Cellmatrix type 1 a collagen

Anti-MT1-MMP (222-1D8) antibody was kindly provided by Prof. Motoharu Seiki (University of Tokyo, Tokyo, Japan), DX-2400 was a gift from Dyax Corp. (Burlington, MA). Anti-MT1-MMP antibody (EP1264Y) was from Abcam (Cambridge, UK), anti-DDR2 antibody (AF2538) was from R&D Systems (Abingdon, UK), anti-actin antibody (C-19) was from Santa Cruz Biotechnology (Santa Cruz, CA), anti-β1 integrin antibodies (AB1952, 6S6, and P4G11) were from Millipore (Watford, UK). Anti-phosphotyrosine (4G10) and anti-mouse and anti-goat alkaline phosphatase-conjugated antibodies were from Sigma-Aldrich, and anti-rabbit alkaline phosphatase-conjugated antibody was from Promega (Southampton, UK). SMARTpool ON-TARGETplus siRNAs for MT1-MMP, DDR2, integrin β1 subunit (ITGB1), and siNT were from Thermo Scientific (Northumberland, UK). Recombinant TNF-α and IL-1β were from Peprotech (London, UK), PureCol collagen type I was from Advanced BioMatrix (Leimuiden, The Netherlands), CellMatrix type I-A collagen was from Nitta-Gelatin Inc. (Osaka, Japan), human and bovine type II collagens were from Chondrex Inc. (Redmond, WA), dasatinib was from LC Laboratories (Woburn, MA), trypsin was from Sigma-Aldrich, and DDR1-IN-1 was synthesized as reported previously (21 (link)).

Collagen film degradation was carried out as described previously [61 (link),62 (link)]. Briefly, PureCol (bovine collagen type-I, Advanced Biomatrix, San Diego, CA, USA) and Cellmatrix type I-A collagen (Nitta Gelatin, Osaka, Japan) were mixed in the ratio of 1:1. This mixture was neutralized and diluted to 2 mg/ml. 12-well multi-well plates were coated with 100 μl collagen solution/well and set for gelation. Cells were cultured on the collagen film for 72 h in the presence or absence of GM6001 (10 μM), TIMP-1 (200 nM), or DX-2400 (200 nM). Cells were removed extensively by trypsin/EDTA (Lonza), and the remaining collagen layer was fixed with 4% formaldehyde in PBS and stained with R-250 Coomassie Brilliant Blue (Thermo Fisher). Representative images were acquired using the 10X dry lens (NA = 0.3) on the Nikon TE2000-E microscope equipped with an ORCA-ER CCD camera (Hamamatsu Photonics) operated by Volocity Acquisition module software (Improvision, PerkinElmer). Collagen degradation was quantified using Fiji by measuring the integrated density of the collagen layer. Mean results were plotted using Prism, and statistical significance was calculated using one-way ordinary ANOVA with Tuckey's multiple comparison test.

To evaluate the antitumor effect of oncolytic HSVs, surgical sections of gastric cancer tissue were cultured in collagen medium for a short time (within 72 h), as previously described [29 (link)]. This ex vivo experimental study received approval by the Wakayama Medical University Human Ethics Review Committee. Human gastric cancer specimens derived from radical gastrectomy were incubated ex vivo on collagen gel immediately after resection. In particular, the mixture of cell matrix type I-A collagen (3 mg/ml; Nitta Gelatin, Japan), reconstitution buffer comprising 2.2% (w/v) NaHCO₃, 0.2 M HEPES, and 50 mM NaOH, and 10× RPMI-1640 medium was poured into 24-well dishes (0.5 ml/well). After preparation of gastric cancer specimens, 2 mm³ cancer pieces placed on collagen gel. Each well was treated with PBS (–), T-01, or T-SCOCS3 at dose of 1 × 10⁹/ml for 1 hr. After 72 hr of incubation at 37°C, the cells viability of gastric cancer tissues was assessed by use of a Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, USA). For H&E staining, virus-treated cancer tissues were embedded in paraffin, and then histological examination was performed according to the instruction manual.