Enhanced cytotoxicity of T-DM1 in HER2-low carcinomas via autophagy inhibition

Jinghui Zhang; Xusheng Chang; Yingcheng Bai; Xiancai Ge; Kai Yin; Qun Xin

doi:10.1371/journal.pone.0322029

Peer Review History

Original SubmissionNovember 19, 2024
19 Nov 2024 Author Response https://doi.org/10.1371/journal.pone.0322029.r001
15 Dec 2024 Decision Letter - Ruo Wang, Editor PONE-D-24-51611Enhanced Cytotoxicity of T-DM1 in HER2-low Carcinomas via Autophagy InhibitionPLOS ONE Dear Dr. Xin, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Jan 29 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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We notice that your supplementary figures are uploaded with the file type 'Figure'. Please amend the file type to 'Supporting Information'. Please ensure that each Supporting Information file has a legend listed in the manuscript after the references list. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No ****** 3. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript explores the potential of combining T-DM1, an antibody-drug conjugate, with autophagy inhibitors to enhance its cytotoxicity in HER2-low gastric carcinoma cells. The authors concluded that T-DM1 alone exhibits limited cytotoxicity in HER2-low carcinoma cells due to the activation of cytoprotective autophagy. However, inhibiting autophagy pharmacologically significantly increases T-DM1’s cytotoxic effects by promoting its lysosomal fusion and facilitating the release of its active cytotoxic agent, emtansine. Comments: The study largely repurposes previously explored concepts by combining T-DM1 with autophagy inhibitors, an approach that has already been examined in other contexts. It does not provide groundbreaking insights or novel mechanisms specific to HER2-low gastric cancer. While the manuscript mentions the involvement of the Akt/mTOR pathway and autophagy modulation, it fails to thoroughly investigate the molecular interactions or provide sufficient evidence to establish causal relationships between autophagy inhibition and the enhanced efficacy of T-DM1. The figures and results sections include excessive repetition, particularly in the presentation of apoptotic and autophagy-related assays. Streamlining this information would improve clarity and enhance the manuscript's readability. The manuscript lacks detailed descriptions of the statistical analyses performed, including criteria for significance and the sample sizes used for each experiment. This omission undermines the reproducibility and robustness of the findings. The study does not adequately compare the efficacy of the proposed combination therapy with existing treatment options for HER2-low gastric cancer, limiting its clinical relevance and applicability. Phrases such as "significantly enhanced cytotoxicity" are used without providing clear quantitative benchmarks, leaving the practical significance of the findings unclear. The discussion fails to place the findings within the broader landscape of HER2-low gastric cancer research. Conflicting evidence or alternative interpretations are not sufficiently addressed, leaving gaps in the contextual analysis. The authors assert that autophagy inhibition may have universal applications across various tumor types but provide no supporting evidence beyond the context of HER2-low gastric cancer cells. This weakens the broader impact of the conclusions. Reviewer #2: In the manuscript entitled: “Enhanced cytotoxicity of T-MD1 in HER2-low carcinomas via autophagy inhibition”, Zhang et al. showed that T-DM1 induces autophagy and combination of T-DM1 with autophagy inhibitors increases T-DM1 efficacy in HER2-low gastric cancer. A potential expansion of the use of T-DM1 and may be other HER2-targeted therapies to tumors with low HER2 expression is significant since the HER2-low patient subpopulation usually exhibits an aggressive disease progression. Despite that, there are several major points that need to be addressed as listed below: Major points: 1- All the IF images need quantification. 2- In Fig. 1B, it was claimed that endocytosis happens at 6 hrs, however, it is not visible from the image provided. 3- Is there any change in T-DM1 internalization upon combination of T-DM1 with autophagy inhibitors. 4- It is not clear how the apoptotic cells were quantified. Were they the Annexin V+ and PI- cells corresponding to early apoptosis or Annexin V+ PI+ cells corresponding to late apoptosis? 5- Is DM1 treatment also inducing autophagy induction upon mTOR inhibition? 6- The reduction of cell viability upon combination of T-DM1 with the autophagy inhibitors is not convincing. 7- Since the major mechanism of action of T-DM1 is mitotic arrest, it is suggested to examine the mitotic arrest markers in HER2-low cells treated with T-DM1 and autophagy inhibitor combination. 