br Western blotting br Cell lysates were prepared
2.7. Western blotting
Cell lysates were prepared using an SDS-based buffer, and subse-quent protein concentration was determined using a standard bicinchoninic Coelenterazine (BCA) assay. 40 μg of proteins were separated using SDS-PAGE and transferred to Immun-Blot PVDF Membrane (Biorad, Hercules, CA, USA). After blocking in a 5% milk TBS-Tween solution, the membrane was blotted overnight at 4 °C with primary antibodies directed against cytosine deaminase, Fc specific human IgG, and GAPDH before being probed with secondary antibodies conjugated to horseradish peroxidase for 1 h at RT (Supplementary Table 1). After in-cubation with ECL substrate (Fisher Scientific), luminescence was de-tected using a ChemiDoc XRS+ (Bio-rad) and quantified using Image Lab 3.0.1 Beta 2 (Bio-rad).
OPG concentration was measured in culture supernatants of MSC using a human osteoprotegerin/TNFRSF11B DuoSet ELISA (R&D Sys-tems) according to the manufacturer's recommendations. Each su-pernatant was assessed undiluted, diluted 1:10, and diluted 1:50 to be quantified in the linear range, and dosed in duplicates. As a control, 1 ng/mL of recombinant human OPG was included in the assays.
2.9. Flow chamber assay
5 × 105 P-2 HUVEC were seeded onto 35 × 10 mm Petri dishes (Corning) coated with 1% gelatin (Sigma) and incubated for one day to reach confluence. HUVEC were stimulated using 50 ng/mL human TNF-α 6 h prior to the assay to induce selectin expression. A flow cham-ber assay was performed using a PicoPlus syringe pump (Harvard Appa-ratus, Holliston, MA, USA) set at four different flow rates (4.42 μL/min to create 1 dyn/cm2 shear force, 8.84 μL/min for 2 dyn/cm2, 17.68 μL/min for 5 dyn/cm2, and 35.36 μL/min for 10 dyn/cm2), and a vacuum pump (Welch, Mt. Prospect, IL, USA) collecting post-chamber efflux. Briefly, 1 × 106 cells/mL of each cell type to be assayed were stained with 2.5 μM CellTrace™Calcein Green dye for better quantification using fluores-cence, suspended in fresh EGM-2, and loaded in a 1 mL syringe attached to the syringe pump. A flow chamber gasket (GlycoTech, Gaithersburg, MD, USA) was attached to the top of the HUVEC-seeded petri dish, a Silastic™ (GlycoTech) fluid line was fixed to the tip of the syringe and connected to the inflow port of the flow chamber gasket, and a drain line was run from the outflow port of the gasket to the waste chamber of the vacuum pump. 300 μL of cell suspension was injected across the HUVEC layer for each run.
On day 0, MSC were engineered using mRNA transfection. On day 1, MDA-MB231 LucF/RFP cells were respectively plated in a 96-well plate at 1.5 × 104 cells per well and in a 24-well plate at 1 × 105 cells per well, to reach 90% confluency on day 2. On day 3, depending on the assay con-ditions, 5-FC or 5-FU was added, and different ratios of MSC were plated on top of cancer cells in MSC culture media. On day 9, the 24-well plate was imaged using brightfield and fluorescence to visualise the co-culture and MDA-MB-231 expressing RFP. The 96-well plate was incu-bated with AlamarBlue™ (Fisher Scientific) at a 1:10 dilution in 100 μL fresh culture medium to measure the viability of the co-culture. After 5 to 6 h, the absorbance was measured at a wavelength of 570 nm (the peak absorbance for the reduced form) and 600 nm (peak absorbance for the oxidised form) using a plate reader (Synergy HT, Biotek, Winooski, VT, USA). Briefly, the metabolic growth (and thereby the viability) of the cells was determined by subtracting the ab-sorbance of the oxidised form from that of the reduced form. Data were normalised to the control (untreated cells).
Concentrated media enriched for osteoprotegerin was generated by centrifuging 15 mL of conditioned media from MSC (day 3 post-engineering) using a 30 kDa Amicon Ultra-15 centrifugal filter unit (EMD Millipore, Temecula, CA, U.S.A.) at 4000g for 15 min. MDA-MB-231 cells were plated in a 96-well plate at a density of 1.5 × 104 cells/ well and treated with recombinant human TRAIL in addition to rhOPG or concentrated MSC supernatant containing an equivalent concentra-tion of OPG (as determined by ELISA).