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: 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: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: B7-H3 is highly expressed on pediatric solid tumors. A, Pediatric tumor microarrays were stained by IHC for the expression of B7-H3. Representative images of Ewing sarcoma (3+), glioblastoma multiforme (3+), medulloblastoma (2+), and alveolar rhabdomyosarcoma (3+, 2+, and 1+) samples are shown. H-scores were generated by multiplying the % cells positive × intensity seen for each core. H-scores are shown for pediatric sarcomas (B), neuroblastoma and Wilms tumor (C), and pediatric CNS tumors (D). RMS, rhabdomyosarcoma; EWS, Ewing sarcoma; DIPG, diffuse intrinsic pontine glioma.

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: Staining, Expressing, Generated, Wilms Tumor Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Expression of B7-H3 on pediatric tumors by IHC

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: Expressing, Staining, Wilms Tumor Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Systemically administered B7-H3 CAR T cells induce regression of osteosarcoma xenografts. A, B7-H3 CAR T cells produce IFNγ, TNFα, and IL2 following 24-hour in vitro coculture with MG63.3 osteosarcoma. Representative results of four experiments with 3 different PBMC donors are shown. B, Mouse model of orthotopic osteosarcoma: 1e6 MG63.3 tumor cells were injected into the periosteum of the tibia in NSG mice. Two to three weeks later, following onset of measurable tumors, 1e7 B7-H3 CAR+ T cells or irrelevant control CD19 CAR T cells were intravenously administered. C, Tumor growth was measured biweekly by digital caliper and tumor area was calculated. Values for individual mice, as well as mean values per treatment group (inset) are shown. D, Survival curves of mice treated as in B. Representative results of three experiments with 3 different PBMC donors are shown. E, Metastatic model of osteosarcoma: MG63.3-derived tumors were allowed to grow and metastasize before the mouse underwent amputation followed by administration of intravenous 1e7 B7-H3 CAR+ T cells. F, Survival curves of mice treated as in E. Representative results of four experiments with 3 different PBMC donors are shown. Error bars, SEM. P values were calculated as described in Materials and Methods.

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: In Vitro, Injection, Derivative Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Systemically administered B7-H3 CAR T cells induce regression of Ewing sarcoma xenografts. A, Mouse model of orthotopic Ewing sarcoma: 2e7 EW8 tumor cells were injected into the periosteum of the tibia in NSG mice. Two weeks later, 1e7 B7-H3 CAR+ T cells or irrelevant control CD19 CAR T cells were intravenously administered. B, Tumor growth was measured twice weekly by digital caliper and tumor area was calculated. Values for individual mice, as well as mean values per treatment group (inset) are shown. C, Survival curves of mice treated as in A. Representative results of two experiments with 2 different PBMC donors are shown. Error bars, SEM. P values were calculated as described in Materials and Methods.

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: Systemically administered B7-H3 CAR T cells can clear medulloblastoma xenografts. A, B7-H3 CAR T cells were cocultured in vitro with medulloblastoma cell lines and patient-derived DIPG cell cultures and, 24 hours later, IFNγ, TNFα, and IL2 were measured in the supernatant. Representative results of three experiments with 3 different PBMC donors are shown. B, Orthotopic xenograft model of medulloblastoma: NSG mice were autochthonously injected with luciferase expressing DAOY medulloblastoma tumor cells. Following evidence of tumor engraftment by IVIS imaging, animals received 1e7 B7-H3 CAR+ T cells or CD19 CAR T cells intravenously. C, In vivo imaging of DAOY tumors treated with B7-H3 or CD19 CAR T cells. D, Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Values for individual mice, as well as mean values per treatment group (inset) are shown. Representative results of three experiments with three different PBMC donors are shown. E, Orthotopic xenograft model of c-myc–amplified medulloblastoma: D425 tumor cells expressing luciferase were autochthonously injected into NSG mice. Mice were treated with 1e7 B7-H3 CAR+ T cells or CD19 CAR T cells after 3–4 days, at which point tumor was detectable by IVIS imaging. F, Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Values for individual mice, as well as average values of living mice per treatment group (inset) are shown. G, In vivo imaging of D425 tumors treated with B7-H3 or CD19 CAR T cells. H, Survival curves of mice shown in G. Representative results of three experiments with three different T cell donors are shown. I, Confocal images of brains from D425-GFP+ tumor bearing mice treated with B7-H3 CAR-mCherry or CD19 CAR-mCherry T cells, harvested at two different time points after T-cell infusion. Representative image of two mice at two time points in one experiment. Error bars, SEM. P values were calculated as described in Materials and Methods.

