Journal: Scientific reports

Article Title: Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays.

doi: 10.1038/s41598-024-64559-7

Figure Lengend Snippet: Figure 1. Schematic representation of our hybrid-SELEX method for selection of B7H3-specific ssDNA aptamer. (a) Retinoblastoma cell-SELEX to develop aptamers against retinoblastoma using Weri-RB1 cells and target cell and Mio-M1 as control cells. (b) We have screened the RB cell-SELEX enriched pools on recombinant B7H3 protein by dot-blot and chose the cell-SELEX enriched pool-15 (CSEP-15) as the starting library for the B7H3 hybrid SELEX. In our experiment, the hybrid-SELEX process is divided into (c) the B7H3 protein- based SELEX selection and (d) cell-based SELEX enrichment. The CSEP-15 is incubated with B7H3 protein immobilized on magnetic beads for positive selection and empty magnetic beads for counter selection for each cycle in protein-SELEX. The pool enriched from protein SELEX is incubated with Weri-RB1 for positive selection and Mio-M1 for counter selection in cell-SELEX. After 9 rounds of selection, the enriched aptamer pools were sequenced by NGS. SELEX, Systematic Evolution of Ligands by EXponential enrichment.

Article Snippet: Immunohistochemistry was performed to assess the binding of B7H3 aptamer (VRF-HS_B7H3-03) to RB primary tumor as a positive control, lysed blood spiked with Weri-RB1 cells (leukocytes as a negative control), and cross-validated with IHC using B7H3 antibody (Cell Signaling technology, 14058, 1: 100 dilution).

Techniques: Selection, Control, Recombinant, Dot Blot, Incubation, Magnetic Beads

Journal: Scientific reports

Article Title: Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays.

doi: 10.1038/s41598-024-64559-7

Figure Lengend Snippet: Figure 2. Monitoring the enrichment of the DNA libraries during hybrid-SELEX by flow cytometry and dot- blot. (a) Fluorescence intensities of target cells (Weri-RB1) incubated with FITC-labelled ssDNA pools from the initial library to the ninth-selection round. (b) Fluorescence intensities of negative control cells (Mio-M1) incubated with FITC-labelled ssDNA pools from the initial library to the ninth-selection round. (c) Represented is the dot-blot assay showing the fluorescence intensities of ssDNA pools from the initial library to the ninth- selection round, bound to the recombinant B7H3 protein.

Article Snippet: Immunohistochemistry was performed to assess the binding of B7H3 aptamer (VRF-HS_B7H3-03) to RB primary tumor as a positive control, lysed blood spiked with Weri-RB1 cells (leukocytes as a negative control), and cross-validated with IHC using B7H3 antibody (Cell Signaling technology, 14058, 1: 100 dilution).

Techniques: Flow Cytometry, Dot Blot, Fluorescence, Incubation, Selection, Negative Control, Recombinant

Journal: Scientific reports

Article Title: Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays.

doi: 10.1038/s41598-024-64559-7

Figure Lengend Snippet: Figure 3. Binding ability, secondary structures and dissociation constants of selected B7H3 aptamers. (a) Binding affinity of FITC-labelled aptamers VRF-HS_B7H3-01, VRF-HS_B7H3-02, VRF-HS_B7H3-03, VRF-HS_B7H3-04 and VRF-HS_B7H3-05 to Weri-RB1 cells assessed by flow cytometry. (b) Binding ability of FITC-labelled aptamers VRF-HS_B7H3-01, VRF-HS_B7H3-02, VRF-HS_B7H3-03, VRF-HS_B7H3-04 and VRF-HS_B7H3-05 to Mio-M1 cells assessed by flow cytometry. (c–g) Predicted secondary structures for five aptamer candidates, VRF-HS_B7H3-01, VRF-HS_B7H3-02, VRF-HS_B7H3-03, VRF-HS_B7H3-04 and VRF-HS_B7H3-05 selected for further. The presented predicted secondary structures were the ones with lowest ΔG. Constant sequence regions are highlighted in black, and green represents the random regions. (h–l) Binding curve of aptamers VRF-HS_B7H3-01, VRF-HS_B7H3-02, VRF-HS_B7H3-03, VRF-HS_B7H3-04 and VRF-HS_B7H3-05 with Weri-RB1 and Mio-M1 cells assessed by flow cytometry and recombinant B7H3 protein by dot-blot. Equilibrium dissociation constants (Kd) (nM) were calculated using GraphPad Prism 7, under the non-linear fit model, one-site non-competitive binding to fluorescent population ratio at used aptamer concentrations.

