越来越多的研究表明，将免疫检查点抑制剂（ICIs）与抗血管生成药物相结合可以增强其抗肿瘤效果。本研究将三种抗血管生成剂DC101（作用于VEGFR 2）、SAR131675（作用于VEGFR 3）和fruquintinib（作用于多个靶点的小分子抑制剂）分别给予B16F1-OVA负载的C57BL/6小鼠进行实验。评估肿瘤组织中免疫细胞浸润、血管正常化和高内皮静脉（HEV）形成，以提供药物联合使用的证据。和SAR131675相比，DC101和fruquintinib均显著减缓了黑色素瘤的生长，增加了CD3+和CD8+ T细胞浸润比例，并且DC101的效果更为明显。此外，DC101和fruquintinib提高了干扰素γ和穿孔素水平，同时DC101增加了粒缘蛋白B水平，而fruquintinib和SAR131675没有。只有fruquintinib处理组显示出调节性T细胞浸润的降低。我们发现，在DC101处理组中，肿瘤细胞和CD45+免疫细胞中PD-L1表达上调，CD3+ T细胞上调PD-1表达。然而，fruquintinib仅增加了肿瘤中的PD-L1表达。DC101和fruquintinib都降低了CD31+血管的比例，而DC101增加了α-SMA+/CD31+细胞比例，并且降低了HIF-1α的表达比fruquintinib更多。此外，DC101增强了树突状细胞和B细胞的浸润以及局部HEV的形成。综上，我们的数据表明，DC101可能是ICIs和抗血管生成剂联合临床应用的更好选择。

There is an increasing interest in combining immune checkpoint inhibitors (ICIs) with anti-angiogenic drugs to enhance their anti-tumor effects. In this study, three anti-angiogenic agents, DC101 (acting on VEGFR2), SAR131675 (acting on VEGFR3), and fruquintinib (a small-molecule inhibitor acting on multiple targets) were administered to B16F1-OVA-loaded C57BL/6 mice. Immune cells infiltration in the tumor tissues, vascular normalization, and high-endothelial venule (HEV) formation were assessed to provide evidence for drug combination. Both DC101 and fruquintinib significantly slowed the melanoma growth and increased the proportion of CD3+ and CD8+ T cells infiltration compared with SAR131675, of note, the effect of DC101 was more pronounced. Moreover, DC101 and fruquintinib increased the interferon-γ and perforin levels, meanwhile, DC101 increased the granzyme B levels, whereas fruquintinib and SAR131675 did not. Only the fruquintinib-treated group showed decreased regulatory T cells infiltration. We found upregulation of PD-L1 expression in tumor cells and CD45+ immune cells in DC101-treated group as well as upregulation of PD-1 expression on CD3+ T cells. However, fruquintinib only increased PD-L1 expression in tumors. Both DC101 and fruquintinib reduced the proportion of CD31+ vessels, while DC101 increased the ratio of α-SMA +/CD31+ cells and reduced the expression of HIF-1α more than fruquintinib. Moreover, DC101 enhanced the infiltration of dendritic cells and B cells, and local HEV formation. In conclusion, our data indicate that DC101 may be a better choice for the combined clinical application of ICIs and anti-angiogenic agents.Copyright © 2023 Elsevier Inc. All rights reserved.