Journal: Journal of Cellular and Molecular Medicine

Article Title: NR1D1 ‐transactivated lncRNA NUTM2A‐AS1 promotes chemoresistance and immune evasion in neuroblastoma via inhibiting B7‐H3 degradation

doi: 10.1111/jcmm.18360

Figure Lengend Snippet: NUTM2A‐AS1 is associated with B7‐H3 and inhibits B7‐H3 ubiquitination level. (A,B) The graphs depict the protein scores of the 11 proteins identified through RNA pull‐down and LC–MS analysis. (C,D) The graphs illustrate the exponentially modified protein abundance index (emPAI) of the 11 proteins identified by RNA pull‐down assays and LC–MS. (E) B7‐H3 was specifically pulled down by biotin‐labelled sense NUTM2A‐AS1 (S) but not NUTM2A‐AS1 anti‐sense (AS) RNA in the indicated cells. (F) RNA immunoprecipitation (RIP) assays were conducted using anti‐B7‐H3 antibodies with extracts from neuroblastoma (NB) cells. Relative enrichment represents the RNA levels associated with B7‐H3 relative to an input control, comparing anti‐B7‐H3 antibody immunoprecipitation with IgG antibody. GAPDH mRNA served as the negative control. (G) B7‐H3 mRNA expression in NUTM2A‐AS1 knockdown cells was assessed by qRT‐PCR assay. (H,I) B7‐H3 protein expression in NUTM2A‐AS1 knockdown cells was examined by western blot assay. (J) NB cells with NUTM2A‐AS1 knockdown and control cells were treated with or without MG132 (5 μM) for 12 h, and cell lysates were analysed by western blotting. (K) NB cells with or without siRNAs specific for NUTM2A‐AS1 were treated with 20 μg/mL cycloheximide (CHX) or a vehicle for the indicated periods, and B7‐H3 levels were analysed by western blotting. (L) Western blot analysis of the ubiquitination of B7‐H3 in NUTM2A‐AS1 knockdown SK‐N‐SH cells. ***p < 0.001.

Article Snippet: Antibodies for western blot analysis are as follows: B7‐H3 (R&D Systems, #AF1027, 1:250) and GAPDH (Abcam, #EPR16891, 1:10000).

Techniques: Ubiquitin Proteomics, Liquid Chromatography with Mass Spectroscopy, Modification, Quantitative Proteomics, RNA Immunoprecipitation, Control, Immunoprecipitation, Negative Control, Expressing, Knockdown, Quantitative RT-PCR, Western Blot

Journal: Journal for Immunotherapy of Cancer

Article Title: Multiomic analysis of colorectal adenocarcinoma reveals a new subtype of myofibroblastic cancer-associated fibroblasts that express high levels of B7-H3 and have poor-prognosis value

doi: 10.1136/jitc-2025-013356

Figure Lengend Snippet: Expression and prognostic factor of B7-H3 expression in CRC samples. ( A ) Box plot representing B7-H3 expression in normal colon tissue compared with primary CRC tumor in bulk primary CRC dataset. Significance between groups was defined by Student’s t-test. ( B ) Box plot representing B7-H3 expression in CRCassigner subclassification of CRC samples in bulk primary CRC dataset. Red frame highlights the CRCassigner subclass the most enriched in B7-H3 expression, whereas the green one highlights the CRCassigner subclass the less enriched in B7-H3 expression. Significance was defined using an ANOVA Tukey Honestly Significant Difference (HSD) test. ( C ) Box plot representing B7-H3 expression in CMS subclassification of CRC samples in bulk primary CRC dataset. Red frame highlights the CMS subclass the most enriched in B7-H3 expression, whereas the green one highlights the CMS subclass the least enriched in B7-H3 expression. Significance was obtained using an ANOVA Tukey HSD test. ( D ) Kaplan-Meier survival curve representing the disease-free survival of B7-H3 high (red) group and B7-H3 low (black) group in regard to time (months after diagnosis). Significance was defined using a log-rank test. ( E ) Kaplan-Meier survival curve representing the disease-free survival according to CMS subclasses (CMS1 in yellow, CMS2 in blue, CMS3 in pink and CMS4 in green) segregated between B7-H3 high (solid line) and B7-H3 low (dash line), in regard to time (months after diagnosis). Significance was defined using a log-rank test. ANOVA, analysis of variance; CMS, consensus molecular subtype; CRC, colorectal cancer; DFS, disease-free survival.

