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