MicroRNAs Modulating Cancer Immunotherapy Mechanisms and Therapeutic Synergies

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review covers the roles of miRNAs in cancer immunotherapy, from molecular mechanisms to clinical applications, including miRNA-mediated regulation of immune checkpoints, tumor microenvironment remodeling, T cell and macrophage polarization, therapeutic synergies with immune checkpoint blockade, and recent advances in delivery systems.
However, the manuscript suffers from redundancy and a largely descriptive style, lacking critical synthesis and balanced evaluation. 

Major concerns:
1. The manuscript lists the actions of individual miRNAs one by one, rather than integrating them mechanistically (e.g., “shared miRNA-mediated PD-L1 regulation” or “network features of immune tolerance”).

2. Although delivery systems such as LNPs, exosomes, and aptamers are mentioned, key safety and translational issues are not adequately discussed — e.g., immune-related toxicity (as in the MRX34 trial termination), dose control, off-target effects, and host immune recognition of delivery vehicles.

3. The clinical trial section is mainly descriptive (e.g., MRX34, TargomiR, Cobomarsen) without mechanistic insight into the reasons for success or failure and lessons learned from early miRNA trials.

4. Some statements are too assertive relative to the supporting evidence. For instance, I think the claim that “engineered CAR-miR cells represent a paradigm shift toward multifunctional T cells” overstates the current evidence. 

5. miR-21 may have both oncogenic and tumor-suppressive roles depending on the context, and miR-155 can promote tumorigenesis under chronic inflammation.

6. While some sections (e.g., miR-138-5p with anti–PD-1) clearly specify “preclinical models,” others mix findings from cell lines, animal models, and clinical observations without consistent labeling.

Minor Concerns
1. Figure 2 indicates “miR-34a → M1 macrophage,” yet the text never mentions miR-34a’s role in macrophage polarization. 
2. The roles of miR-34a and miR-155 are repeated across sections (mechanistic vs. therapeutic synergy); merging or cross-referencing would improve conciseness.

Author Response

Response to Reviewer 1 comments

Query (Q)1: The manuscript lists the actions of individual miRNAs one by one, rather than integrating them mechanistically (e.g., “shared miRNA-mediated PD-L1 regulation” or “network features of immune tolerance”).

Response(R)1: We thank the reviewer for this insightful observation. We respectfully note that the primary objective of this review is to provide a mechanistically detailed, miRNA-by-miRNA mapping of their roles in cancer immunotherapy. Because many miRNAs exert highly context-specific, target-specific, and sometimes opposing effects, a gene-by-gene structure allows readers to clearly distinguish each regulatory mechanism without conflating them. The manuscript already embeds mechanistic integration within each subsection for example, shared PD-L1 regulatory motifs, common PTEN/PI3K–AKT-mediated immune-evasion pathways and overlapping control of antigen presentation and macrophage polarization. These integrative themes emerge organically throughout the text rather than as a standalone summary. We therefore believe the existing format preserves clarity, mechanistic precision, and conceptual readability, especially for researchers seeking specific miRNAs relevant to their therapeutic or biomarker interests. We hope the reviewer agrees that this structure provides a balanced combination of mechanistic depth and accessibility.

Q2. Although delivery systems such as LNPs, exosomes, and aptamers are mentioned, key safety and translational issues are not adequately discussed — e.g., immune-related toxicity (as in the MRX34 trial termination), dose control, off-target effects, and host immune recognition of delivery vehicles.

R2: We appreciate the reviewer’s focus on translational considerations. We respectfully note that the scope of this review is to summarize mechanistic roles of miRNAs in immunotherapy, with delivery platforms presented as supporting examples rather than the primary subject of the article. Nevertheless, the manuscript already includes key translational aspects for instance, it discusses the immune-mediated toxicities observed in the MRX34 trial, challenges in nuclease degradation, off-target biodistribution, immunogenic recognition of RNA/lipid constructs by TLR receptors, and dose-limiting concerns related to delivery vehicle clearance and innate immune activation. Given the already substantial length and mechanistic emphasis of the review, we aimed to maintain focus on miRNA biology rather than provide an in-depth engineering or pharmaceutical analysis of delivery vehicles. We believe the current level of coverage sufficiently frames the major translational barriers while preserving the manuscript’s central theme. We hope the reviewer agrees that this balance maintains the clarity and scientific focus of the paper.

