Systemic therapy for hepatocellular carcinoma (HCC) has undergone a paradigm shift in recent years, and immune checkpoint inhibitor (ICI)-based combinations are now established as the standard of care in the first-line setting [
1-
3]. The cancer-immunity cycle, a highly regulated and sequential process that orchestrates antitumor immune responses, is essential to understanding ICI-based combinations deeply [
3]. The cycle consists of two major functional phases: the priming phase and the effector phase. The priming phase begins with the release of tumor-associated antigens (Step 1), followed by antigen presentation by professional antigen-presenting cells such as dendritic cells, and leads to the activation of naïve T lymphocytes. CD4
+ helper T cells play a pivotal role in this phase by supporting antigen presentation and facilitating the priming of CD8+ cytotoxic T cells. The effector phase involves the trafficking of activated T cells to the tumor, their infiltration into the tumor immune microenvironment (TIME), the recognition of malignant cells, and ultimately their elimination through cytotoxic activity, which is mediated by molecules such as Granzyme B. However, the presence of immunosuppressive components, including FOXP3
+ regulatory T cells and tumor-associated macrophages, can attenuate these effector functions and limit effective tumor rejection. Therapeutic strategies that activate both phases of this cycle are crucial for converting immunologically “cold” tumors into “hot” ones.
Several ICI-based combinations have demonstrated clinical efficacy in patients with unresectable HCC [
1,
2,
4,
5]. Atezolizumab plus bevacizumab, which combines an anti-programmed death-ligand 1 (PD-L1) antibody with an antivascular endothelial growth factor (VEGF) antibody (PV), was the first immunotherapy-based regimen approved for HCC [
6-
8]. In contrast, two additional regimens include antibody therapies that target cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) as a central component. The STRIDE regimen consists of durvalumab and a single dose of tremelimumab, which together target PD-L1 and CTLA-4 (PC) [
9]. Nivolumab plus ipilimumab combines antibodies against PD-1 and CTLA-4 [
10]. These CTLA-4-based combinations, broadly classified as immune-oncology (IO) plus IO regimens, have each demonstrated survival benefit in phase 3 or pivotal clinical trials [
9,
10]. However, the distinct alterations in TIME induced by these three immunotherapy combinations are not fully understood [
11]. In the present study, we sought to characterize the differences in TIME induced by PV and PC therapy.
Blockade of PD-L1, CTLA-4, and VEGF is known to affect different steps of the cancer-immunity cycle: effector T-cell activation, priming of naïve T cells, and modulation of tumor vasculature and immunosuppressive cells, respectively. Considering these complementary mechanisms, simultaneous inhibition of all three pathways could provide the most comprehensive activation of the cycle. On this basis, we hypothesized that targeting PD-L1 (or PD-1), CTLA-4, and VEGF, referred to as triplet therapy, might maximize antitumor immunity. To test this hypothesis, we conducted a sequential treatment experiment in which PV and PC therapies were administered in two distinct orders: PV followed by PC (PV-PC), and PC followed by PV (PC-PV). These regimens were evaluated in a syngeneic subcutaneous HCC mouse model, and the composition of the TIME was assessed by immunohistochemical analysis.
A syngeneic subcutaneous tumor model was established by inoculating Hep53-4 murine hepatoma cells (2×106; Cell Line Service GmbH, Oppenheim, Germany) into the right flank of 5-week-old female C57BL/6J mice (Kyudo, Japan). When tumors reached approximately 100 mm³, mice were randomized into five groups (n=6 per group): vehicle (VT), anti-PD-L1 plus anti-VEGF therapy (PV), anti-PD-L1 plus anti-CTLA-4 therapy (PC), PV followed by PC (PV-PC), and PC followed by PV (PC-PV). Antibodies were administered intraperitoneally three times per week. The neutralizing anti-PD-L1 antibody (clone 10F.9G2, Bio X Cell, Cat# BE0101), anti-VEGFA antibody (clone BL512810; BioLegend), and anti-CTLA-4 antibody (clone 9D9, Bio X Cell, Cat# BE0164) were used, based on prior murine validation. In sequential groups, the initial regimen (PV or PC) was given for three doses in one week, followed by the alternate regimen for three additional doses. Mice were euthanized at the endpoint, and tumors were excised for histological analysis. All procedures for animal experiments were approved by the Ethics Committee of Kurume University School of Medicine (20220327).
