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: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: ATPIF1 overexpression enhances CAR-T cell persistence but impairs in vivo antitumor activity. (A, B) Flow cytometric quantification of GFP + Her2 CAR-T and Her2-IF1 CAR-T cells in mouse peripheral blood mononuclear cells (PBMCs) and spleens. NCG mice (n=4 in each group) were intravenously injected with Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Three weeks later, the mice were euthanized, the peripheral blood and spleen were collected, respectively. The flow cytometry was used to detect the percentage of GFP + CAR-T cells. (C) Tumor growth inhibition by Her2 CAR-T and Her2-IF1 CAR-T cells in SKBR-3 xenografted NCG mice. NCG mice were xenografted with SKBR3 cells (n=5 or 6 in each group). When tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Three weeks later, the mice were euthanized, tumors were excised and weighed. *P < 0.05; **P < 0.01. (D, F) The memory phenotype of Her2 CAR-T and Her2-IF1 CAR-T cells under 21% O 2 and 1% O 2 after stimulated with the conditional medium of SKBR-3 cells. (E, G) The exhaustion markers of PD-1, LAG-3 and TIM-3 determination in Her2 CAR-T and Her2-IF1 CAR-T cells under 21% O 2 and 1% O 2 after stimulated with the conditional medium of SKBR-3 cells. *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: The human breast cancer cell line SKBR-3 (HTB-30, ATCC) was cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S; Sigma-Aldrich, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Over Expression, In Vivo, Activity Assay, Injection, Flow Cytometry, Inhibition

Journal: Frontiers in Immunology

Article Title: The critical role of Atpif1 in Her2-targeted CAR-T cell therapy for solid tumor via modulation of metabolism and mtDNA-STING signal pathway

doi: 10.3389/fimmu.2026.1733753

Figure Lengend Snippet: Immunofluorescence and multiplex immunohistochemistry analyses. (A, B) Enhanced CD3 + staining in group of Her2-shIF1 CAR-T in SKBR-3 xenografted tumor tissues. NCG mice were xenografted with SKBR3 cells (n=5 in each group); when tumors reached ~100 mm³, mice were intravenously injected with untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T, or Her2-shIF1 CAR-T cells. Four days later, Mice were euthanized, and tumors were excised for immunofluorescence staining. (C) Multiplex immunohistochemistry staining of 18 formalin-fixed paraffin-embedded (FFPE) human breast cancer tissue sections, showing ATPIF1 (yellow), CD3 (green), HIF-1α (orange), and DAPI (blue). (D, E) Quantitative analysis of the correlation between CD3 + staining and ATPIF1 + cells (D) , and between HIF-1α + and ATPIF1 + cells (E) from the results of Multiplex immunohistochemistry staining in (C) respectively. **P<0.01, ***P<0.001.

Article Snippet: The human breast cancer cell line SKBR-3 (HTB-30, ATCC) was cultured in RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (P/S; Sigma-Aldrich, USA) at 37 °C in a humidified atmosphere containing 5% CO 2 .

Techniques: Immunofluorescence, Multiplex Assay, Immunohistochemistry, Staining, Injection, Formalin-fixed Paraffin-Embedded