Journal: Frontiers in Immunology

Article Title: Robust CD8 + T cell responses induced by an mRNA-LNP vaccine encoding rat HER2 extracellular domain confer prophylactic tumor protection

doi: 10.3389/fimmu.2026.1737558

Figure Lengend Snippet: mRNA-LNP vaccine development via integrated mechanism, preparation, and characterization. (A) Schematic of the mRNA-LNP vaccine mechanism. Intramuscularly injected mRNA-LNPs are delivered to DCs. The encoded antigen is expressed and processed through two major pathways: the immunoproteasome/MHC class I pathway activates CD8 + T cells, while the endosomal/MHC class II pathway activates CD4 + T cells. Activated CD4 + T cells provide help to B cells for antibody production, orchestrating a coordinated adaptive immune response. (B) Schematic microfluidic process of mRNA-LNPs preparation. (C) Plasmid map of rat HER2 ECD and rat HER2 ECD-IFNγ. (D) Agarose gel electrophoresis of plasmid and linearized plasmid DNA. (E) Agarose gel electrophoresis of transcribed mRNA and total mRNA for rat HER2 ECD and rat HER2 ECD-IFNγ. M, 1kb ladder; 1, Plasmids with rat HER2 ECD sequence and 2, rat HER2 ECD-IFNγ sequence; 3, Linearized plasmid DNA with rat HER2 ECD sequence and 4, rat HER2 ECD-IFNγ sequence; 5, Agarose gel electrophoresis images of transcribed mRNA and 6, total mRNA of rat HER2 ECD; 7, Agarose gel electrophoresis images of transcribed mRNA and 8, total mRNA of rat HER2 ECD-IFNγ.

Article Snippet: Splenocytes (2 × 10 5 cells/well) were stimulated in complete RPMI 1640 medium with or without 2 μg/mL rat HER2 protein (#80079-R08H, Sino Biological) for 12 hours at 37 °C.

Techniques: Bioprocessing, Injection, Plasmid Preparation, Agarose Gel Electrophoresis, Sequencing

Journal: Frontiers in Immunology

Article Title: Robust CD8 + T cell responses induced by an mRNA-LNP vaccine encoding rat HER2 extracellular domain confer prophylactic tumor protection

doi: 10.3389/fimmu.2026.1737558

Figure Lengend Snippet: Therapeutic efficacy of HER2 ECD mRNA-LNP vaccines in 4T1-HER2 tumor-bearing mice. (A) Treatment schedules and dosing intervals of the 4T1-HER2 tumor model. (B) Serum anti-HER2 antibody kinetics after 5 μg or 10 μg rHER2 ECD mRNA-LNP vaccination. (C) Changes in serum HER2 antibody concentration over time after administration after rHER2 ECD mRNA-LNP or rHER2 ECD-IFNγ mRNA-LNP vaccination. (D) Growth curve of 4T1-HER2 tumors. (E) Representative images of tumor volume. N = 3. Data are presented as mean ± standard error of the mean (SEM); two-sided t-test. (* P < 0.05 and *** P < 0.001).

Article Snippet: Splenocytes (2 × 10 5 cells/well) were stimulated in complete RPMI 1640 medium with or without 2 μg/mL rat HER2 protein (#80079-R08H, Sino Biological) for 12 hours at 37 °C.

Techniques: Drug discovery, Vaccines, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Robust CD8 + T cell responses induced by an mRNA-LNP vaccine encoding rat HER2 extracellular domain confer prophylactic tumor protection

doi: 10.3389/fimmu.2026.1737558

Figure Lengend Snippet: Safety profiles of HER2 ECD mRNA-LNP vaccines in 4T1-HER2 tumor-bearing mice. (A) Body, heart, liver and lung weights of mice. (B) Hepatic function, renal function and cardiac function on day 68. N = 3. Data are presented as mean ± SEM; two-sided t-test. No significant intergroup differences were observed.

Article Snippet: Splenocytes (2 × 10 5 cells/well) were stimulated in complete RPMI 1640 medium with or without 2 μg/mL rat HER2 protein (#80079-R08H, Sino Biological) for 12 hours at 37 °C.

Techniques: Vaccines

Journal: Frontiers in Immunology

Article Title: Robust CD8 + T cell responses induced by an mRNA-LNP vaccine encoding rat HER2 extracellular domain confer prophylactic tumor protection

doi: 10.3389/fimmu.2026.1737558

Figure Lengend Snippet: Memory T cell subsets and antitumor activity. (A) Representative flow cytometry plots of T CM cells (CD44 + CD62L + ), T EM cells (CD44 + ) and naive cells (CD44 - CD62L + ) within CD4 + and (B) CD8 + T cells. (C) Rate of inhibition of 4T1-HER2 cells by vaccinated mouse splenic lymphocytes. (D) Concentration of IFNγ antibody in serum after administration. N = 3. Data are presented as mean ± SEM; two-sided t-test. (* P < 0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: Splenocytes (2 × 10 5 cells/well) were stimulated in complete RPMI 1640 medium with or without 2 μg/mL rat HER2 protein (#80079-R08H, Sino Biological) for 12 hours at 37 °C.