Tumors were formalin-fixed and paraffin-embedded. Sections (5 μm) were subjected to antigen retrieval, incubated overnight at 4°C with primary antibodies, and visualized using HRP-conjugated secondary antibodies and diaminobenzidine. The antibodies used were anti-CD31 (tumor vasculature), anti-CD8 (cytotoxic T cells), anti-CD4 (helper T cells), anti-FOXP3 (regulatory T cells), anti-F4/80 (macrophages), anti-CD11c (dendritic cells), and anti-Granzyme B (activated cytotoxic cells), as previously validated [
12]. Quantification was performed in five random high-power fields (×200).
All quantitative data are presented as mean±standard error of the mean (SEM). Group comparisons were conducted using one-way ANOVA followed by Tukey’s post hoc test, with significance set at P<0.05 (JMP Pro, version 16.0; SAS Institute, Tokyo, Japan).
PV and PC therapies induced distinct but complementary alterations in the TIME (
Fig. 1). PV significantly reduced CD31-positive vasculature, FOXP3-positive regulatory T cells, and F4/80-positive macrophages, while increasing CD8-positive cytotoxic T cells and Granzyme B-positive cells. The reduction of FOXP3
+ regulatory T cells releases inhibitory cytokines and checkpoints that normally suppress cytotoxic T-cell activity. Likewise, the decrease in tumor- associated macrophages alleviates metabolic and ligand- mediated suppression of T cells. Together with vascular normalization reflected by reduced CD31
+ vessels, these changes create a microenvironment more permissive for effector-phase activity. These findings indicate effector-phase activation and are consistent with the immunomodulatory properties of VEGF blockade [
12]. In contrast, PC therapy increased CD4-positive helper T cells and CD11c-positive dendritic cells. CTLA-4 blockade acts mainly at the priming phase by relieving inhibitory signals during the early activation of naïve T cells, thereby enhancing antigen presentation and T-cell priming. Dendritic cells present tumor antigens and activate CD4
+ helper T cells, which in turn provide essential signals to support the priming and expansion of CD8+ cytotoxic T cells. Thus, although PV and PC act through different mechanisms, both therapies successfully converted the TIME into an immune-hot state.
Sequential administration of PV and PC further enhanced immune activation compared with monotherapies (
Fig. 1). CD8-positive T cells and Granzyme B-positive cells increased in both PV-PC and PC-PV, while immunosuppressive populations were further reduced. Although the two sequences produced comparable outcomes, both induced stronger immune activation than single regimens. Sequence-specific differences were not evident, which may be explained by the persistence of antibody activity due to long half-lives, potentially masking order-dependent variations [
13].
Antitumor efficacy paralleled the immunological findings. Tumor weight was significantly reduced in both sequential groups compared with vehicle treatment, with no difference between PV-PC and PC-PV (data not shown). Notably, the sequential combination of PV and PC therapies appeared to overcome the immunological limitations of each individual regimen. These results provide a preclinical rationale supporting the triplet therapy strategy and underscore the importance of understanding how each therapy modulates the TIME to design effective immunotherapy strategies that maximize antitumor responses. Both anti-PD-L1 plus VEGF therapy and anti-CTLA-4-based therapy are currently approved as first-line treatments, and sequential administration of two first-line regimens is generally not performed. Moreover, given the varying approval status and regulatory restrictions for ICIs across countries, implementing a triplet therapy combining PD-L1, VEGF, and CTLA-4 blockade may be challenging in the current clinical landscape. However, as demonstrated in this study, the ideal strategy to optimize antitumor immunity may involve sequentially integrating these three agents to fully activate both the priming and effector phases of the cancer-immunity cycle.
