human erbb2 her2 protein  (R&D Systems)

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: Scientific Reports

Article Title: Machine learning-driven drug repurposing for HER2-positive breast cancer

doi: 10.1038/s41598-026-45361-z

Figure Lengend Snippet: Mechanism of HER2 in breast cancer.

Article Snippet: Fig. 7 Molecular Dynamics of Timolol maleate complex with wild-type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts, and ( d ) Ligand-Protein Contacts.

Techniques:

Journal: Scientific Reports

Article Title: Machine learning-driven drug repurposing for HER2-positive breast cancer

doi: 10.1038/s41598-026-45361-z

Figure Lengend Snippet: 2D and 3D amino acid interactions of the top 5 selected ligands complexed with the wild HER2 protein (7PCD).

Article Snippet: Fig. 7 Molecular Dynamics of Timolol maleate complex with wild-type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts, and ( d ) Ligand-Protein Contacts.

Techniques:

Journal: Scientific Reports

Article Title: Machine learning-driven drug repurposing for HER2-positive breast cancer

doi: 10.1038/s41598-026-45361-z

Figure Lengend Snippet: 2D and 3D amino acid interactions of the top 5 selected ligands complexed with the mutated HER2 protein (8U8X).

Article Snippet: Fig. 7 Molecular Dynamics of Timolol maleate complex with wild-type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts, and ( d ) Ligand-Protein Contacts.

Techniques:

Journal: Scientific Reports

Article Title: Machine learning-driven drug repurposing for HER2-positive breast cancer

doi: 10.1038/s41598-026-45361-z

Figure Lengend Snippet: Molecular Dynamics of Timolol maleate complex with wild-type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts, and ( d ) Ligand-Protein Contacts.

Article Snippet: Fig. 7 Molecular Dynamics of Timolol maleate complex with wild-type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts, and ( d ) Ligand-Protein Contacts.

Techniques:

Journal: Scientific Reports

Article Title: Machine learning-driven drug repurposing for HER2-positive breast cancer

doi: 10.1038/s41598-026-45361-z

Figure Lengend Snippet: Molecular Dynamics of Timolol maleate complex with mutated type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts & ( d ) Ligand-Protein Contacts.

Article Snippet: Fig. 7 Molecular Dynamics of Timolol maleate complex with wild-type HER2 protein for 1000ns simulation ( a ) RMSD, ( b ) RMSF, ( c ) Protein-Ligand Contacts, and ( d ) Ligand-Protein Contacts.

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: TPP-45142 is a bispecific molecule that binds with a novel epitope of HER2. A, Schematic representation of TPP-45142. Green, two HER2-binding NANOBODY domains; orange, anti-TCRαβ NANOBODY domain; and gray, Fc domain with effectorless function. B, Cryo-EM structure of the complex HER2–29E09–Fab was obtained at 2.78 Å resolution. Left, colored electron density map. Right, full model. C, Cryo-EM structure of the 27A05–HER2–47D05–Fab complex was obtained at 2.66 Å resolution. Left, colored electron density map. Center, full model. Right, 27A05–HER2 interface. D, Structural superposition showing the relative location of pertuzumab and trastuzumab (based on PDB 6OGE) versus 27A05 and 29E09 as observed using cryo-EM. E, Structural superposition of 29E09 and 27A05. [ A, Created in BioRender. Vintem, A.P. (2026) https://BioRender.com/lk4spzo .]

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: Cytotoxicity of TPP-45142 and its mechanism of action toward HER2-low breast cancer cell lines. A–D, TDCC of TPP-45142 for three T-cell donors compared with that of non-HER2 negative control (TPP-45161); co-cultures of human T cells with HCC1954, ZR-75-1, BT-20, or BT-549 cells were used at an E:T ratio of 5:1. E, TDCC of TPP-45142 for three T-cell donors compared with that of TPP-45161 in a co-culture of human T cells with BT20 3D spheroids at an E:T ratio of 1:5. F, T-cell activation induced by TPP-45142 as measured by expression of CD25 and CD69 expression on both CD4 + and CD8 + T cells as per FC analysis of ZR-75-1 and BT20 cells. G, Production of IFN-γ, IL2, IL6, IL8, IL10, and TNF-α cytokines in the culture supernatants obtained in the T-cell activation assay was measured using electrochemiluminescence assays.

