Batimastat

Spheroids were treated upon plating with a panel of antagonists or agonists at the following final concentrations: 20 nM batimastat (R&D 2961/10), 40 μM ciliobrevin D (Sigma 250,401), 10 μM defactinib (Med Chem HY-122899), 30 nM pemigatinib (Med Chem HY-109099), 1 μM volsertib (Selleck S2235), 4 mM 2-Deoxy-Glucose (Sigma D8375), 10 μM GC7 (EMD Millipore 259,545), 1 μg/mL follistatin (R&D 769-FS-025), 1 μg/mL collagen XV (9854-CL-050), 5 μg/mL collagen I (Sigma C2249), and 10 ng/mL activin-A (R&D 338-AC-010). For integrin inhibition, PDAC-MSC multicellular spheroids were treated with 2 mM EGTA or the mAb13 anti-ITGB1 antibody (Sigma MABT821) at a 1:10 dilution.

Collagen I infiltration and migration assay was assessed by seeding 2 × 10⁶ PC013, BxPC-3, or CAPAN2 cells in 10% FBS DMEM overnight. The medium was replaced the next day with fresh serum-free DMEM supplemented with 2 μCi/ml [6-³H] thymidine. Collagen I matrix gels were prepared by diluting bovine collagen I (Life Technologies, Carlsbad, CA) in serum-free control medium and supernatants from PSCs cultured in the presence of control (no supplements), IL-1α (1 ng/ml), and TGFβ (2 ng/ml) as single agents or in combination, and 10 μM of the broad-spectrum MMP inhibitor batimastat (R&D Systems Europe, Ltd, Abingdon, England). Cancer cells were added to a final concentration of 1 × 10⁶ cells/ml and a collagen I concentration of 0.3%. Fifty microliters of collagen I gel mixture containing 5 × 10⁴ cells was distributed to cell culture inserts (pore size 8.0 μM) placed in 24-well plates. The inserts were incubated at 37°C for 30 minutes before adding DMEM supplemented with 10% FBS to the bottom compartment. After 24 hours of incubation at 37°C, the cells were harvested by applying a cotton stick on each side of the insert surface, and [6-³H] thymidine incorporation was determined by liquid scintillation (Packard Tri-Carb 1900 TR). Infiltration and migration of cells were calculated as [6-³H] thymidine incorporation detected on the bottom side divided by total incorporation.