Article Snippet: The tissues were permeabilized with 0.5% Triton X-100 (Euromedex, France) in 1X PBS for 15 min and saturated with 5% normal horse serum in 1X PBS for 30 min. An antibody panel targeting B7-H3 (catalog number #66481-1-Ig, clone 1E7D1, uncoupled, diluted to 1:25, Proteintech) and the secondary antibody anti-mouse Cy3 (Jackson ImmunoResearch) in tumor cells (pankeratin, catalog number #53-9003-82, clone AE1/AE3, primary coupled with Alexa Fluor 488, diluted to 1:50, eBioscience) and in CAFs (vimentin, catalog number #130-127-018, clone REAL1008, primary coupled with APC, diluted to 1:50, Miltenyi) was constructed.

Techniques: Expressing, Biomarker Discovery

Journal: Journal for Immunotherapy of Cancer

Article Title: Multiomic analysis of colorectal adenocarcinoma reveals a new subtype of myofibroblastic cancer-associated fibroblasts that express high levels of B7-H3 and have poor-prognosis value

doi: 10.1136/jitc-2025-013356

Figure Lengend Snippet: Enrichment of tumor microenvironment features in regard to B7-H3 level of expression. ( A ) Forest plot representing the enrichment of immunome (Bindea et al ) between B7-H3 high and B7-H3 low groups. P value, Odd ratio (OR) with confidence interval (CI) were represented in an adjacent table. Significant enrichments were represented in blue, darkest being the most significant. ( B ) Forest plot representing the enrichment of cancer-associated signatures (CAF) between B7-H3 high and B7-H3 low groups. P value, OR with CI were represented in an adjacent table. Significant enrichments were represented in blue, darkest being the most significant.

Article Snippet: The tissues were permeabilized with 0.5% Triton X-100 (Euromedex, France) in 1X PBS for 15 min and saturated with 5% normal horse serum in 1X PBS for 30 min. An antibody panel targeting B7-H3 (catalog number #66481-1-Ig, clone 1E7D1, uncoupled, diluted to 1:25, Proteintech) and the secondary antibody anti-mouse Cy3 (Jackson ImmunoResearch) in tumor cells (pankeratin, catalog number #53-9003-82, clone AE1/AE3, primary coupled with Alexa Fluor 488, diluted to 1:50, eBioscience) and in CAFs (vimentin, catalog number #130-127-018, clone REAL1008, primary coupled with APC, diluted to 1:50, Miltenyi) was constructed.

Techniques: Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Multiomic analysis of colorectal adenocarcinoma reveals a new subtype of myofibroblastic cancer-associated fibroblasts that express high levels of B7-H3 and have poor-prognosis value

doi: 10.1136/jitc-2025-013356

Figure Lengend Snippet: B7-H3 expression in epithelial and stromal cells in CRC and normal adjacent tissue. ( A ) Dot plot representing the expression of B7-H3 in cell subtypes of a single-cell dataset ( GSE132465 up panel and GSE144735 down panel) of normal tissue and CRC samples. Circle size represented the percentage of cell enriched in B7-H3 , and the colored scale represented the average expression. Most enriched clusters were framed. T means “Tumor”, B means “Border” and N means “Normal. ( B ) Histologic and projection of B7-H3 expression of B7-H3 in spatial transcriptomic data of Normal tissue (N=2 patients). A colored scale represents the level of expression. Epithelial and stroma regions were defined in the dashed line. ( C ) Histologic and projection of B7-H3 expression in spatial transcriptomic data of CRC (N=3 patients). Colored scale represents the level of expression. Tumor and stroma regions were defined in the dashed line. CRC, colorectal cancer.

