FFPE samples (CRC and adjacent tissues) were sectioned into 4µm thick, and prepared for immunohistochemistry staining. The sections were incubated with rabbit polyclonal anti-FAT4 antibody (ab243591, 1:200, Abcam, UK) at 4 °C overnight. After washing the sections by PBS, the sections were then incubated with goat anti-rabbit secondary antibody (ab205718, 1:2,000, Abcam, UK) at room temperature for 30 min. The peroxidase reactivity was measured by 3,3’-diaminobenzidine (DAB) (7411-49-6, Suzhou Yacoo Chemical Reagent Co., Ltd, China) and counterstained by haematoxylin. The IHC evaluation was conducted according to the description of Kusinska (16 (link)). The score grade of IHC intensity was classified as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). Based on the scores, positive cells were accordingly assigned into four groups, namely, 0 (negative), 1(less than 10%), 2 (from 10% to 50%), and 3 (more than 50%) groups. The score of IHC intensity multiplied by the percentage of positive cells was expressed as the final score. If the final score was lower than or equal to 1, it was considered to be negative, while the score ranging from 2 to 9 was positive.