Fig. 7: In Vitro and In Vivo Effects of STAT6 and MTHFD2 Inhibitors on HCC Growth.

A, B Cell viability of Hep3B cells were treated with DS18561882 (MTHFD2 inhibitor) or AS1517499 (STAT6 inhibitor) with Sorafenib at various concentrations for 24 h. C, D Cell viability of SNU475 cells were treated with DS18561882 or AS1517499 in combination with sorafenib at various concentrations for 24 h. Cell viability was assessed using the CCK8 assay, with DMSO-treated cells defined as 100% viability (blue bar), and all other treatments were normalized accordingly and expressed as percentage viability. Data represents the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001. E Representative images of Hep3B sphere formation following treatment with DMSO, DS18561882, AS1517499, sorafenib, or their combinations. Scale bar, 50 μm. F The expressions of SOX2 and Nanog were accessed by qRT-PCR in sphere culture treated with DS18561882, AS1517499, or sorafenib or combination. Hep3B cells (5000 cells) were seeded in six-well plates coated with polypeptide polyelectrolyte multilayer films (Acrocyte Therapeutics, Taiwan) and cultured in sphere medium for 3 days, then cells were treated with DMSO, DS18561882, AS1517499, sorafenib, or their combinations. Spheres were harvested after 3 days of drug treatment for RNA extraction. Expression of stemness-associated genes was quantified by qPCR, normalized with expression level of 18S (internal control) firstly, then with Hep3B cells grown in 2D culture as baseline (normalized to 1×). The Y-axis shows relative fold-change in expression under sphere-forming conditions compared with 2D culture. Data represents the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01. G PDX Tumor Growth: Tumors were excised from patients and implanted subcutaneously in five-week-old, anesthetized NPG mice. Inhibitors administered two weeks post-implantation that present as Day 0 in X axis, every 3–4 days through IP. Each treatment group included four mice. Tumor volume was measured as length × width × height (mm³). H The tumors were excised 28 days after drug treatment. The tumor weight (g) in control, AS1517499, DS18561882 treated group (right panel). Data represents the mean ± SD from four mice in each group. *P < 0.05. I Proposed model: SOX4 regulates STAT6 epigenetically and forms a complex with STAT6 to regulate MTHFD2, impacting de novo purine synthesis, NADPH production, and HBP metabolites, promoting tumor growth and drug resistance in HCC. This illustration was created with BioRender.com.