Article Snippet: Immunohistochemistry was performed to assess the binding of B7H3 aptamer (VRF-HS_B7H3-03) to RB primary tumor as a positive control, lysed blood spiked with Weri-RB1 cells (leukocytes as a negative control), and cross-validated with IHC using B7H3 antibody (Cell Signaling technology, 14058, 1: 100 dilution).

Techniques: Binding Assay, Flow Cytometry, Sequencing, Recombinant, Dot Blot

Journal: Scientific reports

Article Title: Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays.

doi: 10.1038/s41598-024-64559-7

Figure Lengend Snippet: Figure 4. Affinity of B7H3 aptamers by Dot-blot and western blot analysis. (a) Dot-blot assay with (i–v) FITC labelled and (vi–x) biotin labelled aptamers to demonstrate the capability of the B7H3 aptamers to recognize their target immobilized on PVDF membranes; (i) & (vi)—VRF-HS_B7H3-01, (ii) & (vii)—VRF-HS_B7H3-02, (iii) & (viii)—VRF-HS_B7H3-03, (iv) & (viii)—VRF-HS_B7H3-04 and (v) & (x)—VRF-HS_B7H3-05; 1— B7H3 Recombinant protein, 2—RB tumor protein lysate, 3—Weri-RB1 protein lysate, 4—BSA, 5—Weri-RB1 secretome and 6—Secondary control. (b) Sandwich dot blot assay with (i–iii) biotin labelled and (ii–iv) FITC labelled aptamers to demonstrate the capability of aptamers to recognize different epitopes of their target immobilized on nitrocellulose membranes. (i) & (ii)—unlabelled VRF-HS_B7H3-01 is used as capture and FITC or biotin labelled VRF-HS_B7H3-03 is used for detection, (iii) & (iv) unlabelled VRF-HS_B7H3-03 is used as capture and FITC or biotin labelled VRF-HS_B7H3-01 is used for detection; 1—B7H3 Recombinant protein, 2—RB tumor protein lysate, 3—Weri-RB1 protein lysate, 4—Weri-RB1 secretome and 5—BSA. (c) Comparison of the specificity of B7H3 antibody to VRF-HS_B7H3-03 aptamer in a protein blot analysis (cropped image). Lane 1—B7H3 Recombinant protein, 2—RB tumor protein lysate, 3—Weri-RB1 protein lysate and 4—Mio-M1 protein lysate; (i) Probed with anti-B7H3 Rabbit monoclonal antibody, (ii) Probed with biotinylated VRF-HS_B7H3-03 aptamer and (iii) & (iv) Probed with GAPDH mouse monoclonal antibody. Results of an aptamer blot from a nonreducing SDS polyacrylamide gel in which B7H3 was clearly detected near 90 kDa similar to antibody. However, the recombinant protein manufacturer of the B7H3 (R&D systems) reported a molecular weight of 38–48 kDa for its product which is consistent with the strongly detected band’s weight (Full raw blot included in Supplementary data Fig. S5).

Article Snippet: Immunohistochemistry was performed to assess the binding of B7H3 aptamer (VRF-HS_B7H3-03) to RB primary tumor as a positive control, lysed blood spiked with Weri-RB1 cells (leukocytes as a negative control), and cross-validated with IHC using B7H3 antibody (Cell Signaling technology, 14058, 1: 100 dilution).

