However, spheroid size was significantly smaller (2.1-fold, p < 0.001) in the HCT116 ASNS−/− cell lines compared to the HCT116 ASNS+/+ group seven days after cell seeding under an asparagine-sufficient condition (Fig. 1C), thus highlighting the role of ASNS in initiating CRC cell proliferation and growth using a 3D culture system. Here, ASNS is linked to colorectal carcinoma.