This study provides preclinical evidence that integrating PD-L1, VEGF, and CTLA-4 blockade enhances antitumor immunity in HCC by engaging both the priming and effector phases of the cancer-immunity cycle. The findings highlight the importance of understanding how individual regimens modulate the TIME to design strategies that maximize immune activation. Although the clinical application of a triplet approach may face regulatory and practical challenges, these results establish a rationale for further translational and clinical research exploring the therapeutic potential of triplet therapy in HCC. Although our data provide a preclinical rationale for triplet therapy, clinical translation would inevitably raise concerns regarding overlapping toxicities. A particular concern is immune-related adverse events (irAEs), which are frequently observed with CTLA-4 and PD-(L)1 blockade and can involve multiple organs, including the liver, lung, gastrointestinal tract, endocrine glands, and skin. Triplet therapy may not only enhance antitumor immunity but also increase the risk and severity of irAEs. The potential accumulation of these toxicities highlights the need for careful safety monitoring and management strategies. Therefore, rigorous evaluation of tolerability in prospective clinical trials will be indispensable before considering clinical application. A limitation of this study is that it was conducted in a murine subcutaneous tumor model, which may not fully recapitulate the complexity of human disease, including treatment resistance and long-term immune dynamics. Another limitation is that we did not assess the spatial distribution of immune cells within tumors, particularly differences between the tumor center and periphery. Previous studies have shown that CD8
+ T-cell infiltration into the tumor core, rather than accumulation at the periphery, is more predictive of effective tumor rejection [
14,
15]. Spatial profiling will therefore be necessary in future work to elucidate whether PV, PC, or their sequence differentially affect intratumoral versus peripheral immune-cell localization. Nevertheless, the findings in this study provide an important mechanistic basis to guide future investigations.
In this study, we demonstrated that PV and PC therapies shape the TIME in distinct ways. Sequential triplet therapy activated antitumor immunity more effectively than each monotherapy alone. A deeper understanding of these therapy- specific differences in TIME may pave the way toward more effective sequential and combination strategies in HCC.
FOOTNOTES
-
Authors’ contribution
H.I. conceptualized the study and was responsible for the overall writing. H.K. and T.K. supervised the scientific content and provided critical feedback throughout the manuscript development. All authors reviewed and approved the final version of the manuscript.
-
Acknowledgements
The authors sincerely thank the members of their research and clinical teams for their continuous support.
-
Conflicts of Interest
H.I. has received honoraria (lecture fees) from Eisai Co., Ltd. and Chugai Pharmaceutical Co., Ltd., and research funding from Eisai Co., Ltd. T.K. has received honoraria (lecture fees) from ASKA Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Kowa Company, Ltd., AbbVie GK., Eisai Co., Ltd., EA Pharma Co., Ltd., Nippon Boehringer Ingelheim Co., Ltd., Sumitomo Pharma Co., Ltd., Novo Nordisk Pharma Ltd., Otsuka Pharmaceutical Co., Ltd., Janssen Pharmaceutical K.K. The other authors declare no conflicts of interest relevant to this work.
Figure 1.Representative immunohistochemical staining of the tumor immune microenvironment (TIME). Representative images of immunohistochemical staining for CD31 (tumor vessels), CD8 (cytotoxic T cells), CD4 (helper T cells), FOXP3 (regulatory T cells), Granzyme B (activated immune cells), CD11c (dendritic cells), and F4/80 (macrophages) are shown for each treatment group: Vehicle, PV (anti-PD-L1 plus anti-VEGF therapy), PC (anti-PD-L1 plus anti-CTLA-4 therapy), PV-PC (PV followed by PC), and PC-PV (PC followed by PV). Scale bars represent 100 μm. The bar graphs below each marker indicate the quantitative analysis of positive cells per field. Data are presented as mean±SEM. Statistical significance was determined using one-way ANOVA followed by post hoc tests. *P<0.05, **P<0.01, ***P<0.001. n.s., not significant.
Abbreviations
cytotoxic T-lymphocyte–associated antigen 4
immune checkpoint inhibitor
anti–PD-L1 plus anti–CTLA-4 therapy
programmed death ligand 1
anti–PD-L1 plus anti–VEGF therapy
standard error of the mean
tumor immune microenvironment
vascular endothelial growth factor
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