Q3. The clinical trial section is mainly descriptive (e.g., MRX34, TargomiR, Cobomarsen) without mechanistic insight into the reasons for success or failure and lessons learned from early miRNA trials.

R3: We appreciate the reviewer’s observation. The primary intent of the clinical trial section is to provide a concise overview of human-tested miRNA therapeutics relevant to immuno-oncology. While the section summarizes major trials (MRX34, TargomiR, Cobomarsen), mechanistic insights and translational lessons are already embedded within their descriptions for example, the immune-mediated toxicity and delivery-associated cytokine activation underlying the MRX34 trial termination, the minicell-based delivery specificity influencing TargomiR responses, and the pathway-selective inhibition demonstrated in Cobomarsen. Because this review is mechanistically focused on miRNA regulation of cancer immunity, we intentionally kept the clinical trial discussion brief and avoided extensive pharmacologic analysis. A detailed review can be drafted on this topic later.  We thank reviewer for suggesting a good idea to draft a review article that could potentially fill the gap.

Q4. Some statements are too assertive relative to the supporting evidence. For instance, I think the claim that “engineered CAR-miR cells represent a paradigm shift toward multifunctional T cells” overstates the current evidence. 

R4: Thank you for this helpful suggestion. We agree that the original phrasing may appear too strong given the preclinical stage of CAR-miR research. We have therefore revised the statement to “a promising direction toward multifunctional therapeutic T cells,” which more appropriately reflects the current evidence while retaining the intended scientific meaning.

Q5. miR-21 may have both oncogenic and tumor-suppressive roles depending on the context, and miR-155 can promote tumorigenesis under chronic inflammation.

R5: We thank the reviewer for highlighting the context-dependent behaviour of these miRNAs. We agree that both miR-21 and miR-155 can display dual roles depending on tissue type, inflammatory signals, and tumor microenvironmental context. To acknowledge this complexity, we have added a brief clarifying sentence in the relevant sections noting that miR-21 may exhibit tumor-suppressive effects under specific conditions and that miR-155 can promote tumorigenesis in chronic inflammatory settings.

 Q6. While some sections (e.g., miR-138-5p with anti–PD-1) clearly specify “preclinical models,” others mix findings from cell lines, animal models, and clinical observations without consistent labelling.

R6: Thank you for this thoughtful observation. To improve clarity and ensure consistency across the manuscript, we have added a brief statement at the end of the Introduction clarifying that, unless otherwise specified, the mechanistic findings summarized in the review derive from a combination of cell-based, animal, and early translational studies, with model types highlighted when essential for interpretation. This ensures transparency regarding the evidence source while preserving the flow and readability of the individual miRNA sections.

Minor Concerns
1. Figure 2 indicates “miR-34a → M1 macrophage,” yet the text never mentions miR-34a’s role in macrophage polarization.

Response: Thank you for pointing this out. To ensure consistency between the text and Figure 2, we have added a brief sentence noting that miR-34a promotes M1 macrophage polarization through Notch/STAT1-mediated regulation in myeloid cells.

2. The roles of miR-34a and miR-155 are repeated across sections (mechanistic vs. therapeutic synergy); merging or cross-referencing would improve conciseness.

Response: We thank the reviewer for this insightful observation. We agree that miR-34a and miR-155 appear in both the mechanistic and therapeutic sections. To improve clarity and avoid unnecessary repetition, we have added a concise cross-referencing sentence at the beginning of Section 3.1 (page 11).

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the opportunity to review this manuscript. This is a comprehensive and timely review article. The authors summarized a massive amount of information, covering the topic from basic molecular mechanisms to clinical trials and advanced AI-driven biomarker discovery.

However, the manuscript's greatest weakness is a significant overlap and redundancy across sections. Addressing this structural issue could be streamlined to improve the logical flow and readability of this manuscript. Below are my comments for revision.