Article Snippet: The tissues were permeabilized with 0.5% Triton X-100 (Euromedex, France) in 1X PBS for 15 min and saturated with 5% normal horse serum in 1X PBS for 30 min. An antibody panel targeting B7-H3 (catalog number #66481-1-Ig, clone 1E7D1, uncoupled, diluted to 1:25, Proteintech) and the secondary antibody anti-mouse Cy3 (Jackson ImmunoResearch) in tumor cells (pankeratin, catalog number #53-9003-82, clone AE1/AE3, primary coupled with Alexa Fluor 488, diluted to 1:50, eBioscience) and in CAFs (vimentin, catalog number #130-127-018, clone REAL1008, primary coupled with APC, diluted to 1:50, Miltenyi) was constructed.

Techniques: Expressing, Single Cell

Journal: Journal for Immunotherapy of Cancer

Article Title: Multiomic analysis of colorectal adenocarcinoma reveals a new subtype of myofibroblastic cancer-associated fibroblasts that express high levels of B7-H3 and have poor-prognosis value

doi: 10.1136/jitc-2025-013356

Figure Lengend Snippet: Expression of B7-H3 at the protein level in CRC tissues. ( A–C ) Immunofluorescence pictures of B7-H3 expression in CRC tissues. The whole tissue and an insert were shown. B7-H3 was represented in yellow, fibroblast in pink (VIM), tumor in green (KRT), and nuclei in blue (DAPI). Healthy and tumorous regions were defined when needed by a dashed line. ( D ) Box plot representing B7-H3 MFI of tumor versus CAF compartments in each patient of the cohort, including healthy epithelial and stromal compartments as control. P means patients. ( E ) Box plot representing the global B7-H3 MFI of tumor versus CAF compartments for all patients of the cohort. P value was obtained by paired t-test. CAF, cancer-associated signature; CRC, colorectal cancer; MFI, Mean Fluorescence Intensity.

Article Snippet: The tissues were permeabilized with 0.5% Triton X-100 (Euromedex, France) in 1X PBS for 15 min and saturated with 5% normal horse serum in 1X PBS for 30 min. An antibody panel targeting B7-H3 (catalog number #66481-1-Ig, clone 1E7D1, uncoupled, diluted to 1:25, Proteintech) and the secondary antibody anti-mouse Cy3 (Jackson ImmunoResearch) in tumor cells (pankeratin, catalog number #53-9003-82, clone AE1/AE3, primary coupled with Alexa Fluor 488, diluted to 1:50, eBioscience) and in CAFs (vimentin, catalog number #130-127-018, clone REAL1008, primary coupled with APC, diluted to 1:50, Miltenyi) was constructed.

Techniques: Expressing, Immunofluorescence, Control, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Multiomic analysis of colorectal adenocarcinoma reveals a new subtype of myofibroblastic cancer-associated fibroblasts that express high levels of B7-H3 and have poor-prognosis value

