*2.7. In vitro Scratch Assay*

CaCo-2 and CoLo-205 cells were plated in six-well plates to create a cell monolayer, following the protocol described by C. Liang et al [25]. Briefly, a "scratch" with a p200 pipette tip was created on the cell monolayer, for each sample and experiment. After removal of the debris by washing with 1 mL of culture medium, an ultrafine tip marker was used to create markings on the outer bottom of the plate that represent reference points close to the scratch. For the scratch assay, each cell line was incubated with 3 mL of medium containing free 5-FU, 5-FU/LPs or anti-FZD10/5-FU/LPs at a 5-FU concentration of 2 µmol for 24 or 48 h. The experiments were conducted at 37 ◦C. Untreated cells were used as control for each tested cell line. At 0, 24 and 48 h, the plates were placed under a confocal microscope Nikon Eclipse Ti2, for phase-contrast assessment to acquire the images of the scratch before and after cell incubation with the different samples.
