Live hepatocytes were attached to collagen I-coated 6-well plates (0.6 106 cells/well) for 6 h in hepatocyte attachment medium (Williams E medium, 1% P/S, 2 mM L-glutamine, 1% NEAA and 10% FBS), then cultured in Williams E medium made up of 1% P/S, 0.1 g/ml fungizone, 50 g/ml gentamycin, 2 mM L-glutamine and 0.1 mM NEAA. Secretome profiling through label free quantitative proteomics. New mouse hepatocytes were attached to collagen I-coated 15-cm plates (1.5 107/plate) for 4 h, and cultured in 25 ml serum/phenol red-free DMEM for another 6 h to collect the CM. in steatosis, concomitant with activation and senescence of hepatic stellate cells (HSCs), exhibiting a senescence-associated secretory phenotype (SASP). Depleting senescent HSCs by senolytic treatment with dasatinib/quercetin or ABT-263 inhibits tumour progression. We further demonstrate that FBP1-deficient hepatocytes promote HSC activation by releasing HMGB1; blocking its release with the small molecule inflachromene limits FBP1-dependent HSC activation, subsequent SASP development, and tumour progression. Collectively, these findings provide genetic evidence for FBP1 as a metabolic tumour suppressor in liver cancer and establish a crucial crosstalk between hepatocyte metabolism and HSC senescence that promotes tumour growth. Hepatocellular carcinoma (HCC) remains a leading cause of cancer-related mortality worldwide1. Considerable heterogeneity in HCCs mutational scenery2 makes targeted therapies less broadly effective, and recent studies have focused instead on potentially targeting the liver tumour microenvironment (TME), including fibrosis and chronic inflammation3, 4. Hepatic fibrosis contributes to more than 80% of HCC and results from activation and transdifferentiation of quiescent hepatic stellate cells (HSCs)5, 6. Similarly, various immune cell subsets have been Oleanolic acid hemiphthalate disodium salt identified as key factors for HCC progression4, 7C9. Non-alcoholic fatty liver disease (NAFLD), caused by aberrant liver metabolism and lipid accumulation, has also emerged as an important predisposing factor for HCC10C12. Overall, the crosstalk between deregulated hepatocyte metabolism and stromal cells within the HCC TME remains to be fully elucidated. The rate-limiting gluconeogenic enzyme FBP1 has been increasingly implicated as a tumour suppressor. FBP1 antagonizes glycolysis through its cytosolic catalytic activity13, 14, while nuclear FBP1 directly interacts with hypoxia inducible factors (HIFs) in clear cell renal carcinoma (ccRCC), inhibiting their transcriptional activity impartial of its enzymatic properties15. Despite extensive studies, robust genetic evidence for FBP1 as a tumour suppressor has been lacking. In contrast to frequent (genetic mouse model, Oleanolic acid hemiphthalate disodium salt and uncover a previously unrecognized mechanism in which FBP1 loss and subsequent hepatic metabolic deregulation promote liver cancer through an HSC senescence secretome. We also provide proof-of-principle that targeting senescence in HCCs TME has potential as a promising liver cancer therapy. Results expression is lost during liver tumour progression Through a metabolic gene set analysis of The Malignancy Genome Atlas (TCGA) RNA-sequencing data2, we identified the carbohydrate storage group as one of the most underexpressed gene sets in HCC (Extended Data Fig. 1a). Within this group, all three rate-limiting gluconeogenic genes were downregulated (Fig. 1a), with mRNA levels significantly decreased in stage I tumours relative to normal tissues, and further reduced along disease progression (Fig. 1b). Accordingly, immunohistochemical (IHC) staining of human tissue array revealed high FBP1 protein abundance in normal human livers and lower levels in liver tumours (Fig. 1c, ?,dd and Extended Data Fig. 1b, ?,cc). Open in a separate windows Physique 1 a, Box plots of gluconeogenic gene RNA-seq reads in normal liver and tumour tissues from TCGA dataset. n=50 for normal livers, n=374 for tumour samples. b, Box plots of RNA-seq reads in normal liver and Sema3g stage I-Ill tumour tissues in TCGA dataset. n=50 for normal, n=173 for stage I, n=88 for stage II, n=85 for stage III specimens. c, d, Representative IHC staining (c) and statistical analysis (d) of FBP1 protein in human liver tissue array. n=5 for normal, n=10 for grade 1, n=27 Oleanolic acid hemiphthalate disodium salt for.