EGFR-Targeted Extracellular Vesicles Potentiate Doxorubicin-Induced Apoptosis and Tumor Suppression in Colorectal Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the manuscript titled "EGFR-Targeted Extracellular Vesicles Potentiate Doxorubicin-2 Induced Apoptosis and Tumor Suppression in Colorectal 3 Cancer”, Li et al. described the design, preparation and anticancer activity of novel EGFR-tEVs for delivery of doxorubicin. In general, the manuscript is well written and the information about the tEVs is provided in detail, and the in vivo anticancer activity of novel EVs shows the potential therapeutic benefit of novel tEVs compared to traditional doxorubicin formulations. However, there are some major improvements that need to be made in order for this article to be accepted for publication.

• Some of the references are a bit outdated. The authors should include references regarding prevalence, survival rates, and treatment with a more recent date (ideally from the past 1-2 years).
• In the conclusion, the authors state “In vitro and in vivo analyses confirmed increased p53 and Bax expression, decreased Bcl-2 levels […]”, however, no data on the effect of novel EVs on the levels of Bcl-2 was presented.
• It would be interesting to see whether the novel EVs affect the protein levels of some more colon cancer specific protein levels, rather than just proteins whose levels are overexpressed in variety of different cancers.
• It would be better if the information about the antibodies used for western blot analyses were listed in the “Materials and Methods” section of main text, rather than mentioning it only in the supplementary.

 

After these minor changes, I would recommend the article for publication in this journal.

Author Response

Response: We would like to thank the reviewer for the insightful comments and for recommending our article for publication. We have addressed the concerns as follows:

Point 1: Outdated references regarding prevalence, survival rates, and treatment.

Response: We sincerely appreciate the reviewer’s feedback. We have extensively revised the first paragraph of the Introduction to provide a more accurate and well-referenced overview of the current clinical landscape of colorectal cancer (CRC).

Specifically, we have:

1. Updated Epidemiological Data: Incorporated the latest global statistics (approximately 1.9 million new cases and 900,000 deaths annually) with recent citations from 2024–2025.
2. Refined Clinical Statistics: Clarified the prognosis for metastatic CRC (mCRC) by citing the specific 5-year survival rate (below 15%) to avoid overgeneralization.
3. Elaborated on Treatment Limitations: Rather than simply stating treatments are ineffective, we now specify the challenges of "dose-limiting toxicities," "lack of tumor specificity," and "high recurrence rates" in advanced cases, supported by recent clinical reviews.

These changes ensure a more balanced and evidence-based introduction to the clinical "unmet needs" that our study aims to address. Please see Introduction (Introduction, Lines 33–43) in the revised manuscript.

 

Point 2: Mention of Bcl-2 in the conclusion without supporting data.

Response: We apologize for this oversight. Since the Bcl-2 data was not included in our results, we have removed the mention of "decreased Bcl-2 levels" from the Conclusion section to maintain consistency with the presented data.

 

Point 3: Effects on colon cancer-specific proteins.

Response: We agree that evaluating colon cancer-specific markers would further strengthen the study. While our current study focused on the universal p53-Bax apoptotic pathway to demonstrate the fundamental anti-tumor efficacy of EGFR-tEVs+Dox, we have added a discussion regarding the need to investigate CRC-specific markers (such as CEA or COX-2) in future studies. This has been addressed in the Discussion section (Discussion, Lines [346-354]).

 

Point 4: Moving antibody information to the Materials and Methods section.

Response: In accordance with your suggestion, we have moved the detailed information for all primary and secondary antibodies (manufacturer, catalog numbers, etc.) from the Supplementary Information to the "Materials and Methods" section (Section 4.9, Lines 481-488).

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled “EGFR-Targeted Extracellular Vesicles Potentiate Doxorubicin-Induced Apoptosis and Tumor Suppression in Colorectal Cancer” presents an interesting strategy for enhancing targeted drug delivery using engineered extracellular vesicles. The study combines in vitro and in vivo experiments to demonstrate improved uptake, apoptosis induction, and tumor suppression in EGFR-overexpressing colorectal cancer models. The topic is timely and relevant, particularly in the context of developing more selective and less toxic therapeutic approaches. However, while the overall concept is promising, several aspects of the study require clarification and strengthening, particularly regarding mechanistic validation, experimental rigor, and the depth of analysis needed to support the translational significance of the findings.

