Dear Editor,
We thank Zhuo and colleagues for their careful reading of our Research Letter and for their constructive comments [
1,
2]. In their letter, they raise three significant points that are highly relevant to the translational interpretation of our findings, namely the limitations of relying on a single subcutaneous syngeneic model in the context of the heterogeneous tumor immune microenvironment (TIME) of hepatocellular carcinoma (HCC), the need for functional immune profiling beyond immunohistochemical cell quantification, and the possibility that the long half-lives of therapeutic antibodies may result in pharmacologic overlap between sequential regimens. We agree that all of these considerations are important and should be taken into account when interpreting preclinical data and designing future translational studies.
In our Research Letter, we used a subcutaneous syngeneic tumor model established with a murine hepatocellular carcinoma cell line (Hep53-4) to compare how an anti-programmed death-ligand 1 (PD-L1) plus anti-vascular endothelial growth factor (VEGF) regimen and an anti-PD-L1 plus anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) regimen modulate the tumor immune microenvironment (TIME). We quantitatively assessed TIME modulation by immunohistochemical profiling of significant immune cell populations and a cytotoxic activation marker. Although both regimens shifted tumors toward an immunehot TIME, they generated distinct immune cell compositions. We further evaluated the sequential administration of these regimens. We observed that exposure to anti-PD-L1, anti-CTLA-4, and anti-VEGF was associated with a higher level of immune activation than either regimen alone. In contrast, sequence-specific differences were not evident under the treatment intervals used.
We agree that HCC is biologically heterogeneous and that TIME can vary substantially depending on etiology and underlying liver condition [
3-
9]. We also agree that a subcutaneous syngeneic model has inherent constraints, because it does not fully recapitulate the hepatic immune microenvironment. Specifically, among currently approved combination immunotherapies, an anti-PD-L1 plus anti-VEGF regimen and an anti-PD-L1 plus anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) regimen can both drive tumors toward an immune-hot state, but it remains unclear whether they shape the TIME through distinct processes. We further asked whether sequencing an anti-PD-L1 plus anti-VEGF regimen with an anti-PD-L1 plus anti-CTLA-4 regimen could induce a more immune-hot TIME than either regimen alone. We did not intend to claim direct clinical generalizability across all disease contexts. Consistent with this intention, we explicitly described the use of a murine subcutaneous tumor model as a limitation. Accordingly, as they pointed out, broad validation in diverse clinical and translational settings will be essential to clarify how these combination immunotherapies shape the TIME and how such changes may relate to treatment response and resistance.
We agree that quantification of immune cells by immunohistochemistry alone does not fully capture immune function. In our study, we used immunohistochemistry to quantify significant immune populations, including CD8-positive cytotoxic T cells, CD4-positive helper T cells, FOXP3-positive regulatory T cells, F4/80-positive macrophages, and CD11c-positive dendritic cells, and we also evaluated Granzyme B-positive cells as a marker of activated cytotoxic cells. However, as they pointed out, a comprehensive understanding of the TIME requires integrated information on which cells are present, where they are localized within tumors, what functional states they exhibit, and which mediators they produce. Our study primarily addressed cellular composition; we did not evaluate spatial distribution in detail, functional states at the single-cell level, or secreted mediators. Accordingly, we agree that additional approaches, including multiparameter flow cytometry, multiplex and spatial immune profiling, and single-cell transcriptomic analyses, will be critical in future studies to define how these regimens modulate functional immune states within the TIME.
We agree that the long half-lives of therapeutic monoclonal antibodies can lead to pharmacologic overlap between sequential regimens, which may obscure order-dependent effects. This overlap may be one reason we did not observe differences between the sequences of an anti-PD-L1 plus anti-VEGF regimen followed by an anti-PD-L1 plus anti-CTLA-4 regimen and the reverse sequence, as noted in our Research Letter. Importantly, however, the key finding of our study is that exposure to the three antibodies, namely anti-PD-L1, anti-CTLA-4, and anti-VEGF, induced stronger immune activation than either regimen alone. We agree that further studies are warranted to determine the most appropriate sequencing strategy, including study designs that minimize unintended overlap and allow a more precise assessment of order-dependent effects.
We again thank Zhuo and colleagues for their constructive letter. In recent years, multiple combination immunotherapies have become available for the treatment of HCC, and these regimens are commonly considered within the same therapeutic class of immune-based combinations in clinical decision-making [
10-
12]. Through this preclinical analysis, we aimed to highlight that these regimens can shape the TIME in distinct ways and to explore whether exposure to anti-PD-L1, anti-CTLA-4, and anti-VEGF could enhance immune activation beyond either regimen alone. We hope that a deeper understanding of the TIME will help clinicians select and optimize combination strategies and will ultimately contribute to the development of more effective treatments for HCC.
FOOTNOTES
-
Authors’ contributions
H.I. was responsible for the overall writing. H.K. and T.K. supervised the scientific content and provided critical feedback throughout the manuscript development.
-
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.
Abbreviations
cytotoxic T-lymphocyte-associated protein 4
programmed death-ligand 1
tumor immune microenvironment
vascular endothelial growth factor
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