Journal: Frontiers in Oncology

Article Title: Association of B7H3 with HIF-1α nuclear expression indicates poor prognosis and therapeutic potential in gastric cancer

doi: 10.3389/fonc.2026.1744341

Figure Lengend Snippet: (A) Analysis of B7H3 and HIF-1α gene expression levels in gastric cancer (GC) and adjacent noncancerous tissues. (B) Analysis of the correlation between B7H3 and HIF-1α in gastric adenocarcinoma using the GEPIA database. (C) Analysis of the correlation between B7H3 and HIF-1α in adjacent noncancerous tissues using the GEPIA database. (D) Survival curve of patients with gastric cancer based on B7H3 expression. (E) Survival curve of patients with gastric cancer based on HIF-1α expression. (F) Survival curve of patients with gastric cancer based on combined B7H3 and HIF-1α expression. The red line indicates the high expression group, the blue line indicates the low expression group, and the yellow line indicates the other groups. **** indicates P < 0.001.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with mouse anti-human B7H3 antibody (R&D Systems, MN, USA, #AF1027, 1:200) or rabbit anti-human HIF-1α antibody(R&D Systems, MN, USA, #MAB19352,Clone #2443C,1:200).

Techniques: Gene Expression, Expressing

Journal: Frontiers in Oncology

Article Title: Association of B7H3 with HIF-1α nuclear expression indicates poor prognosis and therapeutic potential in gastric cancer

doi: 10.3389/fonc.2026.1744341

Figure Lengend Snippet: (A) Representative immunohistochemical (IHC) images of B7H3 and HIF-1α expression in gastric cancer tissues from 268 clinical patients with gastric cancer. (B) Representative IHC images of B7H3 and HIF-1α nuclear expression in gastric cancer tissues from 268 clinical patients with gastric cancer. Scale bars: 100 μm; 25 μm. (C) resentative multiplex fluorescence immunohistochemistry (mIHC) images of high B7H3 expression and HIF-1α nuclear expression in gastric cancer tissues. Scale bar: 100 μm.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with mouse anti-human B7H3 antibody (R&D Systems, MN, USA, #AF1027, 1:200) or rabbit anti-human HIF-1α antibody(R&D Systems, MN, USA, #MAB19352,Clone #2443C,1:200).

Techniques: Immunohistochemical staining, Expressing, Multiplex Assay, Fluorescence, Immunohistochemistry

Journal: Frontiers in Oncology

Article Title: Association of B7H3 with HIF-1α nuclear expression indicates poor prognosis and therapeutic potential in gastric cancer

doi: 10.3389/fonc.2026.1744341

Figure Lengend Snippet: (A) Survival curve of gastric cancer patients based on B7H3 expression levels. (B) Survival curve of patients with gastric cancer based on HIF-1α expression levels. (C) Survival curve of patients with gastric cancer based on whether HIF-1α was expressed in the nucleus. (D) Survival curve of patients with gastric cancer based on the co-expression of B7H3 and HIF-1α. (E) Survival curve of patients with gastric cancer based on the co-expression of B7H3 and nuclear HIF-1α.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with mouse anti-human B7H3 antibody (R&D Systems, MN, USA, #AF1027, 1:200) or rabbit anti-human HIF-1α antibody(R&D Systems, MN, USA, #MAB19352,Clone #2443C,1:200).

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: Association of B7H3 with HIF-1α nuclear expression indicates poor prognosis and therapeutic potential in gastric cancer

doi: 10.3389/fonc.2026.1744341

Figure Lengend Snippet: (A) HIF-1α protein expression in HGC-27 cells under gradient concentrations of cobalt chloride induction. (B) B7H3 protein expression in HGC-27 cells under gradient concentrations of cobalt chloride (40 μmol/L, 200 μmol/L, 1000 μmol/L) induction. (C) HIF-1α protein expression in HGC-27 cells with overexpressed or knocked-down B7H3 under hypoxic and normal conditions. (D) HIF-1α expression in nuclear and cytoplasmic proteins of HGC-27 cells with overexpressed or knocked-down B7H3 under cobalt chloride induction. (E) Fluorescence immunohistochemistry of HGC-27 cells with B7H3 knockdown (SHB),B7H3 overexpression (LVB) and control group (SHN/LVN). Blue fluorescence represents DAPI, and red fluorescence represents HIF-1α. Scale bar: 10 μm.

Article Snippet: Subsequently, the sections were incubated overnight at 4°C with mouse anti-human B7H3 antibody (R&D Systems, MN, USA, #AF1027, 1:200) or rabbit anti-human HIF-1α antibody(R&D Systems, MN, USA, #MAB19352,Clone #2443C,1:200).