Techniques: Activity Assay, Flow Cytometry, Inhibition, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Robust CD8 + T cell responses induced by an mRNA-LNP vaccine encoding rat HER2 extracellular domain confer prophylactic tumor protection

doi: 10.3389/fimmu.2026.1737558

Figure Lengend Snippet: Antitumor efficacy of combination therapy. (A) Treatment schedules and dosing intervals of the 4T1-HER2 tumor model. (B) Changes in serum HER2 antibody concentration over time after administration after rHER2 ECD mRNA-LNP vaccination, with and without anti-PD-1. (C) Growth curve of 4T1-HER2 tumors. (D) Representative images of tumor tissue. (E) Tumor volume. N = 6;Data are presented as mean ± SEM. Two-sided t-test. (* P < 0.05 and *** P < 0.001).

Article Snippet: Splenocytes (2 × 10 5 cells/well) were stimulated in complete RPMI 1640 medium with or without 2 μg/mL rat HER2 protein (#80079-R08H, Sino Biological) for 12 hours at 37 °C.

Techniques: Concentration Assay

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 downregulates HER2, p95HER2, HER3 and AKT expression. (A) Immunoblot analysis of HER2, p95HER2 and p-HER2 (Y1221/1222) in JIMT-1 cells treated with EBA for 48 h. (B) Immunoblot analysis of HER3, p-HER3 (Y1289), AKT and p-AKT following treatment with EBA (48 h) in JIMT-1 cells. (C) Immunoblot analysis of HER2, HER3 and EGFR following IP with anti-HER2 antibody in JIMT-1 cells treated with EBA. In silico molecular docking of EBA with the crystal structure of HER2-KD. (D) Surface map of lipophilic and hydrophilic properties at the ATP-binding site of HER2-KD (red, hydrophobic; blue, hydrophilic). (E) 2D interaction diagram showing intermolecular interactions between EBA and HER2-KD. Key amino acid residues within the binding pocket are shown. (F) Predicted binding pose of EBA (purple stick model) within the tyrosine kinase domain of HER2 (blue ribbon). (G) 293T cells were treated with DMSO or EBA for 1 h at 37°C, followed by heating for 3 min. Soluble fractions were collected following centrifugation and analyzed by immunoblotting using an anti-HER2 antibody. EBA, ebastine; p-, phosphorylated; IP, immunoprecipitation; IB, immunoblotting; KD, kinase domain PCB, protein complex binding; TM, transmembrane; a.a., amino acid.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Expressing, Western Blot, In Silico, Binding Assay, Centrifugation, Immunoprecipitation

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: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

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 downregulates HER2, ICD-HER2, HER3, ALDH1A1, CD44 and vimentin in JIMT-1 xenograft tumors. Immunofluorescence staining of JIMT-1 xenograft tumor tissue for (A) full-length HER2 (green), (B) ICD-HER2 (green) and (C) HER3. Immunohistochemical analysis of (D) ALDH1A1 (green) and (E) CD44 (red) in tumor tissue. (F) Tumor sections were immunostained for vimentin (red). Magnification, x500. Fluorescence intensities were quantified. Serum biochemical analysis for (G) liver and (H) kidney function in EBA-treated or CTL mice (n=5). Serum levels of ALT, AST, TBL, BUN and creatinine were assessed. *** P<0.001, **** P<0.0001. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; EBA, ebastine; ICD, intracellular domain; ALDH, aldehyde dehydrogenase; CTL, control; NS, not significant.

Article Snippet: Primary antibodies were as follows: Ki-67 (cat. no. ab16667), CD31 (cat. no. ab28364), ALDH1A1 (Abcam; cat. no. ab52492), Bcl-2 (Abcam; cat. no. ab692) and CD44 (all Abcam; cat. no. ab254530); HER2 (Cell Signaling Technology, Inc.; cat. no. 2165), HER3 (Cell Signaling Technology, Inc.; cat. no. 12708), phosphorylated (p-)HER2 (Y1221/1222; Cell Signaling Technology, Inc.; cat. no. 2243), p-HER3 (Y1289; Cell Signaling Technology, Inc.; cat. no. 2842), Akt (Cell Signaling Technology, Inc.; cat. no. 9272), p-Akt (S473; Cell Signaling Technology, Inc.; cat. no. 4060), PARP (Cell Signaling Technology, Inc.; cat. no. 9542), cleaved PARP (Cell Signaling Technology, Inc.; cat. no. 5625), caspase-3 (Cell Signaling Technology, Inc.; cat. no. 7148), -7 (Cell Signaling Technology, Inc.; cat. no. 12827) and -8 (Cell Signaling Technology, Inc.; cat. no. 4790), cleaved caspase-3 (Cell Signaling Technology, Inc.; cat. no. 9664), -7 (Cell Signaling Technology, Inc.; cat. no. 8438) and -8 (Cell Signaling Technology, Inc.; cat. no. 9496), Bax (Cell Signaling Technology, Inc.; cat. no. 2772) and vimentin (Cell Signaling Technology, Inc.; cat. no. 5741); anti-intracellular domain (ICD) HER2 clone 4B5 (Ventana Medical Systems; cat. no. 790-4493) and GAPDH (Invitrogen; Thermo Fisher Scientific, Inc.; cat. no. MA5-15738).

Techniques: Immunofluorescence, Staining, Immunohistochemical staining, Fluorescence, Control