Dear Editor,
The study by Iwamoto et al. focused on the regulatory mechanisms of the tumor immune microenvironment (TIME) in triple immunotherapy for hepatocellular carcinoma (HCC), providing valuable preclinical data for sequential combination treatment strategies [
1]. However, considering the latest advancements in HCC research and the requirements for clinical translation, we believe that there is room for improvement in this study regarding the representativeness of models, the depth of mechanism analysis, and the rigor of the core design.
The high heterogeneity of HCC serves as a fundamental premise for individualized decision-making in immunotherapy. This heterogeneity is not only reflected at the molecular level but is also closely associated with etiological factors such as hepatitis B virus, hepatitis C virus, alcohol consumption, and non-alcoholic steatohepatitis. HCC driven by different etiologies exhibits intrinsic differences in the composition of the TIME and the functional states of immune cells [
2]. A recent single-cell analysis based on human HCC samples further confirms that different etiological backgrounds are significantly associated with distinct immune cell compositions and functional states [
3]. A synergistic effect observed in a single cell line model may not be extrapolatable to the clinically prevalent and more immunosuppressive subtypes of HCC. Furthermore, subcutaneous models lack the liver-specific immune ecosystem, including Kupffer cells and liver sinusoidal endothelial cells, which play indispensable roles in regulating intrahepatic immune tolerance and T cell infiltration [
4]. Therefore, we recommend that future studies incorporate orthotopic HCC models with diverse etiological backgrounds and molecular subtypes for validation, in order to assess their generalizability in more clinically relevant heterogeneous contexts.
The second point is that the study’s assessment of the tumor immune microenvironment is limited to cell quantities, failing to reveal crucial changes in the functional states of immune cells. The study quantified the density of various immune cells, such as CD8 (cytotoxic T cells) and FOXP3 (regulatory T cells), using immunohistochemistry, which provides fundamental information about changes in immune landscape composition. However, the key determinant of anti-tumor immune efficacy lies not in cell quantity but in their functional state. Indeed, in HCC, T cell exhaustion is one of the primary mechanisms leading to immunotherapy failure, characterized by the co-expression of multiple inhibitory receptors and the loss of effector function [
5]. Merely increasing the number of intratumoral CD8 may not translate into effective tumor clearance unless their exhausted state is simultaneously reversed. This study did not assess critical functional indicators such as T cell exhaustion markers, cytokine production capacity, or proliferative potential. Consequently, it remains unclear whether the observed increase in immune infiltration represents an expansion of functionally competent effector cells or merely an accumulation of dysfunctional cells. Future research urgently needs to integrate single-cell RNA sequencing or multiplex flow cytometry to deeply dissect the functional subsets and exhaustion states of T cells. This should be combined with technologies such as intravital imaging to validate genuine improvements in vascular function, thereby more accurately assessing the true biological effects of this therapeutic approach.
Finally, in this study, the design of the sequential treatment may lead to biased interpretation of the results due to insufficient consideration of the long half-lives of the antibody drugs involved. Therapeutic monoclonal antibodies, such as anti-programmed death ligand 1 (PD-L1) antibodies, can exhibit a half-life of several weeks in vivo [
6]. This means that when switching immediately to the anti–PD-L1 plus anti–CTLA-4 therapy regimen after administering the anti–PD-L1 plus anti–VEGF therapy treatment in the first week, the previously administered antibodies remain at effective pharmacological concentrations in the bloodstream and tissues. Therefore, the so-called sequential therapy is essentially closer to a delayed overlapping combination therapy, rather than a truly sequential treatment where two mechanisms are distinctly and consecutively engaged. This makes it challenging to determine whether the observed immune enhancement effect stems from a specific “priming-effect” sequence or is merely the result of the additive effects of the three drugs co-existing in the mouse body. Future mechanistic studies could consider using drug forms with significantly different half-lives (such as small molecule inhibitors) or implementing longer washout periods to more clearly dissect whether treatment sequence dependency genuinely exists.
In summary, the work by Iwamoto et al. represents a crucial step forward in exploring triple immunotherapy for HCC. We believe that by adopting model systems more representative of clinical heterogeneity, incorporating in-depth evaluation methods that better reflect true biological function, and designing studies more rigorously to clarify the actual mechanisms of drug synergy, we will be able to more reliably assess and ultimately optimize this highly promising therapeutic strategy.
FOOTNOTES
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Authors’ contribution
Han Zhuo: conceptualization, data curation, writing–original draft preparation, and supervision; Yue Teng: conceptualization, data curation, writing–reviewing and editing; Zhongming Tan: validation, writing–original draft preparation, and writing – reviewing and editing. All authors read and approved the final manuscript.
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Acknowledgements
This work was supported by Basic Research Program of Jiangsu (SBK2015041078 and SBK20250202967), National Natural Science Foundation of China (81972675 to Z.T.) and Beljing Chen Jumel Foundation (Z2025100248 to H.Z.).
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Conflicts of Interest
The authors have no conflicts to disclose.
Abbreviations
non-alcoholic steatohepatitis
programmed death ligand 1
single-cell RNA sequencing
tumor immune microenvironment
REFERENCES
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Citations
Citations to this article as recorded by
- Correspondence to letter to the editor on “Distinct tumor immune microenvironment modulation by anti-PD-1/PD-L1, VEGF, and CTLA-4 blockade provides a rationale for triplet therapy in hepatocellular carcinoma”
Hideki Iwamoto, Hironori Koga, Takumi Kawaguchi
Clinical and Molecular Hepatology.2026; 32(3): e424. CrossRef