Techniques: Expressing, Fluorescence, Immunohistochemistry, Knockdown, Over Expression, Control

Journal: Nature Communications

Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity

doi: 10.1038/s41467-025-58740-3

Figure Lengend Snippet: a Schematic diagram of human B7H3-NQ mutant constructs. WT B7H3 contains four pairs of Asn N-glycosylation sites at amino-acid positions 91 (N91) / 309 (N309), 104 (N104) / 322 (N322), 189 (N189) / 407 (N407) and 215 (N215) / 433 (N433) in the nearly exact tandem duplication of IgV-IgC domain. Potential asparagine N-glycosylation sites (N) were mutated to glutamine (Q). b HEK293T-B7H3KO cells were transiently re-expressed with the same amount of plasmids as indicated. Flow cytometry measuring B7H3 protein on the cell membrane in the indicated cell lines. Left, representative images of membrane B7H3. Right, median Fluorescence Intensity (MFI) of B7H3 ( n = 3 biological independent samples). c MDA-MB-231-B7H3KO cells and A549-B7H3KO were stably rescued with human B7H3-WT and B7H3-NQ mutant cDNA individually. Flow cytometry measuring B7H3 protein on the cell membrane in the indicated cells. d Schematic diagram of mouse B7H3-NQ mutant constructs. WT B7H3 contains four Asn N-glycosylation sites at amino-acid positions 91 (N91), 104 (N104), 189 (N189) and 215 (N215) in the single IgV-IgC domain. Potential asparagine N-glycosylation sites (N) were mutated to glutamine (Q). e 4T1-B7H3KO cells, B16-B7H3KO cells and E0771-B7H3KO cells were stably rescued with mouse B7H3-WT and B7H3-NQ mutant cDNA individually. Flow cytometry measuring B7H3 protein on the cell membrane in the indicated cell lines. f The indicated cell lines were treated with MG132 in the presence of CHX at indicated intervals. The intensity of B7H3 protein was quantified using ImageJ software. g The indicated cell lines were treated with MG132 for 6 h. Immunoprecipitation analysis of Flag-tagged B7H3 ubiquitination with the indicated antibodies. The p -value in ( b , f ) was determined by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean ± SD. Data in ( b – g ) are representative of three independent experiments.

Article Snippet: In addition, compared with the commercial anti-B7H3 antibody (Cat. 17-2769-42, eBioscience), Ab-82 preferentially recognized WT and N91/302/104/322 B7H3 rather than 8NQ and N91/302/104/322Q B7H3 (Fig. ).

Techniques: Mutagenesis, Construct, Glycoproteomics, Flow Cytometry, Membrane, Fluorescence, Stable Transfection, Software, Immunoprecipitation, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity

doi: 10.1038/s41467-025-58740-3

Figure Lengend Snippet: a Confocal microscopy of MDA-MB-231-B7H3KO and A549-B7H3KO cells reconstituted with human B7H3-WT and B7H3-NQ mutant cDNA. Green, Actin; Red, B7H3. Scar bar, 50 µm. b Confocal microscopy of MDA-MB-231-B7H3KO and A549-B7H3KO cells co-expressed with human mNegoGreen-B7H3-WT cDNA or mNegoGreen-B7H3-NQ mutant cDNA, and KDEL-mRuby cDNA. BF, bright field; Green, B7H3; Red, ER marker (KDEL). Scale bars, 20 μm. c Immuno- fluorescence staining with antibodies against B7H3(red) and ERGIC (green). Scale bars,100 μm. d Immunofluorescence staining with antibodies against B7H3 (red) and TGN38 (green) in the indicated cell lines. Scale bars,100μm. DAPI and Hoechst: nuclear counterstaining. Data are representative of three independent experiments.

Article Snippet: In addition, compared with the commercial anti-B7H3 antibody (Cat. 17-2769-42, eBioscience), Ab-82 preferentially recognized WT and N91/302/104/322 B7H3 rather than 8NQ and N91/302/104/322Q B7H3 (Fig. ).

