Proteoglycan 4 and regorafenib synergistically reduce HCC progression in an orthotopic model
Background: Hepatocellular carcinoma (HCC) is a major therapeutic challenge, with limited long-term benefits from treatments such as regorafenib due to their efficacy and adverse effects. Proteoglycan-4 (PRG4) has previously shown promise in enhancing anti-proliferative effects of multi-kinase inhibitors in simple in vitro two-dimensional (2D) HCC models. Objective: We evaluated the adjuvant effect of PRG4 in enhancing regorafenib's anti-tumor efficacy in preclinical more complex in vitro three-dimensional (3D) HCC tumoroids, and in vivo HCC models. Design: HLC19 and HLF HCC cell lines were engineered to stably overexpress PRG4. Anti-proliferative effects of PRG4, either alone or in combination with regorafenib, were evaluated in 2D monolayers, 3D Matrigel embedded spheroids and orthotopic xenograft mouse models established via HCC cells intrahepatic injection. RNA sequencing was done to assess the transcriptomic profiles of PRG4 overexpressing spheroids. Results: PRG4 expression partially inhibited HCC tumor growth in vivo, but enhanced regorafenib treatment leading to a near-complete tumor regression. PRG4 expression significantly impaired cell growth in both 2D and 3D HCC models in vitro. In an angiogenesis in vitro model, PRG4 hindered endothelial tubulogenesis both alone and in combination with regorafenib. By transcriptomic analysis of matrigel-embedded HCC cell spheroids exposed to PRG4 and/or regorafenib, PDGF pathway emerged as a target of PRG4 + regorafenib, corroborating the role of PRG4 in impairing angiogenesis. G0/G1 phase of cell cycle resulted more delayed in spheroids exposed to PRG4 and regorafenib than those treated with regorafenib only, compared to untreated cells. Conclusions: PRG4 enhances the anti-tumor activity of regorafenib across various HCC models. These findings support PRG4 as a promising adjuvant candidate to improve clinical outcomes in HCC. Overall design: HCC cells (HLC19) transduced with an empty vector (EV), and PRG4-expressing vector (PV) were pre-assembled as tumorspheres and embedded in Matrigel®. Spheroids were then treated with DMSO (vehicle) or regorafenib (5 µM) for 4 days. At the end of treatment, total RNA was isolated from spheroids and NGS was performed to profile differential expressed genes (DEGs) as a result of exposure to regorafenib, PRG4, and combined PRG4 + regorafenib.