Extended Data Fig. 9: Effect of MAPK signalling and drug addiction variants on antigen presentation and sensitivity to T cell killing.

a) Flow cytometry assessment of B2M and HLA-A,B,C expression in HT-29 ABE cells harbouring drug addiction variants. Data represent the mean ± SD of biological triplicates. IFN-gamma treatment serves as a positive control (48 h, 400 U/ml). ****P-value < 0.0001; ***P-value = 0.0003 (HLA) or 0.0005 (B2M); **P-value = 0.002; *P-value = 0.037; unpaired, two-tailed Student’s t-test comparing to non-targeting gRNA (NT) condition. Genotyped variants are shown. b) Flow cytometry assessment of B2M and HLA-A,B,C expression in CRC-9 ABE tumour organoid cells harbouring drug addiction variants. Cells were treated with DMSO (control) or the MEK inhibitor trametinib (25 nM) for 48 h before analysis. Data represent the mean ± SD of two independent experiments. IFN-g treatment serves as a positive control (48 h, 400 U/ml). ****P-value < 0.0001; ***P-value = 0.0015; **P-value = 0.0066 (B2M or 0.0012 (HLA); *P-value = 0.031; unpaired, two-tailed Student’s t-test comparing non-targeting gRNA (NT) condition. Genotyped variants are shown. c) Representative flow cytometry gating used for CRC-9 tumour organoids to assess HLA-A,B,C and B2M cell surface protein expression. Single, live cells with mApple (ABE) and BFP (gRNA) expression were gated for analysis. d) Co-competition flow cytometry assays of WT (GFP – NT gRNA expressing cells) and drug resistant CRC-9 tumour organoids (BFP – gRNA expressing) at 72 h. Data represent the mean ± SD of biological triplicates. ****P-value < 0.0001; ***P-value = 0.0001; **P-value = 0.0018; *P-value = 0.031; unpaired, two-tailed Student’s t-test comparing to non-targeting gRNA (NT) condition. Genotyped variants are shown. e) Co-culture assay of primary, autologous, anti-tumour T cells with CRC-9 tumour organoids harbouring different drug addiction variants. Cancer cells were pre-treated with the MEK inhibitor trametinib (25 nM) for 48 h before washing and plating the co-culture assay plate. Flow cytometry assessment of absolute cell numbers (measured by counting beads) following 72 h co-culture. Data are expressed as the percentage of live cells remaining as compared to the relevant condition in the absence of T cells and represent the mean ± SD of biological triplicates. ***P-value = 0.013, **P-value = 0.025, *P-value = 0.037; unpaired, two-tailed Student’s t-test. Genotyped variants are shown.