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Negative Control, Co-Culture Assay, Activation Assay, Expressing, Electrochemiluminescence

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: PK profiles and antitumor efficacy of TPP-45142 in the ZR-75-1 HER2-low breast cancer mouse model. A, TPP-45142 PK behavior in the ZR-75-1 xenograft model. Human T cells were administered to female NGS mice bearing intramammary ZR-75-1 tumors, and they were treated once with 89 Zr-TPP-45142 or the non-HER2 negative control 89 Zr-TPP-45161 ( n = 3). Microsamples (5 µL/time point) of blood were collected, and radioactivity was measured extemporaneously using a gamma counter (time: after radiolabeled-compound injection). B, Tumor accumulation of 89 Zr-TPP-45142 or non-HER2 negative control 89 Zr-TPP-45161 as measured by PET/CT imaging ( n = 3; time: after radiolabeled-compound injection). C, Antitumor activity of TPP-45142 in the ZR-75-1 xenograft model. Human T cells (10 × 10 6 ) were administered to female NSG mice bearing ZR-75-1 tumors, and they were treated on days 22 and 29 with TPP-45142 (500, 100, 50, and 10 μg/kg) and non-HER2 negative control TPP-45161 (500 μg/kg; n = 10 per group). ID, injected dose; MAD, median absolute deviation.

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Negative Control, Radioactivity, Injection, Positron Emission Tomography-Computed Tomography, Imaging, Activity Assay

Journal: Molecular Cancer Therapeutics

Article Title: TPP-45142—an Anti-HER2 T-cell Engager—Designed for Selective HER2-Low Cancer Immunotherapy

doi: 10.1158/1535-7163.MCT-25-0654

Figure Lengend Snippet: Safety profile of TPP-45142. A, T cell–mediated lysis of human cardiomyocytes was measured by impedance using xCELLigence. For donor 3, nine doses were tested in the range of 3 × 10 −11 to 5 × 10 −7 mol/L, whereas for donors 1 and 2, only the highest three doses were tested. B and C, HER2 distribution on the cell surface of BT-549 and HCM, respectively, via immunofluorescence. Arrowheads point to large HER2-enriched areas. D, Comparison of HER2 distribution between BT-549 ( B ) and HCM ( C ) cells via frequency distribution analysis of objects sorted by area. E, Heatmap of cytokine concentration as measured using the Luminex multiplex array after MIMIC CRA.

Article Snippet: Human, cyno, and mouse HER2 Fc proteins were immobilized on a ProteOn GLC sensor chip (BioRad Laboratories, Inc. cat. #176-5011; 20 μg/mL, 10 mmol/L acetate pH 4.0, 120 seconds, 30 μL/minute).

Techniques: Lysis, Immunofluorescence, Comparison, Concentration Assay, Luminex, Multiplex Assay

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 knockdown impairs in vitro antitumor activity and rewires the metabolism of CAR-T cells. (A) Schematic diagrams of lentiviral vector constructs for Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells. a U6 promoter was inserted downstream of the Her2 CAR sequence to force the shRNA of ATPIF1. (B, C) ATPIF1 protein expression in CAR-T cells analyzed by Western blotting. (D) ATPIF1 mRNA levels in CAR-T cells determined by RT-qPCR. (E) Flow cytometric analysis of CAR-positive cells: CAR-T cells were first stained with biotin-labeled Her2 ectodomain protein, followed by incubation with PE-conjugated anti-biotin antibody for FACS detection of CAR + cells, the Y-axis was the count of cell numbers. (F) In vitro proliferation of CAR-T cells. The Her2 CAR-T, Her2-IF1 CAR-T and Her2-shIF1 CAR-T cells were cultivated with IL-2 stimulation (50 IU/ml) for 12 days, the numbers of CAR + cells were determined every two days. (G-I) Basal oxygen consumption rates (OCR) of resting Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells. Spare Respiratory Capacity (SRC) was calculated and plotted for both resting and activated cell states. (J, M) Cytolytic activity of untransfected T cells (UNT), Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells against SKBR3-Luc + cells at different effector-to-target (E:T) ratios following 24-hour co-culture under 21% O 2 and 1% O 2 conditions, respectively. (K, N) IL-2 concentration in co-culture supernatants from the experiment in (J, M) , respectively. (L, O) IFN-γ concentration in co-culture supernatants from the experiment in (J, M) , respectively. The ELISA method was used to determine the concentration of IL-2 and IFN-γ. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001..

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Knockdown, In Vitro, Activity Assay, Plasmid Preparation, Construct, Sequencing, shRNA, Expressing, Western Blot, Quantitative RT-PCR, Staining, Labeling, Incubation, Co-Culture Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

