24 ultra low attachment plates

Pancreatic cancer spheres were generated and expanded in CSCs media composed of: Advanced DMEM:F12 (GIBCO) supplemented with 1 × glutaMAX (GIBCO), 1 × B-27 (GIBCO), 1 × N2 (GIBCO), 20 ng/ml bFGF (basic fibroblast growth factor) (Invitrogen), and 50 ng/ml EGF (epidermal growth factor) (Peprotech, London, UK). Five hundred cells per 500 µl of sphere medium were seeded in 24 ultra-low attachment plates (Corning B.V., Schiphol-Rijk, Netherlands) as described previously [20 ]. After 7 days of incubation, spheres were typically > 75 µm large. For serial passaging, 7-day-old spheres were harvested using 40 µm cell strainers, dissociated into single cells with trypsin, and then regrown for an additional 7 days. Cultures were kept no longer than 4 weeks after thawing from frozen stocks (passage 3–4). Statistical significance was assessed by Student's t-test.

Cells (2 × 10⁵ cells/well into 6-well plates) were treated with sTN145, sTN58, sTN29 or Scr (400 nM-final concentration). After 24 h of treatment, cells were trypsinized, resuspended in Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F-12, Sigma-Aldrich) serum-free medium, and counted. Cells (5 × 10³ cells/well) were plated in 24 ultralow attachment plates (Corning Incorporated) and grown in DMEM/F-12 serum-free medium, supplemented with B27 (1:50, Invitrogen), 20 ng/ mL basic fibroblast growth factor and 20 ng/ mL EGF (Sigma-Aldrich). After 10 days, the size and number of primary mammospheres (P0) were analyzed under phase-contrast microscopy (Leica DMI3000 B apparatus). All spheres with a diameter >40 μm were counted and measured in at least 10 fields per condition. Primary mammospheres (P0) were then collected, disaggregated into single-cell suspension mechanically after a short incubation with trypsin (Sigma-Aldrich), and reseeded (5 × 10³ cells/well into 24 ultralow attachment plates) in a stem culture medium to form a secondary mammosphere population (P1). Self-renewal activity was evaluated by counting the number of P1 mammosphere/cell-seeded × 100. Cells treated in the same way except without the aptamers (mock-treated) were used as control.

For 3D heterotypic tumor spheroids, 2 × 10³ cancer cells were mixed with 8 × 10³ MSCs (ratio 1:4) and seeded in 24-ultralow attachment plates (Corning Incorporate, Corning, NY) in Dulbecco's Modified Eagle Medium (DMEM)/F-12 medium (D8437 Sigma-Aldrich), supplemented with 2% Matrigel Basement Membrane Matrix Growth Factor Reduced (Corning Incorporate), B27 (1:50, Gibco™ by Invitrogen, Carlsbad, CA), 20 ng/ml basic fibroblast growth factor (Sigma-Aldrich) and 10 ng/ml epidermal growth factor (Sigma-Aldrich). For homotypic cultures, 2 × 10³ cancer cells or 8 × 10³ MSCs were seeded alone. Spheroid formation was checked daily using a phase-contrast microscopy (Leica DMI3000 B apparatus), images were captured and the diameter of spheroids was measured.