Review for 10.1186/s12876-025-03768-4 (2025)

Title: To Verify the Biological Characteristics of Disulfidptosis Associated Gene ADORA2B
in Esophageal Cancer
Version: 1 Date: 05 Jan 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-The term "disulfide death" or "disulfidptosis" is introduced without clear explanation. While
the concept may be novel, it would benefit from a more thorough definition for readers who
may not be familiar with the term.
-Similarly, the term SLC7A11 is mentioned, but it is not fully explained for a broader audience.
Given that this is a cell-specific marker involved in disulfide stress, more context on its
significance and its relationship to ADORA2B would clarify the biological relevance.
While the introduction provides background, it lacks a direct, clear research hypothesis or
question. It is implied that ADORA2B’s role in esophageal cancer needs exploration, but it could
be strengthened by articulating a more explicit research aim or hypothesis that guides the study.
-Although ADORA2B is discussed in other cancers, the introduction does not sufficiently
establish the current gaps in understanding ADORA2B’s role specifically in ESCC. A more
explicit mention of the literature gap or lack of studies on ADORA2B in this specific cancer
type would underscore the novelty of the research.
-The combination of TCGA-ESCA and GSE196756 datasets is described briefly, but it would
benefit from more detail on how batch effects were controlled and the statistical methods used
to combine the data. The use of the Harmony R package is mentioned but would benefit from
a brief explanation of why this specific method was chosen.
-Normalization in single-cell RNA-seq: The mention of normalization through Seurat’s
NormalizeData function is good, but additional details about how potential biases (e.g.,
sequencing depth or batch effects) were controlled would enhance the robustness of the
methodology.
-While ADORA2B knockdown in ESCC cell lines is performed, there is no mention of whether
appropriate control groups (e.g., non-targeting siRNA or scrambled siRNA) were used in all
assays. This would help ensure that the effects observed are specifically due to ADORA2B
knockdown.
-For Western blot and qRT-PCR, it is important to specify whether housekeeping genes were
used as internal controls for normalization. GAPDH is mentioned for Western blot, but there is
no mention of its use in qRT-PCR normalization, which should be clarified.
-The flow cytometry experiment is described in general terms, but further detail on how
apoptosis was quantified would be helpful. Specifically, which phase of the cell cycle was
analyzed (e.g., early vs. late apoptosis), and whether any gating strategies or specific apoptosis
markers (e.g., Annexin V/PI) were used for flow analysis?
-The phrase "mRNA related to COR Result and mRNA were defined with R > 0.4 as the limit"
is unclear. It seems to describe a Pearson correlation analysis, but the term “COR RESULT”
is undefined. It would help to clarify this phrase and explain the context of the R-value more
explicitly (e.g., correlation coefficient threshold).
-Similarly, the "DEseq" analysis for differential expression is mentioned, but it’s not clear what
the exact methods or cutoffs were for identifying significant DEGs (differentially expressed
genes). Adding more detail on the filtering process (e.g., FDR adjustment, log2 fold-change
cutoffs) would enhance the transparency of the analysis.
-The TIMER2.0 analysis and ssGSEA correlations with immune infiltration are interesting, but it
is not entirely clear how these correlations impact the functional behavior of the immune system
in ESCA. For instance, the negative correlations with CD8 T cells and eosinophils suggest
immune suppression, but more in-depth analysis could elucidate whether ADORA2B directly
inhibits immune activation or whether it plays a more indirect role in immune modulation.
-The results based on the GSE196756 scRNA-seq dataset are significant, but the data are
presented somewhat briefly. The differentiation of cell populations into macrophages, B cells,
malignant tumor cells, and T cells could benefit from additional explanation.
-Specifically, SOX2, VCAN, and CD163 are mentioned as markers for specific cell types, but
the rationale for selecting these markers and the relevance of these cell types in the tumor
microenvironment (TME) should be briefly discussed.
-The functional assays (CCK8, Transwell, EDU, flow cytometry) are briefly mentioned, but
additional details on the methodology and statistical significance of these results would improve
the section. For example, providing quantitative data or discussing specific effects observed
(e.g., percentage reduction in cell proliferation or migration) would help the reader assess the
strength of the experimental findings.
-Additionally, the discussion would benefit from a deeper interpretation of the mechanistic
pathways involved in ADORA2B’s effect on cell behavior. For instance, how exactly does
ADORA2B affect migration and invasion—is it through modulation of the extracellular matrix,
adhesion molecules, or signal transduction pathways?
-While the knockdown of ADORA2B is discussed as a potential therapeutic strategy, the
translation of this into clinical practice is not fully addressed. Could ADORA2B inhibition
lead to side effects due to its immune-modulating properties, particularly in the TME? More
information on how ADORA2B-targeting therapies could be safely integrated into clinical
settings would strengthen the discussion.
-Additionally, the clinical relevance of ADORA2B as a biomarker for early diagnosis or
prognostic assessment could be discussed in more depth. What would be the practical use of
measuring ADORA2B levels in clinical settings (e.g., as a diagnostic test, or as a prognostic
factor for ESCC staging)?