对于肝细胞癌（HCC）患者对免疫检查点阻断（ICB）的有限反应强调了扩大当前免疫治疗方法范围的紧迫需要。联合微创介入和解剖结合治疗来源于新的免疫治疗。本研究调查了这两种治疗药物的最佳组合方法和其基本机制。在免疫缺陷和免疫功能正常的小鼠模型中评估了三种不同剂量lenvatinib促进组织灌注和血管正常化的效果。通过分析内皮细胞和包细胞的血管形态学来研究其潜在机制。通过流式细胞术和免疫荧光成像进一步评估了最佳剂量lenvatinib的增强免疫浸润以及lenvatinib与抗PD-1抗体的协同效应。在免疫缺陷和免疫功能正常的小鼠模型中，存在一种最佳剂量，能够更好地正常化肿瘤血管和增加免疫细胞浸润。适当浓度的lenvatinib通过诱导NRP-1-PDGFRβ复合物的形成和激活Crkl-C3G-Rap1信号通路在人脐静脉内皮细胞中加强了其完整性。此外，它通过诱导包细胞中的酪氨酸磷酸化来促进内皮细胞与包细胞之间的相互作用。此外，最佳剂量的lenvatinib和抗PD-1抗体的联合明显抑制了肿瘤生长。我们的研究提出了一个解释最佳剂量lenvatinib如何引发血管正常化的机制，并确认了它与ICBs的协同增强效应。 版权所有2023年 Yang、Guo、Song、Pan、Zhao、Huang、Han、Ouyang、Yang、Chen、Di、Tang、Zhu、Wang、Li、He、Weng、Xiang和Xia。

The limited response to immune checkpoint blockades (ICBs) in patients with hepatocellular carcinoma (HCC) highlights the urgent need for broadening the scope of current immunotherapy approaches. Lenvatinib has been shown a potential synergistic effect with ICBs. This study investigated the optimal method for combining these two therapeutic agents and the underlying mechanisms.The effect of lenvatinib at three different doses on promoting tissue perfusion and vascular normalization was evaluated in both immunodeficient and immunocompetent mouse models. The underlying mechanisms were investigated by analyzing the vascular morphology of endothelial cells and pericytes. The enhanced immune infiltration of optimal-dose lenvatinib and its synergistic effect of lenvatinib and anti-PD-1 antibody was further evaluated by flow cytometry and immunofluorescence imaging.There was an optimal dose that superiorly normalized tumor vasculature and increased immune cell infiltration in both immunodeficient and immunocompetent mouse models. An adequate concentration of lenvatinib strengthened the integrity of human umbilical vein endothelial cells by inducing the formation of the NRP-1-PDGFRβ complex and activating the Crkl-C3G-Rap1 signaling pathway in endothelial cells. Additionally, it promoted the interaction between endothelial cells and pericytes by inducing tyrosine-phosphorylation in pericytes. Furthermore, the combination of an optimal dose of lenvatinib and an anti-PD-1 antibody robustly suppressed tumor growth.Our study proposes a mechanism that explains how the optimal dose of lenvatinib induces vascular normalization and confirms its enhanced synergistic effect with ICBs.Copyright © 2023 Yang, Guo, Song, Pan, Zhao, Huang, Han, Ouyang, Yang, Chen, Di, Tang, Zhu, Wang, Li, He, Weng, Xiang and Xia.