# Proteoglycan-4 potentiates the antitumor efficacy of regorafenib in an orthotopic model of hepatocellular carcinoma

## TL;DR

Proteoglycan-4 boosts the effectiveness of regorafenib in treating liver cancer in both lab and animal models.

## Contribution

PRG4 enhances regorafenib's antitumor effects in 3D and in vivo HCC models, suggesting a novel adjuvant therapy.

## Key findings

- PRG4 overexpression combined with regorafenib caused near-complete tumor regression in mice.
- PRG4 and regorafenib together delayed the cell cycle and inhibited angiogenesis in HCC models.
- Transcriptomic analysis revealed PDGF pathway involvement in PRG4's anti-angiogenic effects.

## Abstract

Hepatocellular carcinoma (HCC) presents a significant therapeutic challenge, as current treatment options provide limited long-term benefits due to issues surrounding their effectiveness and associated adverse effects. Our previous research demonstrated that Proteoglycan-4 (PRG4) enhances the anti-proliferative effect of the multi-kinase inhibitor regorafenib in simple in vitro two-dimensional HCC models. In this study, we aimed to investigate the potential adjuvant role of PRG4 in improving the efficacy of regorafenib within both three-dimensional in vitro and in vivo HCC models.

Human HCC cells were engineered to stably overexpress PRG4. The effects of PRG4 on cell proliferation, both alone and in combination with regorafenib, were tested in monolayer cultures, Matrigel-embedded spheroids, and an orthotopic xenograft HCC mouse model. Additionally, transcriptomic profiling of spheroids generated from control or PRG4-overexpressing HCC cells, either untreated or treated with regorafenib, was performed.

PRG4 expression partially inhibited HCC tumor growth in vivo and enhanced regorafenib antiproliferative activity, leading to a near-complete tumor regression. This synergistic PRG4 + regorafenib interaction in impairing HCC cell growth was further confirmed in 2D and 3D HCC models in vitro. In addition, PRG4 restrained angiogenesis by hindering endothelial tubulogenesis in vitro. 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. The G0/G1 phase of the cell cycle was more delayed in spheroids exposed to both PRG4 and regorafenib compared to those treated with regorafenib alone, relative to untreated cells.

PRG4 demonstrated antitumor activities in vivo and shows promise as an adjuvant to enhance therapeutic interventions in HCC.

The online version contains supplementary material available at 10.1186/s13046-025-03575-5.