Spheroid Formation Assay for CRC Cells

To investigate the capacity of CRC cells to grow under non-adherent conditions, cells were seeded at a density of 1.5 × 10⁶ cells in 10 cm plates. After 24 h of recovery, the cells were treated with increasing concentrations of SARB (10, 20, 50 μM) or Combi 35 µM. Incubation continued for another 48 h, then the cells were washed with PBS, trypsinized and 5 × 10⁴ cells were then seeded in ultra-low attachment (ULA) six-well plates (Corning) in 2 ml of CSC medium (PromoCell) and 40 μl Matrigel with growth factors (Corning). Spheroid formation was monitored 7 days after seeding and then the spheroids were transferred to 15 ml falcon tubes, washed with PBS, trypsinzed, and 2.5 × 10⁴ cells were seeded in ULA six-well plates with 2 ml CSC medium and 40 μl Matrigel. Cells were let grow for another 14 days and fresh medium was added every 2–3 days. At day 21, the spheroid growth was documented by light microscopy using a Nikon Eclipse Ti–S microscope or Leica DMI1 light microscope. The images were then analyzed using a macro (given in SI) which was imported to ImageJ (modified from ref. ^{58 (link)}) to determine spheroid number.