Techniques: Dot Blot, Western Blot, Recombinant, Control, Comparison, Molecular Weight

Journal: Scientific reports

Article Title: Development of DNA aptamers targeting B7H3 by hybrid-SELEX: an alternative to antibodies for immuno-assays.

doi: 10.1038/s41598-024-64559-7

Figure Lengend Snippet: Figure 5. Immunohistochemistry of B7H3 antibody and VRF-HS_B7H3-03 to RB tumour sections and retina. (a,d) H and E staining, (b,e) IHC with B7H3 antibody, and (c,f) IHC with biotin-labelled VRF-HS_B7H3-03 of primary retinoblastoma tumour and retina respectively.

Article Snippet: Immunohistochemistry was performed to assess the binding of B7H3 aptamer (VRF-HS_B7H3-03) to RB primary tumor as a positive control, lysed blood spiked with Weri-RB1 cells (leukocytes as a negative control), and cross-validated with IHC using B7H3 antibody (Cell Signaling technology, 14058, 1: 100 dilution).

Techniques: Immunohistochemistry, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Generation of B7 ‐ H3 isoform regulated by ANXA2 / NSUN2 / YBX1 axis in human glioma

doi: 10.1111/jcmm.18575

Figure Lengend Snippet: Characterization of different isoforms of B7‐H3 expression. (A) Cartoon displays the transcript length and basic structure information of the different isomers of B7H3, as well as primers, antibodies and probes used in this study. The table at the bottom right gives the length of the different isoform PCR products, which can be used as a reference for all subsequent RT‐PCR results. (B) RT‐PCR show the PCR products of B7‐H3 isoform in five glioma cell lines used by P1 primers (top), P2 primers (middle) and β‐actin primers (bottom). (C) WB assay shows the protein bands of B7‐H3 isoform, ANXA2, NSUN2 and YBX1 in five glioma cell lines. The band locations of 4Ig and 2Ig and the absence of 3Ig are inferred by RT‐PCR. (D) WB assay shows the protein bands of B7‐H3, ANXA2, NSUN2 and YBX1 in 24 glioma tissues. The double bands represent 4Ig and 2Ig according to the protein size in (C). (E) Venn diagram shows the binding proteins on B7‐H3 pre‐mRNA via probe pull down and mass spectrum. YBX1, NSUN2 and ANXA2 are highlighted in different intersections. (F) Enrichment of ANXA2 on B7‐H3 in five glioma cells by RIP‐qPCR. The statistical significances compared between other four cells and U373MG via one‐way ANOVA are indicated by asterisk. **** p < 0.0001, *** p < 0.001 and ** p < 0.01.

Article Snippet: Primary antibodies against B7‐H3 (1: 2000, Cat. no. 58798, CST, USA), ANXA2 (1: 2000, Cat. no. 8235, CST), NSUN2 (1: 2500, Cat. no. 44056, CST), YBX1 (1: 2000, Cat. no. 9744, CST), β‐actin (1: 5000, Cat. no. 4967, CST) and Flag (1: 1000, Cat. no. 14793, CST) were used.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Binding Assay

Journal: Molecular Cancer Therapeutics

Article Title: Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

doi: 10.1158/1535-7163.mct-21-0836

Figure Lengend Snippet: Figure 1. Integrative proteomic and transcriptomic surfaceome profiling of osteosarcoma. A, The workflow of the integrative proteomic and transcriptomic approach used to identify immunotherapeutic targets in osteosarcomas. B, Expression profile of the cell-surface proteins identified by mass spectrometry in osteosarcoma cell lines and PDX models. C, Expression profile of the 209 overexpressed surface protein-encoding genes in 98 patients with osteosarcoma from the TARGET database (TARGET OS) and 17 osteosarcoma cell lines that we analyzed (OSC). The 11 candidate surface proteins and the 4 candidates that overlapped with existing drug targets are marked. The 4-candidate targets (MT1-MMP, MRC2, CD276, and LRRC15) were highly expressed in most of the patient samples and cell lines.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500; ref. 27), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Expressing, Mass Spectrometry

