpaired t -tests, with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 indicating significant differences compared to the DMSO control. (B) Transwell migration and invasion assay of MDA-MB-231 and 4T1 cells with Dig and AA treatment for 24 h. Representative images from randomly selected fields of transwell inserts, and Scalebar = 100 μm. (C) Quantitative data from the Transwell migration and invasion assays. Cell numbers were calculated and are expressed as the mean ± SEM of three independent experiments. * p < 0.05,** p < 0.01, *** p < 0.001 and **** p < 0.0001, as determined by unpaired t -tests. (D) Diagrammatic representation of tumor volume measurement. The diagram illustrates the measurement method, including caliper-based measurements of length and width used to calculate tumor volume (Volume = 1/2 × length × width^2). (E) Tumor sizes at day 14. (F) The body weight changes of mice in the period of 14 days after different treatments. The body weight of mice was monitored every 2 days after Dig and AA treatment. Data are expressed as the mean ± SEM. No significant changes in body weight were observed, suggesting that the treatments did not cause overt toxicity in mice.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: Inhibition, Concentration Assay, CCK-8 Assay, Control, Migration, Invasion Assay

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: Dig combined with PD-1 immunotherapy can promote immune stimulation of in situ 4T1 breast tumors. (A) HE staining of mouse tumor tissues (scale bar = 100 μm). (B) Representative flow cytometry plots showing tumor-associated macrophages (TAMs) (CD45.2+, CD11b+, F4/80+) were obtained after different treatments. (C) Representative flow cytometry plots showing tumor immune cells after different treatments, including CTLs (CD45+, CD3+, CD8+) and Th cells (CD45+, CD3+, CD4+). (D) Representative flow cytometry plots demonstrating tumor-infiltrating LAG-3+ exhausted T cells (CD3+, CD8+, LAG-3+) after different treatments. (E) Representative flow cytometry plots demonstrating tumor-infiltrating TIM-3+ exhausted T cells (CD3+, CD8+, TIM-3+) after different treatments. (F) Representative flow cytometry plots demonstrating tumor-infiltrating PD-1+ exhausted T cells (CD3+, CD8+, PD-1+) after different treatments. (G) The levels of TAMs were quantified through flow cytometry analysis (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.0033; Saline vs Dig: 0.0106; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.2039; Saline vs AA+PD-1: 0.9935; PD-1 vs Dig: 0.9961; PD-1 vs Dig+PD-1: 0.4395; PD-1 vs AA: 0.4330; PD-1 vs AA+PD-1: 0.0009; Dig vs Dig+PD-1: 0.2080; Dig vs AA: 0.7273; Dig vs AA+PD-1: 0.0028; Dig+PD-1 vs AA: 0.0109; Dig+PD-1 vs AA+PD-1: <0.0001; AA vs AA+PD-1: 0.0715. (H) The levels of CTLs were quantified by flow cytometry analysis (n = 5). The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.7941; Saline vs Dig: 0.2550; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.0161; Saline vs AA+PD-1: <0.0001; PD-1 vs Dig: 0.9237; PD-1 vs Dig+PD-1: 0.0013; PD-1 vs AA: 0.2253; PD-1 vs AA+PD-1: 0.0016; Dig vs Dig+PD-1: 0.0136; Dig vs AA: 0.7542; Dig vs AA+PD-1: 0.0164; Dig+PD-1 vs AA: 0.2253; Dig+PD-1 vs AA+PD-1: >0.9999; AA vs AA+PD-1: 0.2576. (I) Flow cytometry analysis quantified the levels of LAG-3+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. Flow cytometry analysis quantified the levels of LAG-3+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.0006; Saline vs Dig: 0.0030; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.5950; Saline vs AA+PD-1: >0.9999; PD-1 vs Dig: 0.9841; PD-1 vs Dig+PD-1: 0.0094; PD-1 vs AA: 0.0282; PD-1 vs AA+PD-1: 0.0005; Dig vs Dig+PD-1: 0.0019; Dig vs AA: 0.1152; Dig vs AA+PD-1: 0.0027; Dig+PD-1 vs AA: <0.0001; Dig+PD-1 vs AA+PD-1: <0.0001; AA vs AA+PD-1: 0.5676. (J) Flow cytometry analysis quantified the levels of TIM-3+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: 0.0073; Saline vs Dig: 0.3784; Saline vs Dig+PD-1: <0.0001; Saline vs AA: 0.9296; Saline vs AA+PD-1: 0.3206; PD-1 vs Dig: 0.4016; PD-1 vs Dig+PD-1: 0.0959; PD-1 vs AA: 0.0007; PD-1 vs AA+PD-1: <0.0001; Dig vs Dig+PD-1: 0.0011; Dig vs AA: 0.0698; Dig vs AA+PD-1: 0.0050; Dig+PD-1 vs AA: <0.0001; Dig+PD-1 vs AA+PD-1: <0.0001; AA vs AA+PD-1: 0.8546. (K) Flow cytometry analysis quantified the levels of PD-1+ exhausted T cells (n = 5). Statistical analysis was performed using Tukey’s multiple comparison test. The following adjusted p-values were obtained for each comparison: Saline vs PD-1: <0.0001; Saline vs Dig: 0.2205; Saline vs Dig+PD-1: 0.0276; Saline vs AA: 0.9984; Saline vs AA+PD-1: 0.8260; PD-1 vs Dig: 0.0043; PD-1 vs Dig+PD-1: 0.0469; PD-1 vs AA: <0.0001; PD-1 vs AA+PD-1: <0.0001; Dig vs Dig+PD-1: 0.9030; Dig vs AA: 0.1042; Dig vs AA+PD-1: 0.0181; Dig+PD-1 vs AA: 0.0108; Dig+PD-1 vs AA+PD-1: 0.0015; AA vs AA+PD-1: 0.9632. Data are expressed as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 vs. Saline.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: In Situ, Staining, Flow Cytometry, Comparison, Saline

