Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 3. Rate of activation of MMP-2 by MT2-MMP via the TIMP- 2-independent pathway. Gelatin zymography of conditioned medium of transfected Timp2/ cells expressing (A) hMT2-MMP cultured in the absence of TIMP-2, or (B) hMT1-MMP cultured in the presence of 3 nM TIMP-2, or vector control cells cultured in the presence of 3 nM TIMP-2 as indicated. Cells were cultured in serum-free medium con- taining 5 nM recombinant TIMP-2-free pro-MMP-2 with and without ConA for the times indicated in hours. Medium containing pro-MMP-2 incubated without cells (M) is shown, and the positions of pro-, inter- mediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Activation Assay, Zymography, Transfection, Expressing, Cell Culture, Plasmid Preparation, Control, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 4. Effects of TIMP-2 and TIMP-4 on MMP-2 activation by MT2-MMP. Gelatin zymography of conditioned medium of Timp2/ cells transfected with: A, hMT2-MMP; B, hMT1-MMP; or C, vector alone. Cells were cultured for 24 h in serum-free medium containing 5 nM TIMP-2-free pro-MMP-2, and between 0 and 250 nM TIMP-2 or TIMP-4 was added as indicated. Medium containing MMP-2 incubated without cells (M) is shown, and the positions of pro-, intermediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Activation Assay, Zymography, Transfection, Plasmid Preparation, Cell Culture, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 5. Activation of MMP-2 by MT2-MMP is cell-associated. Gelatin zymography of cell extracts and culture supernatants of Timp2/ cells expressing hMT2-MMP or hMT1-MMP or transfected with the vector alone. Cells were cultured for 24 h in serum-free me- dium with and without 5 nM recombinant TIMP-2-free pro-MMP-2 and 3 nM TIMP-2 as indicated. Culture supernatants were removed, and cells were washed extensively in PBS before being lysed in 0.2% Zwit- tergent. Medium containing pro-MMP-2 incubated without cells (M) is shown, and the positions of pro-, intermediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Activation Assay, Zymography, Expressing, Transfection, Plasmid Preparation, Cell Culture, Recombinant, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 6. Recombinant MMP-2 hemopexin C domain inhibits ac- tivation of MMP-2 by MT2-MMP. Gelatin zymography of cell culture supernatants of Timp2/ cells expressing: A, hMT2-MMP; B, hMT1- MMP; or C, transfected with the vector alone. Cells were cultured for 24 h in serum-free medium containing a 0- to 500-fold molar ratio of recombinant MMP-2 hemopexin C domain (CD) to pro-MMP-2. Cells expressing hMT1-MMP or transfected with the vector alone were incu- bated in the presence of 3 nM TIMP-2 (B and C). Medium containing MMP-2 incubated without cells (M) is shown, and the positions of pro-, intermediate, and active forms of MMP-2 are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Recombinant, Zymography, Cell Culture, Expressing, Transfection, Plasmid Preparation, Incubation

Journal: Journal of Biological Chemistry

Article Title: Cellular Activation of MMP-2 (Gelatinase A) by MT2-MMP Occurs via a TIMP-2-independent Pathway

doi: 10.1074/jbc.m108643200

Figure Lengend Snippet: FIG. 7. MT2-MMP does not activate truncated MMP-2 lacking the hemopexin C domain. Gelatin zymography of culture superna- tants and cell extracts of Timp2/ cells expressing hMT2-MMP or hMT1-MMP or transfected with the vector alone. Cells were cultured for 24 h in serum-free medium containing either recombinant full- length pro-MMP-2 or pro-MMP-2CD as indicated. Cells expressing hMT1-MMP or transfected with the vector alone were incubated in the presence of 3 nM TIMP-2 ( TIMP-2). Clones #4.7 and #42 expressing hMT2-MMP were incubated without TIMP-2. Full-length MMP-2 (lane 1) or MMP-2CD (lane 13) supernatants were treated with 0.5 mM p-aminophenylmercuric acetate as activation standards (Std) and to show that both forms of recombinant MMP-2 could be activated. The positions of MMP-2 and MMP-2CD are indicated.

Article Snippet: Designated wells were incubated with 2.5 g/ml mouse monoclonal antibody against hMT1-MMP (MAB918) (R & D Systems, Inc.), 2.5 g/ml rabbit polyclonal antibody against MT1MMP (AB815) (Chemicon), 20 g/ml mouse monoclonal antibody against MT2-MMP (MAB3220) (Chemicon), 2.5 g/ml affinity-purified rabbit polyclonal antibodies that we raised against the following peptide sequences in hMT2-MMP, 285DVDNFKLPEDDLRG298, 341GGKPERPPKPG351, and 461EETQRGDPGYPK472, using methods described before (21), control mouse immunoglobulin (Dako), control non-immune rabbit serum (Molecular Probes), or TBS-BSA alone, for 2 h at 20 °C and then washed four times with excess TBS.

Techniques: Zymography, Expressing, Transfection, Plasmid Preparation, Cell Culture, Recombinant, Incubation, Clone Assay, Activation 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: (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 osteosarcoma patients 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) ( 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: (A-D) RNA-seq data showed MT1-MMP (A), MRC2 (B), CD276 (C), and LRRC15 (D) were overexpressed in osteosarcoma compared to 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 to 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; TPM Transcripts Per Million, FPKM Fragments Per Kilobase 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) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: RNA Sequencing Assay, Expressing, 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: (A, 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) ( 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: (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. (PD = progressive disease, 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 event-free survival (EFS) compared to 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 to 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) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: RNA Sequencing Assay, Expressing, Immunohistochemistry

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: (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, K) Summary of IHC staining H-score of MT1-MMP, MRC2, and CD276 in the tissue microarray for 37 osteosarcoma patients (J) and 19 PDX models (K). Boxes indicate standard deviation 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) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Staining, Negative Control, Immunohistochemistry, Microarray, Standard Deviation

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: (A) Chemical structure of BT1769, which exhibits high binding affinity to human and mouse MT1-MMP, as determined by SPR. (B) Anti-tumor 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) ( 27 ), and CD276 (R&D Systems, FAB1027P, 1:40) were added.

Techniques: Binding Assay, Activity Assay, Concentration Assay