Journal: Molecular Cancer Therapeutics

Article Title: Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

doi: 10.1158/1535-7163.mct-21-0836

Figure Lengend Snippet: Figure 2. mRNA expression of MT1-MMP, MRC2, CD276,and LRRC15 in osteosarcoma, normal tissues, and other pediatric cancers. A–D, RNA-seq data showedMT1-MMP(A), MRC2 (B), CD276 (C), and LRRC15 (D) were overexpressed in osteosarcoma compared with a range of normal tissues. The boxes represent the Q1 and Q3 of the data. The bars represent the median. E–G, MT1-MMP (E), MRC2 (F), and CD276 (G) had higher expression in osteosarcoma compared with other pediatric cancers (OS, osteosarcoma; MEL, melanoma; RHB, rhabdomyosarcoma; CPC, choroid plexus carcinoma; HGG, high-grade glioma; EPD, ependymoma; ACT, adrenocortical carcinoma; WLM, Wilms’ tumor; NBL, neuroblastoma;LGG,low-grade glioma;RB, retinoblastoma;AML, acutemyeloid leukemia;MLL,mixed-lineage leukemia;MB, medulloblastoma; BALL, B-cell acute lymphoblastic leukemia; TALL, T-cell acute lymphoblastic leukemia). FPKM, fragments per kilobase million; TPM, transcripts per million.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500; ref. 27), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Expressing, RNA Sequencing, Wilms Tumor Assay

Journal: Molecular Cancer Therapeutics

Article Title: Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

doi: 10.1158/1535-7163.mct-21-0836

Figure Lengend Snippet: Figure 3. MT1-MMP, MRC2, and CD276 are highly expressed cell-surface proteins in osteosarcoma. A and B, Western blots of MT1-MMP, MRC2, and CD276 in a panel of osteosarcoma cell lines (n ¼ 8; A) and PDXs (n ¼ 8; B). C, Flow cytometry analysis of 7 osteosarcoma cell lines. Gray plots represent unstained controls, and colored plots represent staining with MT1-MMP, MRC2, and CD276 antibodies.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500; ref. 27), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Western Blot, Flow Cytometry, Staining

Journal: Molecular Cancer Therapeutics

Article Title: Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

doi: 10.1158/1535-7163.mct-21-0836

Figure Lengend Snippet: Figure 4. IHC staining showed high membranous positivity of MT1-MMP, MRC2, and CD276 in most osteosarcoma patient samples and PDXs. A–C, Representative membrane- staining examples of MT1-MMP in a patient sample (A), PDX (B), and testes (negative control; C). D–F, Representative membrane-staining examples of MRC2 in a patient sample (D), PDX (E), and placenta (negative control; F). G–I, Representative membrane-staining examples of CD276 in a patient sample (G), PDX (H), and placenta (mild positive; I). J and K, Summary of IHC staining H-score of MT1-MMP, MRC2, and CD276 in the tissue microarray for 37 patientswith osteosarcoma (J) and 19 PDX models (K). Boxes indicate SD, and error bars represent data range.

Article Snippet: Antibodies for MT1-MMP (R&D Systems, FAB9181A, 1:40), MRC2 (kindly provided by Dr. Niels Behrendt, University of Copenhagen, Copenhagen, Denmark, clone 2h9, 1:500; ref. 27), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Immunohistochemistry, Membrane, Staining, Negative Control, Microarray

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: GB cell line screening for target selection and generation of OE and KO cell line models. (a) Expression of glioma-associated membrane antigens in primary and conventional GB cell lines by RT-qPCR. N = 2 technical replicates (two independent qPCR reactions) per gene per cell line. (b) Protein expression levels of EPHA2, CD276, IL13Ra2 and CD70 (blue histograms) in primary GB cell lines, measured by flow cytometry. (c) Evaluation of EPHA2, CD276, IL13Ra2 and CD70 protein levels (blue histograms) on the surface of generated tumor cell models by flow cytometry. For (b and c), isotype control antibodies (red histograms) were used, and data were gated on live single cells. For (b and c), indicative histograms from N = 3 biological replicates per marker per cell line and N = 3 independent experimental repeats. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Selection, Expressing, Membrane, Quantitative RT-PCR, Flow Cytometry, Generated, Control, Marker

