Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: International Journal of Molecular Medicine

Article Title: Ebastine targets HER2/HER3 signaling and cancer stem cell traits to overcome trastuzumab resistance in HER2-positive breast cancer

doi: 10.3892/ijmm.2026.5751

Figure Lengend Snippet: EBA impairs cancer stem cell-like properties. (A) BT474 and SKBR3 cells were treated with EBA for 48 h, and ALDH1 activity was assessed by flow cytometry using the Aldefluor assay. DEAB was used to define the baseline of Aldefluor-positive fluorescence. (B) BT474 cells (5x10 4 cells/ml) were plated in ultra-low attachment dishes and cultured in the presence or absence of EBA for 5 days. The number and volume of mammospheres were measured by microscopy. (C) Overall survival of patients with breast cancer stratified by the co-expression of ALDH1A1 and CD44. (D) Spearman correlation analysis of ALDH1A1 and CD44 mRNA levels in patients with HER2-positive breast cancer from The Cancer Genome Atlas cohort (n=76). Kaplan-Meier survival analyses of patients with HER2-overexpressing breast cancer stratified by (E) ALDH1A1 and (F) CD44 expression. Patients were divided into high- and low-expression groups based on the median gene expression. Statistical significance was determined using the log-rank test. (G) JIMT-1 cells were treated with EBA (3 μ M) for 48 h and the CD44 high /CD24 low cell populations were identified by flow cytometry. (H) JIMT-1 cells (1.5x10 4 cells/ml) were cultured under serum-free suspension conditions in the presence of EBA (3 μ M) for 8 days. Mammosphere number and volumes were quantified. ** P<0.01 and **** P<0.0001 vs. vehicle-treated control (0 μ M EBA). EBA, ebastine; ALDH, aldehyde dehydrogenase; DEAB, diethylaminobenzaldehyde; CTL, control; ISO, isotype.

Article Snippet: The human breast cancer cell lines SKBR3, BT474, MDA-MB-453 (American Type Culture Collection) and JIMT-1 (Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) were cultured in DMEM, MEM or RPMI-1640 (all Sigma-Aldrich; Merck KGaA) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 U/ml penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO 2 .

Techniques: Activity Assay, Flow Cytometry, Fluorescence, Cell Culture, Microscopy, Expressing, Gene Expression, Suspension, Control

Journal: iScience

Article Title: Characterization of human CD34 + HSPC-derived neutrophils with limited myeloid-derived immunosuppressive cell activity

doi: 10.1016/j.isci.2025.113404

Figure Lengend Snippet: SL-II CD16 + neutrophils retain tumor cell killing capacity but are deficient in immunosuppressive capacity (A) Cartoon of antibody dependent cellular cytotoxicity (ADCC) and myeloid derived suppressor cell (MDSC) activity exerted by mature activated neutrophils (Figure adapted from Aarts et al. ). (B) In vitro ADCC of LAN-1 cells unopsonized and opsonized (+Dinutuximab) ( n = 4) and SKBR3 cells unopsonized and opsonized (+Trastuzumab) by PMNs (blue) ( n = 6) and SL-II CD16 + neutrophils (pink) ( n = 3) in a 1:50 T:E ratio. (C) Representative CFSE plots for CD8 + T cell proliferation of an in vitro MDSC activity assay. T cells were stimulated with anti-CD3/CD28 antibodies to induce proliferation and co-cultured with either unstimulated or TNFα-stimulated PMNs or SL-II CD16 + neutrophils. After 4 days, T cell proliferation was assessed by CFSE dilution ( n = 12 for T cells alone and n = 16 for co-culturing with PMNs or SL-II CD16 + neutrophils). (D) Boxplots of CD8 + T cell proliferation and damaged T cell formation in the in vitro MDSC assay. Unstimulated or TNFα stimulated PMNs (blue) ( n = 12) or SL-II CD16 + neutrophils (pink) ( n = 16) were co-cultured with T cells. After 4 days, FSC/SSC gating was used to assess ‘damaged’ T cell formation next to T cell proliferation. (E) Representative images taken with imaging flow cytometry of trogocytosis carried out by PMNs and SL-II CD16 + neutrophils. The scale bar was set at 10 μm ( n = 3). PMNs and SL-II CD16 + neutrophils were stained with calcein red-orange (orange), and T cells were stained with DiD (red) before co-culturing for 4 h. Data in (B) and (D) is represented as median and interquartile range. p values were calculated using Mann-Whitney U tests and labeled as ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001. n values represent the number of individual donor samples.

Article Snippet: Human Breast Cancer Cell Line SKBR3 , ATCC , .

Techniques: Derivative Assay, Activity Assay, In Vitro, Cell Culture, Imaging, Flow Cytometry, Staining, MANN-WHITNEY, Labeling