Parallel Genome-Wide CRISPR Screens Clarify Executors of Epithelial-Mesenchymal Transition Mediated Immune resistance to CD8+ T cells

To investigate the role of tumor epithelial-to-mesenchymal plasticity in CD8+ T cell cytotoxicity, we generated two types of tumor cells with epithelial-like (Epi) or mesenchymal-like (Mes) features through an inducible EMT system. The parental KPC3-OVA cells were transduced with constitutively kinase active ALK5 cDNA (ALK5 CA) in doxycycline-induced system by Nakao et al. and Itoh et al, which initiated TGF-β signaling induced EMT. In this system, we firstly treated cells with TGF-β (5 ng/mL) for 2 days, then removed TGF-β ligands and added doxycycline to sustain cancer cells with mesenchymal feature. We proved the mesenchymal-like phenotype could maintain more than 16 days, which was long enough for all our experiments. Then we performed RNA-seq to investigate the differences between these two types of KPC3-OVA :: Tet-On ALK5 CA cells co-cultured with OT-1 CD8+ T cells or not. 2 × 10E6 KPC3-OVA cells (Epithelial or Mesenchymal type) were coculture with 1 × 10E6 OT-I T cells for 0, 6 or 24 hours. Each group has three biological independent replicates (three different donors derived OT-I T cells). OT-I T cells and culture medium was removed and washed three times with ice-cold PBS. Cancer cells were collected for RNA samples preparation by the NucleoSpin RNA kit. After mRNA enrichment by Oliogo dT selection and the following library preparation, RNA-seq was performed in the DNBseq platform (Beijing Genomics Institute, BGI, Hongkong).