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vitro evaluation of the mSA2 CAR-T cell specificity and killing potency. (a) In vitro experimental pipeline. (b) mSA2 CAR-T cell activation after co-culture with GB models, determined by flow cytometry. N = 3 biological replicates (three independent co-cultures) per group. Data gated on live single CD3 + cells (NT) or live single CD3 + /EGFP + cells (SFG, mSA2_h28z, mSA2_hBBz). An isotype control antibody was used for gating. An unpaired two-tailed student’s t-test was used to evaluate statistical significance. N = 2 independent T-Cell donors. Representative results from N = 3 independent experimental repeats. (c) Confocal IF images of P3/CD70 (CD70 + /CD276 + /EPHA2 + ) cells co-cultured with mSA2_hBBz cells. For a-EPHA2: t 0 = 0 min, t 1 = 140 min, t 2 = 280 min, t 3 = 420 min, t 4 = 560 min. For a-CD276: t 0 = 0 min, t 1 = 220 min, t 2 = 440 min, t 3 = 660 min, t 4 = 880 min. For a-CD70: t 0 = 0 min, t 1 = 350 min, t 2 = 700 min, t 3 = 1050 min, t 4 = 1400 min. N = 2 biological replicates (two separate co-cultures) per group. N = 1 T-Cell donor. (d) Quantification of tumor cell signal from (c). A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was performed to assess statistical significance at the t = 560 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Activation Assay, Co-Culture Assay, Flow Cytometry, Control, Two Tailed Test, Cell Culture

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Evaluation of the mSA2 CAR-T cell specificity and efficacy in additional primary GB cell lines. (a) mSA2 CAR-T cell activation after co-culture with S24 or T269 cells, measured by flow cytometry. Data gated on live single CD3 + /EGFP + cells. A one-way ANOVA test with a post-hoc Šídák test for multiple comparisons was used. b) Confocal microscopy images after O/N co-culture of S24 cells labeled with an anti-CD276 biotinylated antibody with mSA2 CAR-T cells. Indicative photos from N = 3 biological replicate co-cultures per construct. N = 1 T-Cell donor. (c) Quantification of Annexin-V signal from (b). d) mSA2 CAR-T cell activation after co-culture with S24, P3 and T269 tumor cells, after incubation with targeting, or a non-targeting biotinylated isotype control antibody, measured by flow cytometry. Data gated on live single CD3 + /EGFP + cells. For (c and d), a one-way ANOVA test with a post-hoc Dunnett’s test for multiple comparisons was used. For (a and d), N = 3 biological replicates (three independent co-cultures) per group and N = 3 independent T-Cell donors. Indicative data from N = 2 experimental repeats. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: Activation Assay, Co-Culture Assay, Flow Cytometry, Confocal Microscopy, Labeling, Construct, Incubation, Control

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: Investigation of the mSA2 CAR-T cell capacity to address tumor heterogeneity in vitro . (a) Co-culture pipeline. (b) Apoptotic tumor cell fraction after co-culture with mSA2 CAR-T cells, measured by flow cytometry. Data gated on live single EGFP − cells. N = 1 T-Cell donor. N = 3 biological replicates (three independent co-cultures) per group. A Welch’s ANOVA test with a post-hoc Dunnett T3 test for multiple comparisons was used for statistical significance. (c) Analysis of the Annexin-V-incorporating fraction from (b) by flow cytometry. Data gated on live single EGFP − /Annexin-V high tumor cells. (d) Quantification of Annexin-V incorporation from (c). (e) Confocal live cell if images of P3/CD276_KO : P3/EPHA2_KO cells (left panel) and P3/CD70 : P3/IL13Ra2 cells (right panel) after 48 h co-culture with mSA2_h28z cells, after incubation with combinations of biotinylated antibodies, or a biotinylated isotype control antibody. Indicative images from N = 2 biological replicates (two independent co-cultures) and N = 2 independent T-Cell donors per group. (f) Quantification of tumor cell signal over time from the co-culture in (e). For (d and f), an unpaired two-tailed student’s t-test was used to evaluate statistical significance. For (f), statistical significance was assessed at the t = 750 min mark. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant. Results from independent experiments are shown; no data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vitro, Co-Culture Assay, Flow Cytometry, Incubation, Control, Two Tailed Test