Techniques: Confocal Microscopy, Mutagenesis, Marker, Fluorescence, Staining, Immunofluorescence

Journal: Nature Communications

Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity

doi: 10.1038/s41467-025-58740-3

Figure Lengend Snippet: a IP-MS analysis showing candidates with increased binding to 8NQ or N91/104/309/322Q B7H3 compared to WT or N91/104/309/322Q B7H3. b The indicated cell lines were treated with MG132, NMS-873 (2 μM) or CB-5083 (5 μM) in the presence of CHX at indicated intervals. c The indicated cell lines were treated with NMS-873 or CB-5083. Immunoprecipitation analysis of Flag-tagged B7H3 ubiquitination with the indicated antibodies. d The indicated cell lines were treated with MG132 for 6 h. Flag-tagged B7H3 WT or its NQ mutants were pulled down by anti-Flag beads in the indicated cell lines, followed by western blotting to detect HRD1 and SEL1L. e MDA-MB-231-B7H3KO-N91/104/309/322Q cells were transiently transfected with HRD1 siRNA or SEL1L siRNA for 48 h. Then the cells were treated with CHX at indicated intervals. The intensity of B7H3 protein was quantified using ImageJ software. f MDA-MB-231-B7H3KO-N91/104/309/322Q cells expressing sgRNAs targeting HRD1 or SEL1L were treated with CHX at indicated intervals. The intensity of B7H3 protein was quantified using ImageJ software. g MDA-MB-231-B7H3KO-N91/104/309/322Q cells expressing sgRNAs targeting HRD1 or SEL1L were treated with MG132 for 6 h. Immunoprecipitation analysis of Flag-tagged B7H3 ubiquitination with the indicated antibodies. The p -value in ( e , f ) was determined by a two-tailed unpaired Student’s t -test. Error bars represent mean ± SD. Data in ( b – g ) are representative of three independent experiments.

Article Snippet: In addition, compared with the commercial anti-B7H3 antibody (Cat. 17-2769-42, eBioscience), Ab-82 preferentially recognized WT and N91/302/104/322 B7H3 rather than 8NQ and N91/302/104/322Q B7H3 (Fig. ).

Techniques: Protein-Protein interactions, Binding Assay, Immunoprecipitation, Ubiquitin Proteomics, Western Blot, Transfection, Software, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity

doi: 10.1038/s41467-025-58740-3

Figure Lengend Snippet: a Tumor growth of the indicated 4T1-B7H3KO cells in female 8 week-old BALB/c mice ( n = 7 mice per group). Tumor volumes (left) and tumor weights upon autopsy on day 24 (right) were calculated. Mice were injected with CD8 or CD4 depletion antibody on days -7, -3, 0, +3, +7, and +10 relative to tumor challenge on day 0. b – d FACS analysis of CD4 + T and CD8 + T cells ( b ), IFN-γ + and TNF + in CD8 + T cells ( c ), IFN-γ + and TNF + in CD4 + T cells ( d ) from the isolated TILs in ( a ) (right, n = 7 mice per group). Representative dot plots from a representative mouse for each group (left). e Tumor growth of the indicated E0771-B7H3KO cells in female 8 week-old C57BL/6 mice ( n = 5 mice per group). Tumor volumes (left) and tumor weights upon autopsy on day 42 (right) were calculated. f - g FACS analysis of IFN-γ + and TNF + in CD8 + T cells from the isolated TILs in ( e ) (right, n = 5 mice per group). Representative dot plots from a representative mouse for each group (left). h The correlation between the expression of B7H3 and the normalized tumor infiltrated CD8 + T cells for TCGA cancer patients was analyzed using the R package “ESTIMATE”. NS, not significance. The p -value in ( a – g ) was determined by a two-tailed unpaired Student’s t -test. Error bars represent mean ± SD. The p- value in ( h ) was assessed using the two-sided Pearson correlation test. Data in ( a – g ) are representative of two independent experiments.

Article Snippet: In addition, compared with the commercial anti-B7H3 antibody (Cat. 17-2769-42, eBioscience), Ab-82 preferentially recognized WT and N91/302/104/322 B7H3 rather than 8NQ and N91/302/104/322Q B7H3 (Fig. ).

Techniques: Injection, Isolation, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity

doi: 10.1038/s41467-025-58740-3

Figure Lengend Snippet: a Working flow chart for generation of glycosylated B7H3 antibody. Created in BioRender. Wq, Z. (2025) https://BioRender.com/p93p347 . b Flow cytometry analysis by the different concentrations of Ab-82 to detect the B7H3 expression on the cell membrane in the indicated tumor cell lines ( n = 3 biological independent samples). c Flow cytometry analysis by the different concentrations of Ab-82 to detect the B7H3 expression on the cell membrane in the N91/104/309/322 B7H3 reconstituted tumor cells ( n = 3 biological independent samples). d Flow cytometry analysis by Ab-82 to detect the B7H3 expression on the cell membrane in the N91/104/309/322Q B7H3 reconstituted tumor cells with or without HRD1 deletion ( n = 3 biological independent samples). e Binding affinity (K D ) analysis of Ab-82 by Biacore T200. f Human B7H3-His fusion protein was de-glycosylated by PNGase F and used in Dot blot analysis by Ab-82. g , h Flag-tagged B7H3 WT or its NQ mutants were pulled down by anti-Flag beads, followed by western blotting to detect the binding ability of Ab-82 to them. i Flow cytometry analysis by anti-B7H3 and Ab-82 at the same concentration to detect the B7H3 expression on the cell membrane in the indicated cell lines ( n = 3 biological independent samples). NS, not significance. The p -value in ( b , c ) was determined by one-way ANOVA with Dunnett’s multiple comparisons test. The p -value in ( d , i ) was determined by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean ± SD. Data in b-i are representative of three independent experiments.

