TY  - EJOU
AU  - Huang, Yi-Sian 
AU  - Ma, Chung-Yung 
AU  - Roan, Hsiao-Yuh 
AU  - Chiang, Cheng-Hsiung 
AU  - Tsou, Hsiao-Hui 
AU  - Chen, -Hui 
AU  - Lin, Yi-Fan 
AU  - Wang, Horng-Dar 
AU  - Yuh, Chiou-Hwa 

TI  - Phenotypic Response Surfaces–Guided Optimization (PRS-OPT) of Propolis-Metformin-Regorafenib Combination Therapy for MASLD-Associated Hepatocellular Carcinoma
T2  - Oncology Research

PY  - 
VL  - 
IS  - 
SN  - 1555-3906

AB  - <b>Objectives:</b> Hepatocellular carcinoma (HCC) arising in metabolic dysfunction–associated steatotic liver disease (MASLD) develops under lipid-rich stress and inflammatory remodeling, which can alter therapeutic windows. We aimed to determine whether phenotypic response surface–guided optimization (PRS-OPT) can nominate hepatocyte-sparing propolis–metformin–regorafenib (PMR) dose windows that retain antitumor activity under MASLD-like fatty-acid (FA) stress and translate to an <i>in vivo</i> immune endpoint. <b>Methods:</b> PMR combinations were profiled in hepatoma cell lines (PLC/PRF/5 and HepG2) and non-malignant hepatocytes (THLE-2) under FA-free and FA-enriched conditions. Quadratic response surfaces were fitted and used for constrained dose nomination, followed by <i>in vitro</i> validation. Cell-death contributions were assessed by inhibitor-rescue, Annexin V/PI flow cytometry, and immunoblotting. PRS-OPT was extended to zebrafish MASLD-HCC to optimize dosing against hepatic macrophage accumulation, with longitudinal imaging and qPCR endpoints. <b>Results:</b> FA exposure shifted the feasible efficacy–tolerability landscape, leading PRS-OPT to nominate a distinct FA-optimized PMR window with improved hepatocyte preservation while maintaining tumor suppression. Validation confirmed tumor-preferential activity across hepatoma lines with hepatocyte sparing. Mechanistic assays supported apoptosis with context dependence and evidence consistent with a ferroptosis-related component in HepG2. In zebrafish MASLD-HCC, PRS-OPT–nominated dosing reduced hepatic macrophage accumulation and improved disease-relevant transcriptional and morphological readouts. <b>Conclusions:</b> PRS-OPT enables interpretable, multi-objective dose nomination for MASLD-relevant HCC contexts, and establishes PRS-OPT as an interpretable, multi-objective framework for regimen nomination that is directly extensible to additional phenotypic endpoints for preclinical evaluation.
KW  - hepatocellular carcinoma; metabolic dysfunction–associated steatotic liver disease (MASLD); zebrafish; macrophages; phenotypic response surface-guided optimization (PRS-OPT)

DO  - 10.32604/or.2026.074145