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vivo histological investigation of the anti-GB mSA2 CAR-T cell killing functionality. (a) In vivo pipeline. (b) Evaluation of TIM-3, LAG-3 and PD-1 levels on mSA2 CAR-T cells before in vivo treatment by flow cytometry. Isotype control antibodies were used to determine gating. Data gated on live single CD3 + /EGFP + cells. (c) Tumor signal one day before and 5 days after mSA2_h28z CAR-T cell treatment, measured by BLI. N ≥ 5 animals per group. (d) Tumor signal quantification from (c). A paired t-test (mSA2_h28z, mSA2_h28z + a-CD70) and a Wilcoxon matched-pairs signed-rank test (mSA2_h28z + a-CD276) were used to assess significance. (e) Tumor signal fold-change quantification from (c). Each dot represents an animal. A one-way ANOVA test with a post-hoc Dunnett test for multiple comparisons was performed. (f) IF analysis of brains of treated animals. Representative images from two animals per treatment group. (g) Quantification of CAR-T and cleaved caspase-3 signal in stained sections from (f). Each dot represents an image from different tumor regions of a given animal. A Kruskal-Wallis test with a post-hoc Dunn’s multiple comparisons test was performed to evaluate significance. For (b and c), mSA2 CAR-T cells were produced from N = 1 T-Cell donor. Data presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s., not significant. No data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vivo, Flow Cytometry, Control, Staining, Produced

Journal: Oncoimmunology

Article Title: Utilization of universal-targeting mSA2 CAR-T cells for the treatment of glioblastoma

doi: 10.1080/2162402X.2025.2518631

Figure Lengend Snippet: In vivo evaluation of the potential mSA2 CAR-T cell interaction with endogenous biotin. (a) Screening of mouse brain single cell suspensions for extracellular biotin presence by flow cytometry. Data gated on live single cells. N = 5 mice. In vitro biotinylated or non-biotinylated HEK293 cells were used as positive (blue histogram) and negative (red histogram) control samples respectively. (b) Analysis of CAR-T cell activation after co-culture of mSA2_h28z cells from N = 1 donor with the mouse brain single cell suspensions from (a) by flow cytometry. Data gated on live single CD3 + /EGFP + cells. Representative plots from N = 3 biological replicates (three independent co-cultures) per mouse. Isotype control antibodies were used to determine gating. (c) Quantification of CD137 positive signal from (b). (d) Granzyme-B and IFN-γ secretion levels in the co-culture SN from (b), determined by ELISA. For (b, c and d), P3 cells (CD276 + ) labeled or unlabeled with an anti-CD276 biotinylated antibody and co-cultured with mSA2_h28z cells were used as positive and negative activation control samples respectively. For (b, c and d), a one-way ANOVA test with a post-hoc Dunnett test for multiple comparisons was performed. Data presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; n.s., not significant; n.d., not detected. No data pooling was performed.

Article Snippet: Cells were then incubated with 10 μg/mL biotinylated anti-human CD70 antibody (#MA5–17726, Invitrogen), or a 1:11 dilution of biotinylated anti-human CD276 antibody (#130–095–514, Miltenyi Biotec), or a 1:11 dilution of biotinylated anti-human CD213a2 antibody (a-IL13Ra2; #130–104–503, Miltenyi Biotec), or 7.6 μg/mL biotinylated anti-human EPHA2 antibody (#BAF3035, R&D Systems) or 10 μg/mL of a biotinylated non-targeting control antibody (#13–4714–85, Thermo Fisher Scientific) at 4 °C for 30 min. All antibodies used in this study are listed in Supplementary Table S3.

Techniques: In Vivo, Flow Cytometry, In Vitro, Control, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Labeling, Cell Culture