Journal: Frontiers in Oncology

Article Title: Targeting NANOS1 in triple-negative breast cancer: synergistic effects of digoxin and PD-1 inhibitors in modulating the tumor immune microenvironment

doi: 10.3389/fonc.2024.1536406

Figure Lengend Snippet: NANOS1 Silencing Reduces TNF-α Expression and Cell Invasiveness. (A) Volcano plots of RNA-seq. Differential gene expression analysis was performed using RNA sequencing data from MDA-MB-231 cells with siNANOS1#1 silencing. The volcano plot shows the distribution of genes based on log-fold change versus the negative log-transformed p-value. Significant upregulated and downregulated genes are indicated with colored dots. Genes that meet the threshold of log-fold change (|logFC| > 2) and adjusted p-value < 0.05 are considered differentially expressed. (B) Heatmap of differential gene expression. (C) Chord diagram of GO enrichment results and related genes. (D) Top 15 KEGG pathways. The x-axis represents the gene ratio ( p < 0.05), and the y-axis represents the enriched terms. (E) The knockdown efficiency of siRNA and the expression of NANOS1 and TNF-α following Dig and AA treatment were quantified by qPCR. MDA-MB-231 and 4T1 cells were transfected with siRNA targeting NANOS1, followed by treatment with Dig and AA. Quantitative PCR was performed to assess the expression levels of NANOS1 and TNF-α. The data are expressed as the mean ± SEM, with statistical significance calculated using one-way ANOVA. (F) Dig and AA decreased the expression of NANOS1 protein (n = 3). Data are expressed as the mean ± SEM. Statistical significances were calculated via one-way ANOVA, * p < 0.05, ** p < 0.010, *** p < 0.001 and **** p < 0.0001 vs. DMSO. (G) Perforation migration and invasion assays of MDA-MB-231 and 4T1 cells after 24 h of siRNA treatment, scale bar = 100 mm. Cell numbers were calculated and are expressed as the mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, as determined by unpaired t-tests, were regarded as significant.

Article Snippet: MDA-MB-231 or 4T1 cells were seeded in a 96‐well plate at 2.0×10^5 cells/mL and cultured in medium at 37°C with a 5% CO2 atmosphere for 24 h. After that, the cells were pretreated with various concentrations of TOE for 24 h. At the end of the stimulation, the medium was removed, and 20 μL of CCK-8 reagent (Elabscience, China) was added to each well, and the incubation was continued for 1 h. The absorbance of each well was measured at 450 nm.

Techniques: Expressing, RNA Sequencing, Gene Expression, Transformation Assay, Knockdown, Transfection, Real-time Polymerase Chain Reaction, Migration