doi: 10.1136/jitc-2025-013356

Figure Lengend Snippet: Nature and prognostic value of B7-H3 high fibroblasts in CRC samples. ( A ) Uniform Manifold Approximation and Projection (uMAP) dimplot representation of fibroblast clusters subclusterization of GSE144735 scRNAseq dataset. ( B ) uMAP dimplot representation of fibroblast clusters subclusterization of GSE132465 scRNAseq dataset. ( C ) Dotplot representation of B7-H3 expression in GSE144735 (up) and GSE312465 (down) fibroblasts subclusters. Circle size represented the percentage of cell enriched in B7-H3, and the color represented the average expression. Most enriched clusters were framed. ( D ) Dotplot representation of several CAF-related signatures expression in GSE144735 (left) and GSE132465 (right) B7-H3 high fibroblasts subclusters. Circle size represented the percentage of cell enriched in each signature and the color represented the average expression. “Hmd” means “Homemade” signatures. ( E ) Summary illustration of B7-H3 high ECM-myCAF main characteristics. Created in BioRender. Mamessier, E. (2025) https://BioRender.com/1g3m69k . ( F ) Summary illustration of B7-H3 high pro-fibrotic pericytes main characteristics. Created in BioRender. Mamessier, E. (2025) https://BioRender.com/1g3m69k ( G ) Kaplan-Meier survival curve representing the disease-free survival between CRC samples enriched in the B7-H3 high ECM-myCAF metagene (red), in regard to time (months after diagnosis), or not (black). Significance was defined using Log-rank. ( H ) Kaplan-Meier survival curve representing the disease-free survival between CRC samples enriched in the B7-H3 high pro-fibrotic pericyte metagene (red) in regard to time (months after diagnosis), or not (black). Significance was defined using Log-rank. CRC samples enriched in the metagene were represented in red. CAF, cancer-associated signature; CRC, colorectal cancer; DFS, disease-free survival; myCAF, myofibroblastic CAF.

Article Snippet: The tissues were permeabilized with 0.5% Triton X-100 (Euromedex, France) in 1X PBS for 15 min and saturated with 5% normal horse serum in 1X PBS for 30 min. An antibody panel targeting B7-H3 (catalog number #66481-1-Ig, clone 1E7D1, uncoupled, diluted to 1:25, Proteintech) and the secondary antibody anti-mouse Cy3 (Jackson ImmunoResearch) in tumor cells (pankeratin, catalog number #53-9003-82, clone AE1/AE3, primary coupled with Alexa Fluor 488, diluted to 1:50, eBioscience) and in CAFs (vimentin, catalog number #130-127-018, clone REAL1008, primary coupled with APC, diluted to 1:50, Miltenyi) was constructed.

Techniques: Expressing, Biomarker Discovery

Journal: Journal for Immunotherapy of Cancer

Article Title: Multiomic analysis of colorectal adenocarcinoma reveals a new subtype of myofibroblastic cancer-associated fibroblasts that express high levels of B7-H3 and have poor-prognosis value

doi: 10.1136/jitc-2025-013356

Figure Lengend Snippet: Nature of B7-H3 high fibroblast across CRC oncogenesis. ( A ) Serial uMAP dimplots representing fibroblast sub-clusterizations in the atlas dataset ( GSE161277 ) depending on oncogenesis status (normal, inflamed, polyps, and tumor). ( B ) Dotplot representing B7-H3 expression of fibroblast subclusters depending on oncogenesis status. Circle size represented the percentage of cell enriched in each signature and the colored scale represented the average expression. Most enriched clusters were framed. ( C ) Dotplot representation of several CAF-related signatures expression in the Atlas B7-H3 high fibroblast subclusters. Circle size represented the percentage of cell enriched in each signature and the colored scale represented the average expression. ( D ) Dotplot representation of pericytes-related signatures expression in the Atlas B7-H3 high fibroblast subclusters. Circle size represented the percentage of cell enriched in each signature and the colored scale represented the average expression. ( E ) Proportion of fibroblast subclusters of Atlas dataset over oncogenesis status (normal, inflamed, polyps, and tumor). B7-H3 high fibroblasts were framed. ( F ) Dimplot representation of B7-H3 expression in fibroblasts clusters of CRC samples. The colored scale represented the average expression. ( G ) Trajectory inference of fibroblast clusters, with cluster_c4 defined as origin. The colored scale represented the proximity between clusters. ( H ) Pseudotime gene expression plot representing the expression of the most interesting genes selected from ) and highlighted in the text on cluster_c4, c_13, c_10, and _c5 continuum. CAF, cancer-associated signature; CRC, colorectal cancer; myCAF, myofibroblastic CAF.