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: In Vitro, Derivative Assay, Injection, Luciferase, Expressing, Imaging, In Vivo Imaging, Software, Amplification

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: B7-H3 CAR T cells have limited activity against B7-H3 low expressing K562 xenografts. A, Mouse model of K562 leukemia: NSG mice were inoculated with K562, a myeloid leukemia that expresses low levels of B7-H3, and then treated with 1e7 B7-H3 CAR+ T cells or mock transduced control T cells 5 days later. B, Survival curves of mice treated as in A. Representative results of five experiments with three different PBMC donors are shown. C, Flow cytometric analysis of B7-H3 expression on the surface of K562 (leukemia), MG63.3 (osteosarcoma), EW8 (Ewing sarcoma), DAOY, and D425 (medulloblastoma) human cell lines. D, Number of B7-H3 surface molecules expressed by human tumor cell lines as estimated by Quantibrite kit.

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: Activity Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors

doi: 10.1158/1078-0432.CCR-18-0432

Figure Lengend Snippet: B7-H3 CAR T cells require adequate antigen expression for in vitro and in vivo activity. A, Flow cytometry analysis of B7-H3 expression on single-cell clones derived from Nalm6 expressing different amounts of lentivirally expressed B7-H3. B, Number of B7-H3 surface molecules expressed by Nalm6-B7-H3 cell lines as estimated by Quantibrite kit. C, GFP+ Nalm6-B7H3 clones were cocultured with B7-H3 CAR T cells and tumor cell killing was measured in an Incucyte assay over 72 hours. Representative data of three experiments with three different PBMC donors is shown. D, Percentage of CAR T cells positive (left) and mean fluorescence index (right) for T-cell activation and degranulation markers CD69 and CD107a, as measured by flow cytometry 6 hours after coculture of B7-H3 CAR T cells with tumor cells expressing increasing amounts of B7-H3. Representative results of three experiments with three different PBMC donors are shown. E and F, Cytokine production by CAR T cells cocultured with tumor cells expressing increasing amounts of B7-H3. G, Mouse model for Nalm6-B7H3: 1e6 NALM6 cells expressing either low or medium amounts of B7-H3 were engrafted into mice by tail vein injection. Three days later, mice were injected with 1e7 B7-H3 CAR+ T cells or untransduced control T cells (MOCK). In vivo imaging of mice bearing (H) Nalm6-B7-H3-Medium leukemia or (I) Nalm6-B7-H3-Low leukemia. J and K, Tumor progression was measured by bioluminescence photometry and flux values (photons per second) were calculated using Living Image software. Values for individual mice are shown. Representative results of four (Nalm6-B7-H3-Med) and two (Nalm6-B7-H3-Low) experiments with two different PBMC donors are shown. N6, NALM6; GL, GFP-Luciferase.

Article Snippet: Supernatant was collected and then used in a Human B7-H3 Quantikine ELISA Kit (R&D Systems).

Techniques: Expressing, In Vitro, In Vivo, Activity Assay, Flow Cytometry, Clone Assay, Derivative Assay, Fluorescence, Activation Assay, Injection, In Vivo Imaging, Software, Luciferase

Journal: Diabetes

Article Title: Tissue-specific effects of rosiglitazone and exercise in the treatment of lipid-induced insulin resistance.

doi: 10.2337/db06-1065

Figure Lengend Snippet: FIG. 4. AS160, GLUT4, and isoform-specific skeletal muscle Akt content. Relative protein levels of total Akt 1/2 (A), Akt phosphorylation at Ser473 (B), isoform-specific Akt1 (C) and Akt2 (D), and total AS160 (E) and GLUT4 (F) were quantified using Western blot analysis and densitometry. SDs between groups (P < 0.05) are indicated by the P values listed on the figure; n 7–8/group.

Article Snippet: The membranes were blocked and incubated overnight at 4°C with antibodies specific for phospho-Akt Ser473, Akt, Akt1, Akt2 (1:1,000; 4058, 9272, 2967, and 2962; Cell Signaling), AS160 (21) (a gift from David James, Garvan Institute, Sydney), PPAR coactivator 1 (PGC-1) (AB3242; Chemicon International), PEPCK (10004943; Cayman Chemical), or GLUT4 (46701-704; Biogenesis).

Techniques: Phospho-proteomics, Western Blot