1. The major issue of the manuscript is the repetitive discussion of identical mechanisms across multiple subsections. For example, the regulation of PD-L1 is mentioned in 3 different sections including 2.2, 2.3, and 2.6. Similarly, Repetitive discussion of miR-155 and DCs also be discussed 3 times in 2.7, 2.8.2, and 3.2.2. Also, content in section 5 was already described in detail in earlier text.
2. The overall logic of section 2 is difficult to follow since the paragraphs are very long and dense with numerous distinct examples and points.
3. In several places, e.g., section 2.5 and table 1, a very long sentence or information is supported by only one or two citations
4. Figure 2 is first mentioned in Section 2.4, but its content (exosomes) is not relevant there; it more closely relates to Section 2.7.
5. Figure 2 itself requires significant revision. The title does not fully capture the content. More importantly, the arrows and labels are unclear and do not fully match the caption. Also, some arrows and related descriptions are missing.
6. Roles of miRNAs contained in extracellular vesicles including exosomes in tumor were described in section 2.7 as the modulation of the tumor microenvironment. However, extracellular vesicles play roles not only in tumor microenvironment but also in the systemic level to achieve a distant cell-cell communication. It would be helpful to separate this part as an independent section and elaborate from microenvironment to macroenvironment.
7. Given the complexity of Section 2, the manuscript would be strengthened by a new schematic that summarizes the diverse regulatory roles of miRNAs in antitumor immunity, not just the EVs-mediated trafficking currently shown in Figure 2.
8. The manuscript has minor formatting and language issues, for example, low/high symbols in line 312. There appears to be inconsistent format in the reference list.

Author Response

Response to Reviewer 2 comments

Q1. The major issue of the manuscript is the repetitive discussion of identical mechanisms across multiple subsections. For example, the regulation of PD-L1 is mentioned in 3 different sections including 2.2, 2.3, and 2.6. Similarly, Repetitive discussion of miR-155 and DCs also be discussed 3 times in 2.7, 2.8.2, and 3.2.2. Also, content in section 5 was already described in detail in earlier text.

R1: We appreciate the reviewer’s detailed observation regarding repeated mentions of certain mechanisms, such as PD-L1 regulation, miR-155’s role in dendritic cells, and concepts reiterated in Section 5. We respectfully note that these repetitions arise because several miRNAs converge on the same immunoregulatory pathways, and each subsection is organized around a distinct functional axis (immune checkpoint regulation, T-cell activation, dendritic-cell maturation, therapeutic synergy, etc.). For this reason, certain mechanisms necessarily reappear in different contexts, as their relevance extends across multiple cell types and immunotherapy modalities. We have been careful to avoid verbatim repetition and, where repeated, the content is presented from different mechanistic perspectives. This structure allows each subsection to remain self-contained for readers who may consult specific miRNA pathways independently rather than reading straight through. Section 5 consolidates these concepts intentionally, serving as a synthesis rather than introducing new material.

Q2. The overall logic of section 2 is difficult to follow since the paragraphs are very long and dense with numerous distinct examples and points.

R2: We thank the reviewer for this observation. Section 2 covers a wide range of immune regulatory mechanisms mediated by miRNAs, and the density partly reflects the inherent complexity of these pathways and the substantial amount of literature available. The examples provided in this section were intentionally grouped within broader mechanistic themes (e.g., immune checkpoints, antigen presentation, macrophage polarization) to preserve continuity and avoid fragmenting concepts across multiple smaller subsections. Although some paragraphs are necessarily detailed due to the multifactorial nature of miRNA–immune interactions, the logical flow follows a structured progression from innate to adaptive immune regulation. We believe this organization allows readers to appreciate the interconnectedness of these pathways without excessive subdivision. Nonetheless, we appreciate the reviewer’s comment and will keep this in mind for future refinement.

Q3. In several places, e.g., section 2.5 and table 1, a very long sentence or information is supported by only one or two citations.

R3: Thank you for this thoughtful observation. We respectfully note that in the sections highlighted (such as Section 2.5 and Table 1), the referenced studies are comprehensive and well-established works that individually encompass the multiple mechanistic points summarized in the text. These citations were purposefully selected because they are seminal, high-quality, and integrative studies that themselves consolidate broad mechanistic findings. As such, a single citation in these contexts sufficiently supports the full scope of the statement without requiring additional references.

Q4. Figure 2 is first mentioned in Section 2.4, but its content (exosomes) is not relevant there; it more closely relates to Section 2.7.

R4: We appreciate the reviewer’s careful observation regarding the placement of Figure 2. We agree that the figure’s focus on exosomal miRNA-mediated communication is more appropriately aligned with the mechanisms discussed in Section 2.7. Accordingly, we have relocated the first citation and full placement of Figure 2 to Section 2.7, where the figure now directly complements the discussion of extracellular vesicle–mediated miRNA transfer and its role in shaping the tumor microenvironment.

