We detected 33 genes showing opposite enrichment in 2D and 3D screens in HLF cells, including KEAP1, a known tumor suppressor that also mediates sorafenib, lenvatinib, and regorafenib resistance.28,29 Multiple genes involved in ubiquitination (CUL3, CUL5, UBE2D3, HUWE1, and SPOP) showed a proliferation benefit or were neutral only when knocked out in 3D CRISPR screens, while they were highly essential in 2D conditions, suggesting that ubiquitination plays a restrictive role in HCC growth in 3D conditions. Here, SPOP is linked to hepatocellular carcinoma.