Lenvatinib is a multiple receptor tyrosine kinase inhibitor targeting mainly vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) receptors. We investigated the immunomodulatory activities of lenvatinib in the tumor microenvironment and its mechanisms of enhanced antitumor activity when combined with a programmed cell death-1 (PD-1) blockade. Antitumor activity was examined in immunodeficient and immunocompetent mouse tumor models. Single-cell analysis, flow cytometric analysis, and immunohistochemistry were used to analyze immune cell populations and their activation. Gene co-expression network analysis and pathway analysis using RNA sequencing data were used to identify lenvatinib-driven combined activity with anti-PD-1 antibody (anti-PD-1). Lenvatinib showed potent antitumor activity in the immunocompetent tumor microenvironment compared with the immunodeficient tumor microenvironment. Antitumor activity of lenvatinib plus anti-PD-1 was greater than that of either single treatment. Flow cytometric analysis revealed that lenvatinib reduced tumor-associated macrophages (TAMs) and increased the percentage of activated CD8 + T cells secreting interferon (IFN)-γ + and granzyme B (GzmB). Combination treatment further increased the percentage of T cells, especially CD8 + T cells, among CD45 + cells and increased IFN-γ + and GzmB + CD8 + T cells. Transcriptome analyses of tumors resected from treated mice showed that genes specifically regulated by the combination were significantly enriched for type-I IFN signaling. Pretreatment with lenvatinib followed by anti-PD-1 treatment induced significant antitumor activity compared with anti-PD-1 treatment alone. Our findings show that lenvatinib modulates cancer immunity in the tumor microenvironment by reducing TAMs and, when combined with PD-1 blockade, shows enhanced antitumor activity via the IFN signaling pathway. These findings provide a scientific rationale for combination therapy of lenvatinib with PD-1 blockade to improve cancer immunotherapy.

Angiogenesis inhibitors such as lenvatinib and sorafenib, and an immune checkpoint inhibitor (ICI), nivolumab, are used for anticancer therapies against advanced hepatocellular carcinoma (HCC). Combination treatments comprising angiogenesis inhibitors plus ICIs are promising options for improving clinical benefits in HCC patients, and clinical trials are ongoing. Here, we investigated the antitumor and immunomodulatory activities of lenvatinib (a multiple receptor tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor 1‐3, fibroblast growth factor receptor 1‐4, platelet‐derived growth factor receptor α, KIT and RET) and the combined antitumor activity of lenvatinib plus anti‐programmed cell death 1 (PD‐1) antibody in the Hepa1‐6 mouse HCC syngeneic model. We found that the antitumor activities of lenvatinib and sorafenib were not different in immunodeficient mice, but lenvatinib showed more potent antitumor activity than sorafenib in immunocompetent mice. The antitumor activity of lenvatinib was greater in immunocompetent mice than in immunodeficient mice and was attenuated by CD8+ T cell depletion. Treatment with lenvatinib plus anti‐PD‐1 antibody resulted in more tumor regression and a higher response rate compared with either treatment alone in immunocompetent mice. Single‐cell RNA sequencing analysis demonstrated that treatment with lenvatinib with or without anti‐PD‐1 antibody decreased the proportion of monocytes and macrophages population and increased that of CD8+ T cell populations. These data suggest that lenvatinib has immunomodulatory activity that contributes to the antitumor activity of lenvatinib and enhances the antitumor activity in combination treatment with anti‐PD‐1 antibody. Combination treatment of lenvatinib plus anti‐PD‐1 antibody therefore warrants further investigation against advanced HCC.

Background: LEN selectively inhibits the kinase activity of VEGFR1-3, FGFR1-4, KIT, PDGFRα, and RET, which are involved in tumor angiogenesis and proliferation in several cancer types. Currently, Phase 1b/2 clinical trials of the combination of LEN and pembrolizumab (a monoclonal antibody [mAb] that blocks the interaction between PD-1 and its ligands) are ongoing for selected types of cancer including melanoma and renal cancer. In order to understand the antitumor effect and mechanism of action of the combination of LEN and PD-1 blockade treatment, we analyzed immune response in syngeneic murine tumor models. Methods: We examined antitumor activity of combination treatment of LEN (10mg/ kg, qd) and anti-mouse PD-1 mAb (500 µg/mouse, twice weekly) against LL/2 murine lung carcinoma, H22 murine hepatocellular carcinoma, and CT26 murine colon cancer in syngeneic mouse models. For immune population analyses, tumor or spleen samples

