Extended Data Fig. 10: ATF6α activation metabolically renders cancer cells resistant to T-cell-mediated cytotoxicity, Related to Fig. 5.

(a-b) Metabolic flux analysis (a) for extracellular acidification rates (ECAR) of FL83B^WT (n = 3) or FL83B^TG (n = 3) hepatocytes, with quantification (b) for glycolysis and glycolytic capacity. (c) Glucose consumption in FL83B^WT or FL83B^TG hepatocytes cultured for 48 h (n = 6/group). (d) qRT-PCR analysis of glucose-related metabolic genes in FL83B^WT or FL83B^TG cells (n = 6/group). Bold labelling depicts significant genes. (e) NMR spectroscopy-based metabolomics analysis of lactate concentration in FL83B cells with ATF6α expression (FL83B^TG) or deletion (FL83B^KO), normalized to respective controls (n = 6/group). (f) NMR metabolomics PLS − DA with coloured 95% confidence intervals illustrating the clear group separation in metabolic profile between FL83B^WT and FL83B^TG cells (n = 6/group). (g) qRT-PCR analysis of primary hepatocytes harvested from livers of 3-month-old TG^Alb-cre− (n = 4) and TG^Alb-cre+ (n = 6) mice. Bold labelling depicts significant genes. (h) qRT-PCR analysis of primary hepatocytes harvested from livers of CD-HFD-fed Atf6^fl/fl (n = 6) and Atf6^ΔHep (n = 6) mice. Bold labelling depicts significant genes. (i) Scheme of HCC cancer cell line (HLE) stably transfected with control vector (HLE^WT) or nATF6α-expressing (HLE^TG) plasmid and co-cultured with or without cytotoxic MART-1 T-cells to assess immune attack by a real-time cell analyzer (xCELLigence). (j) HLE^WT or HLE^TG cancer cell growth over time with quantification on the right panel (n = 3/group). (k) Killing assay and quantification for HLE^WT or HLE^TG cancer cell growth in co-culture conditions with cytotoxic MART-1 T-cells (n = 3/group). (l) Scheme of Colo800 tumour cells stably transfected with control (Colo800^WT) or nATF6α-expressing (Colo800^TG) plasmid and co-cultured with cytotoxic MART-1-specific T-cells to assess immune attack in a real-time cell analyzer (xCELLigence). (m) Killing assay of Colo800^WT or Colo800^TG tumour cells in co-culture conditions with cytotoxic MART-1-specific T-cells (n = 3/group) in the presence or absence of the lactate dehydrogenase inhibitor galloflavin. (n) Killing assay for Colo800^WT or Colo800^TG tumour cell growth in co-culture conditions with cytotoxic MART-1 T-cells (n = 4/group) in the presence or absence of the lactate efflux inhibitor AZD3965. (o) Glucose consumption of Colo800^WT and Colo800^TG cells cultured for 48 h (n = 6/group). (p) qRT-PCR analysis of indicated mRNAs from Colo800^WT and Colo800^TG cells (n = 6/group). Bold labelling depicts significant genes. (q) NMR metabolomics PLS − DA with coloured 95% confidence intervals illustrating the clear group separation in metabolic profile between Colo800^WT (n = 5) and Colo800^TG (n = 6) cells. (r) Top 15 metabolites and their VIP score based on the PLS − DA regression model. Scatter dot plot data are presented as mean values ± SEM. Line graph data are presented as mean values ± SEM (10a) or mean values ± SD (10j-k, m-n). Data in 10b-d,g,h,o,p were analysed by two-tailed unpaired t-test or Mann-Whitney test based on data normality distribution. Data in 10e were analysed by one-way ANOVA. Data in 10j,k were calculated for the area under the curve and analysed by two-tailed Student’s t-test. Data in 10m,n were calculated for the area under the curve and analysed by one-way ANOVA. In vitro co-culture schematics were created in BioRender. Heikenwälder, M. (2026) https://BioRender.com/lgjnsy9.

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