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: 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: mRNA expression of MT1-MMP, MRC2, CD276, and LRRC15 in osteosarcoma, normal tissues, and other pediatric cancers. A–D, RNA-seq data showed MT1-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, acute myeloid 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: 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 = 8; A) and PDXs (n = 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: 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 patients with 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: 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: Chemical structure, surface plasmon resonance, and pharmacokinetic analysis of BT1769. A, Chemical structure of BT1769, which exhibits high binding affinity to human and mouse MT1-MMP, as determined by SPR. B, Antitumor activity of 3.2 mg/kg BT1769 (IV, QW) in HT1080 xenograft-bearing mice is shown on the left (P < 0.001; two-way ANOVA). BT1769 concentration in plasma and MMAE concentrations in plasma and tumor are shown on the right. The table shows the half-life (T1/2) and systemic clearance (Cl) of BT1769 in a naïve CD-1 mouse after IV dosing of 3 mg/kg BT1769.

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: SPR Assay, Binding Assay, Activity Assay, Concentration Assay, Clinical Proteomics

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: Tumor volume and survival after BT1769 treatment in osteosarcoma preclinical models. A, BT1769 showed objective responses (MCR) in 3 osteosarcoma models, PD1 in 2 models (OS1, OS9), and PD2 in 1 model (OS31). The 2 Ewing sarcoma models (ES1, TC-71) had PD1. (MCR = maintained complete response. See appendix for detailed definitions.) Pale colored lines represent individual mice; dark colored lines show cohort median values. B, BT1769 induced significant improvement in EFS compared with control in all 6 of the osteosarcoma models tested (P < 0.05). C, RNA-seq data showed the expression level of MT1-MMP in the tested PDX models. D, MT1-MMP IHC staining of the OS17 recurrent tumor showed a low MT1-MMP expression level after initial MCR compared with the pretreatment tumor. E, MT1-MMP was negative in the Ewing sarcoma models (ES1, TC-71).

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: Control, RNA Sequencing, Expressing, Immunohistochemistry

Journal: Journal of biomedical science

Article Title: Tumor necrosis factor-inducible gene 6 protein and its derived peptide ameliorate liver fibrosis by repressing CD44 activation in mice with alcohol-related liver disease.

doi: 10.1186/s12929-024-01042-5

Figure Lengend Snippet: Fig. 6 Peptide YJ derived from TSG-6 lessens production and activation of CD44ICD and HSC activation. a 3D structure of the most stable clusters of peptide YJ (cyan), CD44 (green) and MMP14 (magenta) predicted by HADDOCK. Magnified image in right panel presents the interaction site of peptide YJ with catalytic region of MMP14. b Western blot and cumulative densitometric analysis of nuclear CD44ICD in pHSCs treated with YJ. Band densities were normalized to the expression level of LAMIN B1, which was used as an internal control. GAPDH was used for negative control in nuclear fraction. c Representative images of double immunofluorescence staining for CD44ICD (red) and α-SMA (green), and quantitative analysis of nuclear CD44ICD-positive cells. Magnified images of CD44ICD-stained images in bottom panel are shown at X60. DAPI (blue) was used as nuclear counterstaining (Scale bar, 50 μm). d qRT-PCR analysis of fibrotic markers including α-SMA, transforming growth factor-β (TGF-β), COL1α1,tissue inhibitor of metalloproteinase 1 (TIMP1) and connective tissue growth factor (CTGF) in these cells. e Western blot and cumulative analysis for CD44, CD44ICD, TGF-β, α-SMA and glial fibrillary acidic protein (GFAP) in whole lysate of these cells. Band densities were normalized to the expression level of GAPDH, which was used as an internal control. The data shown represent one of three experiments with similar results and are presented as mean ± S.E.M. (*p < 0.05, **p < 0.005). Gray circles represent individual data points

Article Snippet: These cells were washed with TBS and blocked in protein blocking solution (X9090; Dako) for 30 min and incubated with first primary antibody, CD44ICD (KAL-KO601; CosmoBio; Carlsbad, CA, USA) or MMP14 (af918; R&D Systems) at 4 °C overnight and followed by Alexa Fluor 568-conjugated goat anti-rabbit IgG (A10042, Invitrogen) or Alexa Fluor 647-conjugated donkey anti-goat IgG (A21447, Invitrogen) for 30 min. For second primary antibodies, sections were incubated with α-SMA (A5228; SigmaAldrich) or CD44 (ab157107; Abcam) for 2 h at room temperature and followed by Alexa Fluor 488-conjugated chicken anti-mouse IgG (A21200, Invitrogen) or Alexa Fluor 568-conjugated goat anti-rabbit IgG (A10042, Invitrogen) for 30 min. 4’,6-diamidino-2-phenylindole (DAPI) were employed in the counterstaining procedure.

Techniques: Derivative Assay, Activation Assay, Western Blot, Expressing, Control, Negative Control, Double Immunofluorescence Staining, Staining, Quantitative RT-PCR