Article Snippet: The tissues were permeabilized with 0.5% Triton X-100 (Euromedex, France) in 1X PBS for 15 min and saturated with 5% normal horse serum in 1X PBS for 30 min. An antibody panel targeting B7-H3 (catalog number #66481-1-Ig, clone 1E7D1, uncoupled, diluted to 1:25, Proteintech) and the secondary antibody anti-mouse Cy3 (Jackson ImmunoResearch) in tumor cells (pankeratin, catalog number #53-9003-82, clone AE1/AE3, primary coupled with Alexa Fluor 488, diluted to 1:50, eBioscience) and in CAFs (vimentin, catalog number #130-127-018, clone REAL1008, primary coupled with APC, diluted to 1:50, Miltenyi) was constructed.

Techniques: Expressing, Gene Expression

Journal: Cancer discovery

Article Title: Tuning the Antigen Density Requirement for CAR T Cell Activity

doi: 10.1158/2159-8290.CD-19-0945

Figure Lengend Snippet: (a) Schema of first generation CD19 CARs with either a CD8 or CD28 hinge-transmembrane region (CD19-CD8H/T-ζ and CD19-CD28H/T-ζ). (b) NALM6 clones expressing either 963 or 45,851 molecules of surface CD19 were cocultured at a 1:1 ratio with either CD19-CD28ζ, CD19–4-1BBζ, CD19-CD28H/T-ζ or CD19-CD8H/T-ζ CAR T cells and tumor cell killing was measured in an Incucyte assay. Representative of three experiments with different T cell donors. Statistical analysis performed with repeated measures ANOVA between CD19-CD28H/T-ζ and CD19-CD8H/T-ζ. (c) CD19-CD28ζ, CD19–4-1BBζ, CD19-CD28H/T-ζ, and CD19-CD8H/T-ζ CAR T cells were cocultured with NALM6 clones expressing various amounts of CD19 for 24 hours and secreted IL-2 was measured in the supernatant by ELISA. Representative of three experiments with different T cell donors. Statistical comparisons performed with the student’s t-test (two sided) between CD19-CD28H/T-ζ and CD19-CD8H/T-ζ. (d) Schema of a Her2 CAR containing a CD28 hinge-transmembrane region and 4–1BB costimulatory domain (Her2-CD28H/T-4–1BBζ). (e) One million 143b osteosarcoma cells were orthotopically implanted in the hind leg of NSG mice. After seven days, mice were treated with 10 million Her2–4-1BBζ CAR T cells, Her2-CD28H/T-4–1BBζ CAR T cells, or untransduced control T cells (MOCK). Leg measurements were obtained twice weekly with digital calibers. Measurements for individual mice are shown. Statistical analysis performed with repeated measures ANOVA. (f) Survival curves for mice treated as in (e). Statistical analysis performed with the log-rank test. (e-f) are representative of two experiments with different T cell donors (n=5 mice per group). (g) Schema of a B7-H3 CAR containing a CD28 hinge-transmembrane region and 4–1BB costimulatory domain (B7-H3-CD28H/T-4–1BBζ). (h) One million CHLA255 neuroblastoma cells were engrafted into NSG mice by tail vein injection in a metastatic neuroblastoma model. Six days later, mice were injected with 10 million B7-H3–4-1BBζ CAR T cells, B7-H3-CD28H/T-4–1BBζ CAR T cells, or untransduced control T cells (MOCK). Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Representative bioluminescent images are shown. (i) Quantified tumor flux values for individual mice treated as in (h). Statistical analysis performed with repeated measures ANOVA. (j) Survival curves for mice treated as in (h). Statistical analysis performed with the log-rank test. (h-j) are representative of two experiments with different T cell donors. For in vitro experiments, error bars represent SD and for in vivo experiments, error bars represent SEM. p < 0.05 was considered statistically significant, and p values are denoted with asterisks as follows: p > 0.05, not significant, NS; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: B7-H3 expression on tumor lines was assessed with PE conjugated mouse anti-human B7-H3 antibody (R&D, clone MAB1027) and Her2 with PE or APC conjugated anti-human CD340 antibody (Biolegend, clone 24D2).

