ACSL4 and polyunsaturated lipids support metastatic extravasation and colonization

Metastasis is the leading cause of the cancer-related death. To systemically study the dependent factors impacting cancer metastasis, we conducted serial transplantation to obtain ES-2-MC2-Liver cells, a cell line with high metastatic efficiency. We then performed in vivo CRISPR screen using phosphatase and metabolism-focused sgRNA library and ranked differential targeting genes. For further validating the identified gene essentialities in previous results, we generated a mini-library targeting the top-depleted and -enriched genes in metastatic organs and performed in vivo CRISPR screen on ES-2-MC2-Liver cells and SKHEP1 cells. Notably, polyunsaturated lipids synthase, ACSL4, displayed as the top perturbed hits among the lung-specific metastatic mediators. We also evaluated enzymes involved in unsaturated fatty acids degradation were dispensable for metastatic tumor colonization. Taken together, our results systemically study the mediators of cancer metastasis and established the dual functions of PUFA-lipids in tumor progression and metastasis that may be exploitable for therapeutic development. Overall design: 1. We infected the ES-2-MC2-Liver cells with sgRNA from phosphatase- and metabolism-focused CRISPR library. Cells collected before intraperitoneal implantion served as the baseline (n=1). Comparative analyses were then conducted between cancer cells isolated from the metastatic livers (n=5) or lungs (n=3 for the phosphatase library and n=5 for the metabolism library) and subcutaneous tumors. 2. ES-2-MC2-Liver and SKHEP-1cells were utilized to perform in vivo pooled mini-CRISPR screens for validation. Cells collected before implantation served as the baseline (n=4). Comparative analyses were conducted between cancer cells isolated from the metastatic livers (n=4 for ES-2-MC2-Liver cells) or lungs (n=4) and subcutaneous tumors (n=4).