Review for 10.1186/s12876-025-03978-w (2025)

Title: Hypoxia-induced exosomal circNRIP1 activates cancer-associated fibroblasts to promote
esophageal squamous cell carcinoma migration and invasion
Version: 2 Date: 18 Feb 2025
Reviewer:  Anwar Ashraf Abouelnasr
Reviewer's report:
Dear Authors, please address the following points below
-The references for multiple studies, such as the statements on the role of CAFs (e.g., Biffi
and Tuveson, 2021; Yang et al., 2023), appear inconsistent in style, especially with incomplete
citations (e.g., "P, 2021" or "E et al., 2022"). It would be beneficial to correct these references,
ensuring they are properly formatted with full details (authors, titles, journals, etc.).
-The text mentions CXCL1-CXCR2-STAT3 signaling and CAF-derived exosomal miR-889-3p,
which are important but could be briefly elaborated on regarding their connection to hypoxic
exosomes and circNRIP1. This additional explanation would make the relationship between
these factors clearer and strengthen the introduction.
-The method mentions the "hypoxic culture bags" used to generate hypoxia conditions, but
the specifics of oxygen concentration (e.g., 1% O2) could be more clearly defined. Are the
conditions precisely controlled throughout the experiment? More information on how long cells
are exposed to hypoxia could be helpful.
-The isolation of exosomes by ultracentrifugation is well-explained. Still, it could be beneficial
to mention any additional characterization techniques (e.g., nanoparticle tracking analysis,
protein markers) that could be used to confirm the purity of the exosome preparation. This would
add confidence to the results regarding exosome effects on CAFs.
-The methodology lacks any reference to in vivo models. Given the in vitro focus of the study,
including a brief note on how the findings might be validated in xenograft models or patient-
derived xenografts (PDX) would strengthen the translational potential of the research. If animal
experiments are not part of the study, it may be worth mentioning this limitation.
-The process for generating stable knockdown and overexpression cell lines could benefit
from additional information, such as viral titer, transfection efficiency, and confirmation of
gene knockdown/overexpression. This ensures the manipulation of circNRIP1 and TRMT6 is
effective.
-While the manuscript mentions that hypoxic exosomes from ESCC cells promote CAF
activation, the exact mechanism by which exosomal circNRIP1 leads to CAF transformation
could benefit from a clearer, stepwise explanation. For example, further clarification on how
circNRIP1 regulates downstream signaling pathways in CAFs (beyond migration and invasion)
could be beneficial. Does it affect CAF differentiation, inflammatory cytokine secretion, or
matrix remodeling factors directly? This would help further contextualize the findings.
-The interaction between circNRIP1 and TRMT6 is a promising aspect of this work, but
more detail could be provided regarding how TRMT6 influences CAF activation upon
binding with circNRIP1. Does TRMT6 alter the expression of specific proteins that drive
CAF activation? A more detailed molecular description would improve the mechanistic
understanding. Additionally, discussing the impact of m1A modifications on tRNA translation
and how this might relate to CAF function would strengthen this aspect of the discussion.
-While the focus is on circNRIP1, it would be worth mentioning other potential cargos carried
by hypoxic exosomes that could contribute to the CAF activation process. For example, do
exosomal miRNAs or proteins play a synergistic role with circNRIP1 in the activation of CAFs?
-Briefly addressing this could present a more holistic view of the TME's complexity and help
explain how exosomes coordinate the activation of CAFs.
-The discussion could benefit from an expanded explanation of how CAFs contribute to ESCC
progression at a tissue level. What specific ECM remodeling events, cytokine secretion, or
other CAF-related behaviors (such as immunosuppression or angiogenesis) are triggered by
circNRIP1 in the ESCC TME? Connecting these findings to the overall tumor behavior would
be valuable for readers who want to understand the broader implications of CAF activation.
-While circNRIP1 is a promising mediator, it would be helpful to briefly mention if other
factors or molecules (e.g., other circRNAs, miRNAs, or proteins) might play a complementary
or compensatory role in CAF activation. This could highlight the complexity of CAF regulation
and potential challenges for targeting circNRIP1 as a sole therapeutic approach.