Transcriptomic trajectory of hepatocyte ballooning reveals a lipotoxic point of no return and initiates oncogenic regulatory rewiring in MASLD

Background Hepatocyte ballooning is a defining histological marker of progressive steatohepatitis, but its molecular basis remains poorly defined. Objective To identify a molecular signature and mechanistic trajectory underlying hepatocyte ballooning across the MASLD-to-hepatocellular carcinoma spectrum. Methods Liver microarray data from 43 patients stratified by histological ballooning score were combined with machine-learning feature selection and publicly available single-nucleus RNA sequencing data (87,244 hepatocyte nuclei) to derive a gene signature, tested in an independent bulk transcriptomic cohort (GSE130970, n = 75); pseudotime, transcription-factor regulon and cell–cell communication analyses characterised the underlying biology, with in vitro validation in palmitic acid-treated Huh7 cells and prognostic testing in 510 TCGA-LIHC patients. Results An eight-gene signature ( SPP1, MFSD2A, PRAP1, ANXA2, KRT8, MACROH2A2 ( H2AFY2 ), JUN and FABP4 ) characterised ballooned hepatocytes and discriminated moderate-to-severe from absent ballooning with an AUC of 0.768 (95% CI 0.654–0.883). Six of these genes formed a Hepatocyte Ballooning–Hepatocyte injury (HBHi) score that was highest in nuclei from cirrhotic (F4) liver. Pseudotime analysis resolved a four-stage molecular collapse culminating at pseudotime ≈ 25, marked by loss of the chromatin stabiliser MACROH2A2 , co-induction of KRT8 and SPP1 , and replacement of the HNF4A/PPARA -driven programme by the stress factors JUN, ATF3 and EGR1 . Ballooned hepatocytes transmitted pro-fibrogenic signals to stellate cells and macrophages via FN1–CD44 and FN1–(ITGAV + ITGB1) axes; high KRT8, JUN and SPP1 expression was each associated with poorer survival in hepatocellular carcinoma. Conclusions Hepatocyte ballooning is a transcriptionally active, stepwise process with a lipotoxic point of no return and prognostic relevance extending to hepatocellular carcinoma.