Epigenetic silencing via methylation of the ASS1 promoter was previously demonstrated in certain cancer types, so we assessed ASS1 mRNA levels in immortalized normal hepatocyte (Fa2N-4 and Miha cell) spheroids and in HCC spheroids derived from Asian (SUN449, SUN475, SNU878, and SNU398 cells) and Caucasian (Hep3B, Huh6, HepG2, and PLC/PRF/5 cells) patients. This evidence concerns the gene ASS1 and cancer.