 

1. The novelty of EGFR-targeted EVs is not clearly distinguished from prior EV-based targeting studies, so the authors should explicitly compare their approach to existing EGFR-targeted delivery systems and highlight what is mechanistically or technically new.
2. The Introduction overstates clinical limitations without sufficient referencing or nuance, so the authors should balance the discussion with up-to-date citations and clearly define the unmet need addressed by this study.
3. The characterization of EVs lacks rigorous validation (e.g., absence of additional negative markers or purity metrics), so the authors should include more comprehensive EV characterization following MISEV guidelines.
4. The drug loading efficiency is relatively low and not discussed, so the authors should justify whether the reported encapsulation efficiency is sufficient for therapeutic relevance or compare with alternative loading strategies.
5. The uptake study relies heavily on fluorescence imaging without mechanistic validation, so the authors should include blocking experiments (e.g., EGFR inhibition) to confirm receptor-specific uptake.
6. The apoptosis analysis is limited to a few markers, so the authors should strengthen the mechanistic insight by including additional assays (e.g., caspase activity or flow cytometry-based apoptosis quantification).
7. The comparison between cancer and normal cells is insufficiently quantitative, so the authors should include statistical comparisons and possibly dose-response analyses to support selectivity claims.
8. The in vivo study uses a small sample size (n=3–4), so the authors should increase animal numbers or acknowledge this limitation and provide power justification.
9. Tumor targeting is inferred from fluorescence accumulation without quantitative biodistribution analysis, so the authors should include more rigorous quantification (e.g., %ID/g or pharmacokinetics).
10. The toxicity assessment is limited to basic histology and serum markers, so the authors should include more sensitive toxicity endpoints or longer-term evaluation to support safety claims.
11. The statistical analysis section lacks detail, so the authors should clearly state tests used, assumptions, and corrections for multiple comparisons.
12. Some statements in the Results are repetitive or overstated, so the authors should streamline the text and avoid redundant conclusions (e.g., repeated claims of “enhanced uptake”).
13. The Discussion does not critically address limitations (e.g., RES uptake, low loading efficiency), so the authors should include a dedicated limitations paragraph.
14. The translational relevance is not fully developed, so the authors should discuss scalability, manufacturability, and comparison with antibody-based EGFR targeting approaches.

Author Response

Please find the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

1. Figure 3D shows the increased expression of p53, Bax, and cleaved PARP1, which led the authors to conclude that EGFR-tEVs+Dox activates the p53-Bax-cleaved PARP1 apoptotic pathway. However, the apoptotic function of p53 is mainly through transcriptional regulation of pro-apoptotic genes such as Bax and activation of mitochondrial pathways, leading to cytochrome c release and caspase-9/3 cascade activation. Supplemental assays were performed for cytochrome c release (cytosolic fraction) or activation of caspase-9/3 (cleaved caspase-9/3). Or at least explicitly stated in the discussion: caspase activation was not directly examined in this study, and the conclusion of the p53-Bax-PARP1 pathway is an inference based on previous literature and needs to be verified in future studies.
2. in Section 4.7, the LC% was calculated using the formula "Mass of doxorubicin in EVs/Total EV protein mass × 100". But does "Total EV protein mass" represent only EV proteins, or does it contain other proteins? In addition, it was not distinguished whether Dox was encapsulated inside the EV or adsorbed only on the surface, which is crucial for the interpretation of efficacy and release kinetics. It is clearly stated whether the protein mass determined by BCA is derived only from EVs, and it is recommended to supplement protease treatment experiments to distinguish the encapsulated drug from the surface adsorbed drug. Or explicitly acknowledge this limitation in the discussion and suggest more precise quantification using methods such as HPLC in the future.
3. Revise "Figure 6E" to "Figure 5E" in the body and ensure that all figure number references are consistent with the chart number.

Author Response

Response to Reviewer 3

Point 1: Regarding the p53-Bax-PARP1 pathway and missing caspase data.

Response: We agree with the reviewer that cytochrome c release and the caspase cascade are essential components of the p53-mediated mitochondrial apoptotic pathway. While our current results clearly show the upstream (p53, Bax) and downstream (cleaved PARP1) changes, we have now explicitly stated in the Discussion (Lines 296-305) that the caspase activation was not directly examined and that our conclusions are based on established literature. We have acknowledged this as a limitation to be addressed in future studies.

 

Point 2: Regarding LC% calculation and surface-adsorbed vs. encapsulated Dox.

Response: This is a very constructive comment. We acknowledge that the total protein mass measured by BCA may contain non-EV proteins and that our current method does not distinguish between internal encapsulation and surface adsorption of Dox. As suggested, we have added a detailed discussion (Lines 329-341) acknowledging these limitations. We also suggested using protease treatment and HPLC for more precise quantification in future optimization of the system.

 

Point 3: Regarding Figure numbering (Figure 6E to 5E).

Response: We apologize for the typographical error. We have corrected "Figure 6E" to "Figure 5E" and carefully reviewed the entire manuscript to ensure all figure citations are consistent with the final figures.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I am satisfied with the revisions made, have no further comments, and recommend the manuscript for publication.