Q5. Figure 2 itself requires significant revision. The title does not fully capture the content. More importantly, the arrows and labels are unclear and do not fully match the caption. Also, some arrows and related descriptions are missing.

R5: We thank the reviewer for this valuable feedback on Figure 2. In response, we have carefully revised the figure to ensure clarity and consistency. The figure title has been updated to more accurately reflect the scope of the schematic, and the caption has been rewritten to provide a clearer and more comprehensive description of the depicted interactions.

Q6. Roles of miRNAs contained in extracellular vesicles including exosomes in tumor were described in section 2.7 as the modulation of the tumor microenvironment. However, extracellular vesicles play roles not only in tumor microenvironment but also in the systemic level to achieve a distant cell-cell communication. It would be helpful to separate this part as an independent section and elaborate from microenvironment to macroenvironment.

R6: We thank the reviewer for this insightful suggestion. We fully agree that extracellular vesicles (EVs), including exosomes, mediate both local (microenvironmental) and systemic (macroenvironmental) communication. In the present manuscript, Section 2.7 focuses specifically on EV-mediated modulation of the tumor microenvironment because this review section was designed to address microenvironment-level immunoregulation in a mechanistic framework. While systemic EV-mediated communication is indeed important, detailed discussion would require expansion into broader topics such as organotropism, metastatic niche preparation, circulation-mediated immune modulation, and endocrine-like EV signalling, which fall beyond the scope of the mechanistic immune regulation theme of Section 2. Our current structure is therefore intentionally focused on immune interactions within the tumour microenvironment.

Q7. Given the complexity of Section 2, the manuscript would be strengthened by a new schematic that summarizes the diverse regulatory roles of miRNAs in antitumor immunity, not just the EVs-mediated trafficking currently shown in Figure 2.

R7: We appreciate the reviewer’s thoughtful suggestion regarding the addition of an additional schematic figure. We fully agree that Section 2 encompasses a broad range of miRNA-mediated immunoregulatory mechanisms. However, to maintain focus and avoid redundancy with existing figures, we elected to keep Figure 2 centred specifically on EV-mediated pathways, which represent one of the most integrative and visually depictable modes of miRNA communication. The remaining mechanisms in Section 2 span a wide range of cellular contexts, including immune checkpoints, antigen presentation, macrophage polarization, NK-cell activation, and cytokine regulation. Because these pathways are highly heterogeneous and mechanistically distinct, condensing them into a single schematic risks oversimplification and loss of clarity. We therefore felt that retaining individual mechanistic explanations in the text provides more scientific accuracy than a broad, compressed illustration

Q8. The manuscript has minor formatting and language issues, for example, low/high symbols in line 312. There appears to be inconsistent format in the reference list.

R8: We thank the reviewer for pointing out these formatting issues. The low/high symbols have been removed to avoid any ambiguity, and the corresponding sentence has been clarified for readability. We also carefully reviewed the reference list to ensure uniformity throughout the manuscript. We appreciate the reviewer’s attention to detail, which has helped improve the clarity and presentation of the paper.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The author's responses are acceptable.

Author Response

Dear Reviewer, 

Thanks for accepting our response to your comments in the first round of revision itself. We sincerely thank your valuable time and efforts in shaping our manuscript to the best reader clarity. The English language was also checked for grammar and typos. Thanks again 

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for the authors' responses. However, I still maintain that the manuscript requires significant reorganization before publication in its current form, especially concerning Section 2. Besides, Figure 2 are still unclear and do not fully match the caption.

Author Response

Dear Reviewer - Thank you for your constructive suggestion. We carefully revised Section 2 and completely reorganized its structure to improve clarity and logical flow. Specifically, we merged overlapping subsections (previous Sections 2.2 and 2.4) into a single unified section titled “Direct targeting of immune checkpoints and co-stimulatory molecules.” All the corrections were yellow highlighted in the manuscript. 

Regarding Figure 2, we fully redesigned and redrew the figure based on your feedback. The new version is divided into clearly labelled Panels a, b, and c, each matched precisely to the updated caption.
Thanks again for your valuable time and efforts in shaping our manuscript. Additionally the English language was also checked for both grammar and typos - thanks