MASLD-mimicking microenvironment drives an aggressive phenotype and represses IDH2 expression in hepatocellular carcinoma

Abstract

Aim: Hepatocellular carcinoma (HCC) in patients with Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly NAFLD) is expected to be a significant public health issue in the near future. Therefore, understanding the tumor microenvironment interactions in MASLD-induced HCC is crucial, and the development of relevant preclinical models is needed. Hence, we aimed to determine the effects of a MASLD-mimicking microenvironment (ME) on the aggressiveness of HCC cells and identify target genes that drive HCC by developing a 3D-in vitro co-culture system. Methods: A 3D co-culture system mimicking the MASLD-ME was created with LX-2 liver stellate cells embedded in 3D collagen gel in the lower and SNU-449 HCC cells on the upper parts of Boyden chambers, and cells were grown in an optimized metabolic medium (MM). The effects of NAFLD-ME on motility, sphere formation, proliferation, and cell cycle of SNU-449 cells were tested by Boyden chamber, 3D sphere formation, XTT, and Flow cytometry, respectively. The protein expression/activation profiles of motile SNU-449 cells that passed the membrane toward NAFLD-ME or control condition were investigated using a multiplex protein profiling system DigiWest and confirmed with RT-PCR, WB, and Flow cytometry. IDH2 levels were examined in primary human HCC and adjacent liver tissues by IHC and in TCGA and CPTAC cohorts by bioinformatics tools. Results: MM treatment increased fat accumulation, motility, and spheroid formation of both SNU-449 and LX-2 cells. MASLD-ME induced activation of LX2 cells, leading to the formation of bigger colonies with many intrusions compared to related controls. DigiWest analysis showed that metabolism-related proteins such as IDH2 were the most affected molecules in SNU-449 cells that migrated toward the MASLD-ME compared to those that migrated toward the control condition. Downregulation of IDH2 expression was confirmed in SNU-449 cells grown in MASLD-ME, in primary HCC tumor samples by IHC, and in HCC patient cohorts by bioinformatics analysis. Conclusion: This study reports the potential involvement of MASLD-ME in the downregulation of IDH2 expression and promoted motility and colonization capacity of HCC cells. The 3D MASLD model presented in this study may be useful in investigating the mechanistic roles of MASLD-ME in HCC.