The Wnt/β-catenin signaling pathway plays crucial roles in embryonic development and the development of multiple types of cancer, and its aberrant activation provides cancer cells with escape mechanisms from immune checkpoint inhibitors. E7386, an orally active selective inhibitor of the interaction between β-catenin and CREB binding protein, which is part of the Wnt/β-catenin signaling pathway, disrupts the Wnt/β-catenin signaling pathway in HEK293 and adenomatous polyposis coli (APC)-mutated human gastric cancer ECC10 cells. It also inhibited tumor growth in an ECC10 xenograft model and suppressed polyp formation in the intestinal tract of ApcMin/+ mice, in which mutation of Apc activates the Wnt/β-catenin signaling pathway. E7386 demonstrated antitumor activity against mouse mammary tumors developed in mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice. Gene expression profiling using RNA sequencing data of MMTV-Wnt1 tumor tissue from mice treated with E7386 showed that E7386 downregulated genes in the hypoxia signaling pathway and immune responses related to the CCL2, and IHC analysis showed that E7386 induced infiltration of CD8+ cells into tumor tissues. Furthermore, E7386 showed synergistic antitumor activity against MMTV-Wnt1 tumor in combination with anti-PD-1 antibody. In conclusion, E7386 demonstrates clear antitumor activity via modulation of the Wnt/β-catenin signaling pathway and alteration of the tumor and immune microenvironments, and its antitumor activity can be enhanced in combination with anti-PD-1 antibody. Significance: These findings demonstrate that the novel anticancer agent, E7386, modulates Wnt/β-catenin signaling, altering the tumor immune microenvironment and exhibiting synergistic antitumor activity in combination with anti-PD-1 antibody.

X-ray microcomputed tomography (micro-CT) with a respiratory gating system is a useful non-invasive approach to evaluate lung tumor development in living animal models. Here micro-CT was applied for the detection of lung lesions induced by a single intraperitoneal injection (250 mg/kg) of urethane in male A/J mice, at 2-week intervals from 10 to 30 weeks after carcinogen exposure. In micro-CT cross sections, lung tumor images were easily distinguished from surrounding non-tumorous tissues, the smallest detected tumor being approximately 0.5 mm in diameter. All of the urethane-treated mice (n = 15) developed lung tumors and the number of tumors developed in each mouse was 8.6 ± 3.9. Six tumors, determined histopathologically to be adenocarcinomas, were detected, growing at different rates during the experimental period. The most aggressive carcinoma, increasing in diameter from 0.9 to 3. (1) For detecting lung cancer, X-ray fluoroscopy has been used widely in practical screening. X-ray computed tomography (CT) is also used to detect early stage lesions and to evaluate tumor progression and metastasis during clinical treatment.(2) Moreover, X-ray CT is a useful tool for monitoring lung tumor development in living animal models (3) and major efforts have been invested in developing devices for imaging the inner anatomy of small animals. As with CT for human cases, micro-CT for rodents allows evaluation of bones and other calcified changes as well as diagnosis of soft tissue changes, such as lung tumor development.(4,5) Non-invasiveness, the ability to monitor therapeutic effects, the capacity to optimize the experimental period, and a lowering in the number of animals used can all be considered advantages of this novel approach. However, there are also disadvantages in that it is difficult to make a refined evaluation in very small rodents, and the image quality is strongly affected by motion-related artifacts. Several efforts, particularly the development of a method with a respiratory gating system for obtaining sharper images, have been made to overcome these difficulties. (6)(7)(8) Hitherto, the utility of micro-CT has been reported for detecting lung tumors in a metastatic rodent model, a K-ras transgenic lung cancer model, and a urethane-induced mouse lung tumor model. (5,(8)(9)(10) However, in those reports the mice were scanned only once by micro-CT, and then killed for histopathological analysis of lung lesions. Full utilization of the advantages of respiration-gated micro-CT for periodic detection of sizes in lung tumors and their growth over time has not been made.In the present study, urethane-induced tumor development was therefore monitored periodically using respiration-gated micro-CT. The results obtained indicate that tumors grow at markedly varying speeds, which may not directly reflect the histopathological findings after autopsy. The necessity for appropriate scanning methods of micro-CT images, which link to histopathology, is discussed in the text. Materials and MethodsAnimals. Male A/J Jms Sl...