Article Snippet: In addition, compared with the commercial anti-B7H3 antibody (Cat. 17-2769-42, eBioscience), Ab-82 preferentially recognized WT and N91/302/104/322 B7H3 rather than 8NQ and N91/302/104/322Q B7H3 (Fig. ).

Techniques: Flow Cytometry, Expressing, Membrane, Binding Assay, Dot Blot, Western Blot, Concentration Assay

Journal: Nature Communications

Article Title: Targeting site-specific N-glycosylated B7H3 induces potent antitumor immunity

doi: 10.1038/s41467-025-58740-3

Figure Lengend Snippet: a Tumor growth of 4T1-hB7H3 cells in female 8 week-old BALB/c mice following treatment with Ab-82 antibody ( n = 9 mice per group). The treatment protocol is summarized by the arrows. Tumor volume (left) and tumor weight upon autopsy on day 23 (right) were calculated. b , c Quantitative IHC analysis of mouse CD4, mouse CD8, mouse GZMB expression ( b ), and human B7H3 expression ( c ) ( n = 9 mice per group). d Working flow chart for humanization of Ab-82 by using genetic engineering technology. e Binding affinity (K D ) analysis of Hu-Ab-82 by Biacore T200. f Tumor growth of A549 cells in female 7 week-old huPBMC-NOG-MHC I/II-2 KO Mice following treatment with Hu-Ab-82 antibody ( n = 8 tumors per group, 4 Mice bearing established tumors on both flanks). The treatment protocol is summarized by the arrows. Tumor volume (left) and tumor weight upon autopsy on day 26 (right) were calculated. g , h Quantitative IHC analysis of human CD8, human GZMB expression ( g ), and human B7H3 expression ( h ) ( n = 8 tumors per group, 4 Mice bearing established tumors on both flanks). i The indicated cells were treated by Ab-82 at different concentrations for 48 h, followed by western blotting to detect the total B7H3 protein level with anti-human B7H3. j Cell surface B7H3 was bound by Ab-82 for 30 min or not, followed by flow cytometry with commercial anti-B7H3 (Cat. 17-2769-42, eBioscience) ( n = 3 biological independent samples). k A549 cells were treated by Ab-82 at different concentrations for 24 h, followed by flow cytometry with commercial anti-B7H3 (Cat. 17-2769-42, eBioscience) ( n = 3 biological independent samples). l Cell surface B7H3 was labeled by Ab-82 and internalized for 5 min, followed by flow cytometry to measure B7H3 level ( n = 3 biological independent samples). m Cell surface B7H3 was labeled by Ab-82 and internalized at different time points in the presence of 50uM primaquine, followed by flow cytometry to measure B7H3 level ( n = 3 biological independent samples). n Cells were treated by 20 μg/ml pHrodo red-labeled Ab-82 for 24 h, followed by immunofluorescence analysis with LSM880.BF, bright field. o Cells were treated by 20 μg/ml pHrodo red-labeled Ab-82 for different time, followed by flow cytometry to measure Ab-82 internalization level ( n = 3 biological independent samples). p The indicated cells were treated by 20 μg/ml pHrodo red-labeled Ab-82 for different time, followed by flow cytometry to measure Ab-82 internalization level ( n = 3 biological independent samples). NS, not significance. The p -value in ( a – c ) and ( f – h ) and ( o ) was determined by one-way ANOVA with Dunnett’s multiple comparisons test. The p -value in ( p ) was determined by one-way ANOVA with Tukey’s multiple comparisons test. Error bars represent mean ± SD. The data in ( a – h ) are representative of two independent experiments. The data in ( i – p ) are representative of three independent experiments.

Article Snippet: In addition, compared with the commercial anti-B7H3 antibody (Cat. 17-2769-42, eBioscience), Ab-82 preferentially recognized WT and N91/302/104/322 B7H3 rather than 8NQ and N91/302/104/322Q B7H3 (Fig. ).

Techniques: Expressing, Binding Assay, Western Blot, Flow Cytometry, Labeling, Immunofluorescence