Journal: PLoS ONE

Article Title: A Novel 3D Fibril Force Assay Implicates Src in Tumor Cell Force Generation in Collagen Networks

doi: 10.1371/journal.pone.0058138

Figure Lengend Snippet: (A) Flow cytometry was performed on GFP-ca-Src and GFP-wt-Src cells cultured to subconfluence in tissue culture flasks. The GFP-ca-Src and GFP-wt-Src expressing cells were harvested by trypsinization, washed, and then were labeled with anti-MMP-14/MT1-MMP-PE while cells were kept chilled on ice. Following immunostaining, the cells were washed, fixed and then subjected to FACS analysis gating cell fluorescence intensities into four groups: top, hi-mid, low-mid, and bottom 25 percentiles for GFP expression. Controls (at left; no antibody) were compared to anti-MMP-14/MT1-MMP-phycoerythrin (anti-MMP-14-PE, at right). (B) The mean fluorescence intensity per cell was plotted for anti-MMP-14-PE versus control (no antibody).

Article Snippet: A volume of 5 µL Phycoerythrin (PE)-conjugated mouse monoclonal anti-human MMP-14/MT1-MMP (R&D Systems, 100 tests) was added to the suspension and incubated for 30 minutes on ice.

Techniques: Flow Cytometry, Cell Culture, Expressing, Labeling, Immunostaining, Fluorescence

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 Assay, 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

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 Assay, Expressing, Immunohistochemistry