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 the in vitro antitumor activity and oxygen consumption rate (OCR) of Her2-targeted CAR-T cells. (A) Schematic diagrams of lentiviral vector constructs for Her2 CAR-T and Her2-IF1 CAR-T cells. The ATPIF1 gene is integrated in tandem after the Her2 CAR vector. (B) Flow cytometric identification of GFP-positive cells. (C) Cytolytic activity of untransfected T cells (UNT), Her2 CAR-T, and Her2-IF1 CAR-T cells against SKBR3-Luc + cells at different effector-to-target (E:T) ratios following 24-hour co-culture. Lytic function was evaluated via bioluminescent killing assay in 96-well microplates. Data are presented as mean ± standard deviation (SD) of triplicate wells from three independent experiments. (D, E) Cytokines determination. Levels of IL-2 (D) and IFN-γ (E) in co-culture supernatants, measured by enzyme-linked immunosorbent assay (ELISA). (F–H) OXPHOS determination with the Seahorse equipment. Basal OCR of resting Her2 CAR-T and Her2-IF1 CAR-T cells. Spare Respiratory Capacity (SRC) was calculated and plotted for both resting and activated cell states. **P<0.01; ***P<0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Over Expression, In Vitro, Activity Assay, Plasmid Preparation, Construct, Co-Culture Assay, Standard Deviation, Enzyme-linked Immunosorbent Assay

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 CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

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: Her2-shIF1 CAR-T cells exhibit enhanced in vivo antitumor activity. (A, B) In vivo tumor growth inhibition by UNT, Her2 CAR-T, Her2-IF1 CAR-T, and Her2-shIF1 CAR-T cells. NCG mice (n=5 or 6 in each group) were xenografted with SKBR3 cells. When tumors reached ~100 mm³, mice were intravenously injected with UNT, Her2 CAR-T, Her2-IF1 CAR-T cells (1x10 6 CAR-T cells per mouse). Two weeks later, the mice were euthanized, tumors were excised and weighed. (C) Tumor growth curves of xenografted tumors in each treatment group, the tumor size is calculated with the formula 0.5*a*b 2 , a is the length of tumor and b is the width of tumor. (D) Body weight changes of mice during the experimental period. (E) Spleen weights of mice at the end of the experiment. *P < 0.05; ***P < 0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: In Vivo, Activity Assay, Inhibition, Injection

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 CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

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

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 knockdown induces mPTP opening, mtDNA leakage, and STING signaling pathway activation. (A, B) Flow cytometric detection of mPTP opening using Calcein-AM staining. The Her2 CAR-T, Her2-IF1 CAR-T and Her-shIF1 CAR-T cells were treated with or without N-Acetylcysteine (NAC, 10 mM) for 24 hours, and then stained with Calcein-AM, flow cytometry was used to detect the content of Calcein-AM, which indicated the levels of mPTP opening. (C) Western blot analysis of p-STING, total STING, and VDAC protein expression. (D) Quantification of p-STING relative to total STING. (E) Quantification of VDAC relative to β-actin (loading control). (F–H) RT-qPCR analysis of cytoplasmic mtDNA (ND-1 and D-loop) and mRNA levels of IFI44 and IFN-β. Cytoplasmic RNA was extracted for detection. (I) Generation of ρ 0 CAR-T cells via ethidium bromide (EB) treatment for 2 weeks. Total mtDNA was extracted to detect D-loop levels. (J) RT-qPCR analysis of IFI44 mRNA expression. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: Knockdown, Activation Assay, Staining, Flow Cytometry, Western Blot, Expressing, Control, Quantitative RT-PCR

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: The enhanced in vivo antitumor activity of Her2-shIF1 CAR-T cells is dependent on STING activation. (A, B) Transwell migration assay of CAR-T cells under hypoxic conditions (1% O 2 ) with or without H151 inhibition. CAR-T cells were stained with Cell-Tracker Red CMTPX and seeded into the upper Transwell chamber. Migrated cells in the lower chamber were visualized via fluorescence microscopy, with five random fields per well imaged for quantification. (C, D) Excised tumor size and weight following treatment with different CAR-T cells, either alone or in combination with H151. 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 (1x10 6 CAR-T cells per mouse); or treated with or without H151 via intraperitoneal injection (7 mg/kg) as indicated. Two weeks later, the mice were euthanized, tumors were excised and weighed. **P < 0.01; ***P < 0.001.

Article Snippet: The CAR-T cells cultured under 21% O 2 and 1% O 2 concentration were collected, and were stained with biotin-labeled Her2 protein (Cat: 10004-H02H, SinoBiological, China) following the PE conjugated anti-biotin antibody (Cat: 12-9895-82, eBioscience), and with the addition of TMRE in the MMP assay kit (Cat# C2001S, Beyotime Biotechnology Limited Company, Shanghai, China) or the mPTP assay kit (Cat# C2009S, Beyotime Biotechnology Limited Company, Shanghai, China), the TMRM and mPTP was determined with flow cytometry (BD, FACSCalibur) and the results in CAR + T cells were analyzed with the Flowjo software.

Techniques: In Vivo, Activity Assay, Activation Assay, Transwell Migration Assay, Inhibition, Staining, Fluorescence, Microscopy, Injection