Techniques: Clone Assay, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Software, In Vitro, In Vivo

Journal: Communications Biology

Article Title: Dimerization of the 4Ig isoform of B7-H3 in tumor cells mediates enhanced proliferation and tumorigenic signaling

doi: 10.1038/s42003-023-05736-8

Figure Lengend Snippet:

Article Snippet: Anti-B7-H3 antibody was produced in house (MIL33B) or purchased from R&D Systems (AF1397).

Techniques:

Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: Figure 1. The optimized SNIP design, trans SNIP BBz, confers robust control over CAR-T cytotoxicity (A) Schematic illustration of the cis SNIP BBz system (left). CAR-CD3z immunoblots of protein lysates from anti-B7H3 cis SNIP BBz CAR-T cells ± 3 mM GPV (middle). Cytotoxicity of day 10 anti-B7H3 cis SNIP BBz CAR-T cells (right). (B) Schematic illustration of the cis SNIP scFv system (left), flow cytometry of surface B7H3 CAR (middle), and tumor killing (right). (C) Schematic illustration of the trans SNIP BBz system (left) and tumor killing (right). (D) Schematic of various combinations of CD8a and CD28 Tm for the CAR and protease modules (left). Tumor killing in the absence (SNIP OFF, middle) or presence (SNIP ON, right) of GPV. (E) CD3z immunoblots of the 8/8 Tm configuration.

Article Snippet: Recombinant B7H3-, HER2-, and human ROR1-Fc (R&D systems) were used to detection B7H3, HER2, and ROR1 surface CAR, respectively.

Techniques: Control, Western Blot, Cytometry

Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: Figure 2. Leaky cytotoxic activity is driven by high antigen density on target lines and ameliorated by substituting cleavage sites with high catalytic activity (A and B) Tumor BLI of mice inoculated with Nalm6-B7H3 leukemia cells and were then treated with B7H3.BBz CAR or mock T cells 4 days later. SNIP-treated mice were given 50 mg/kg GPV and 25 mg/kg ritonavir (RTV) (SNIP ON) or vehicle control (SNIP OFF) 3 times per day by oral gavage. RTV is a pharmacokinetic enhancer of GPV. (C and D) Tumor BLI of mice inoculated with MED8A cells in the right flank and cerebellum and were then treated with B7H3.BBz CAR or mock T cells 5 days later. (E) Cytotoxic activity of SNIP B7H3.BBz bearing different cleavage sites (CSs) against GFP-labeled tumor cells in the absence of GPV (SNIP OFF). Data are mean ± SEM of triplicate wells.

Article Snippet: Recombinant B7H3-, HER2-, and human ROR1-Fc (R&D systems) were used to detection B7H3, HER2, and ROR1 surface CAR, respectively.

Techniques: Activity Assay, Control, Labeling

Journal: Cell

Article Title: Enhanced safety and efficacy of protease-regulated CAR-T cell receptors.

doi: 10.1016/j.cell.2022.03.041

Figure Lengend Snippet: Figure 3. SNIP CAR-T cells display enhanced antitumor efficacy in orthotopic solid tumor models (A) Schematic of the CHLA255 neuroblastoma kidney capsule tumor model using GD2-specific CARs. (B) Tumor progression of CHLA255 by BLI. (C) Quantification of BLI (left) and survival curves of mice in the CHLA255 neuroblastoma model (right). (D) Schematic of the 143B osteosarcoma tumor model using HER2-specific CARs. (E) Tumor progression in the 143B osteosarcoma was monitored by tumor volume measurements (left) and survival curves in the 143B model (right). (F) Schematic of the MED8A medulloblastoma brain tumor model using B7H3-specific CARs. (G) Tumor progression of MED8A BLI.

Article Snippet: Recombinant B7H3-, HER2-, and human ROR1-Fc (R&D systems) were used to detection B7H3, HER2, and ROR1 surface CAR, respectively.

Techniques: