Immunomodulatory effects of lenvatinib targeting immunosuppressive macrophages in combination with transarterial embolization in a rat hepatocellular carcinoma model.

585 Background: Combination strategies of transarterial chemoembolization (TACE) and systemic therapy have clinically gained attention for treating hepatocellular carcinoma (HCC). Clinical trials have demonstrated the efficacy of TACE combined with lenvatinib (LEN) and/or pembrolizumab (PEM), and the triple regimen (TACE+LEN+PEM) was approved in China in 2025 for unresectable, non-metastatic HCC. While TACE induces a hypoxic immunosuppressive tumor microenvironment (iTME), LEN is expected to enhance antitumor effects through immune activation and anti-angiogenic effect. However, the underlying mechanisms remain unclear. We therefore investigated the antitumor effects and iTME changes following transarterial embolization (TAE), with or without LEN, using a rat HCC model. Methods: An orthotopic N1S1-bearing rat HCC model was divided into four groups: Control (n=8), LEN (n=5), TAE (n=8) and TAE+LEN (n=5). TAE was performed on Day 9. LEN (4mg/kg) was administered for 2 days from Day 7 and resumed for 4 days from Day 10. Liver tumors were harvested on Day 15. Histopathological analysis was performed to assess the necrotic area. Additionally, CD45+ cells were isolated and single-cell RNA sequencing (scRNA-seq) was performed to evaluate the treatment-induced changes in iTME. Results: The necrotic area was significantly greater in the TAE+LEN group (99.9±0.11%) compared with Control (18.1±14.1%), LEN (44.4±12.2%), and TAE (58.6±25.9%) (P < 0.05 for all). scRNA-seq analysis identified 10 distinct cell types, including neutrophil, CD8+ and CD4+ T cells, and macrophages. TAE increased neutrophils and decreased other immune cell populations. The number of CD8+ T cells was decreased in the post-TAE samples, and there was no major difference between TAE and TAE+LEN groups. The gene expression of macrophages showed various alterations in response to TAE and/or LEN administration. Differential gene expression analysis revealed that macrophages in the TAE group significantly expressed Trem2, Spp1, and Lgals1 (P < 0.005 for all), which are immunosuppressive macrophage markers reported in previous research (Tan et al., 2023). Notably, TAE+LEN significantly reduced expression of these markers compared with TAE alone (P<0.005 for all). Conclusions: LEN enhanced the antitumor efficacy of TAE in a rat HCC model. This effect may be mediated by the immunomodulatory action of LEN, which suppresses immunosuppressive macrophage phenotypes induced by TAE.