8- To eliminate the potential cell line-specific effects, it is suggested that the key experiments, e.g., reduced cell viability and apoptosis induction upon combination of T-DM1 with the autophagy inhibitors are repeated in a second HER2-low gastric cancer cell line. Reviewer #3: In this manuscript, Zhang and colleagues aimed to enhance the therapeutic efficacy of Ado-trastuzumab emtansine (T-DM1) in HER2-low gastric carcinoma cells, which typically exhibit limited responsiveness to this treatment. By investigating the role of autophagy in T-DM1 resistance, the authors discovered that T-DM1 induces cytoprotective autophagy via the Akt/mTOR pathway in HER2-low SGC7901 cells. This autophagy activation mitigates the drug's cytotoxic effects. However, pharmacological inhibition of autophagy significantly enhanced T-DM1-induced cytotoxicity and apoptosis by promoting lysosomal degradation of the drug and facilitating the release of its cytotoxic payload, emtansine. These findings highlight a novel strategy for improving T-DM1 efficacy in HER2-low gastric cancer by combining it with autophagy inhibitors, potentially broadening the therapeutic applications of T-DM1. That said, there are several points that should be addressed before the manuscript is suitable for publication: Major Comments: 1. In Figure 1A, the authors demonstrate that the HER2-low cell line exhibits limited responsiveness to T-DM1. To strengthen this claim, it would be beneficial to include a HER2-positive cell line as a control. A direct comparison of T-DM1 cytotoxicity between HER2-high and HER2-low cell lines would provide clearer evidence of the differential sensitivity. 2. The manuscript discusses the Akt/mTOR pathway's involvement in autophagy induced by T-DM1. However, to provide stronger mechanistic insight, the authors should consider using specific inhibitors of this pathway. This would validate whether the Akt/mTOR pathway is indeed a critical mediator of autophagy following T-DM1 treatment, as suggested in Figure 5. 3. While statistical analyses are mentioned, the manuscript lacks consistent information on the specific tests used for each figure. Including detailed descriptions of the statistical tests applied and the rationale for their selection would enhance the credibility and reproducibility of the findings. 4. The discussion could benefit from a more comprehensive comparison with existing studies on T-DM1 and other ADCs in HER2-low cancers, such as breast cancer. Additionally, elaborating on the broader therapeutic implications, such as the potential application of autophagy inhibitors in other HER2-low cancers or ADC therapies, would add significant value. Minor Comments: - The WB analysis for HER2 expression in Supplemental Figure 1 should be prioritized over immunofluorescence (IF) for its quantitative accuracy. Including a HER2-positive GC cancer cell line in the comparison panel would further enhance the relevance of the findings. - There is some inconsistency in the use of terms such as "autophagy inhibition" and "blocking autophagy." Ensuring consistency in terminology throughout the manuscript would improve readability. A professional language review might also be beneficial to refine clarity and conciseness. By addressing these points, the manuscript will be significantly strengthened and better positioned for publication. The study presents important insights into enhancing T-DM1 efficacy in HER2-low gastric cancer, and these revisions would further solidify the impact and rigor of the work. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . 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Attachments Attachment Submitted filename: Comments.pdf https://doi.org/10.1371/journal.pone.0322029.r002
Revision 1
23 Feb 2025 Author Response Responses to the reviewers Referee #1 (Remarks to the Author): The manuscript explores the potential of combining T-DM1, an antibody-drug conjugate, with autophagy inhibitors to enhance its cytotoxicity in HER2-low gastric carcinoma cells. The authors concluded that T-DM1 alone exhibits limited cytotoxicity in HER2-low carcinoma cells due to the activation of cytoprotective autophagy. However, inhibiting autophagy pharmacologically significantly increases T-DM1’s cytotoxic effects by promoting its lysosomal fusion and facilitating the release of its active cytotoxic agent, emtansine. Comments: The study largely repurposes previously explored concepts by combining T-DM1 with autophagy inhibitors, an approach that has already been examined in other contexts. It does not provide groundbreaking insights or novel mechanisms specific to HER2-low gastric cancer. Thank you very much for your constructive comments. Previous studies mainly focused on HER2-positive tumors, but our study focuses on HER2-low gastric cancer initially. In fact, the proportion of HER2-low gastric cancer (21%) is close to HER2-positive gastric cancer (23.2%), and groundbreaking insights or novel mechanisms specific need to be further studied (Nakayama I, Takahari D, Chin K, et al. Incidence, clinicopathological features, and clinical outcomes of low HER2 expressed, inoperable, advanced, or recurrent gastric/gastroesophageal junction adenocarcinoma. ESMO Open. 2023 Aug;8(4):101582.). While the manuscript mentions the involvement of the Akt/mTOR pathway and autophagy modulation, it fails to thoroughly investigate the molecular interactions or provide sufficient evidence to establish causal relationships between autophagy inhibition and the enhanced efficacy of T-DM1. Thanks very much for your valuable suggestion. The Akt/mTOR pathway is an important pathway regulating metabolism, proliferation and survival and plays a key role in maintaining cell homeostasis in cells. This signaling pathway can participate in the occurrence and development of many diseases by mediating autophagy. As a major regulatory molecule of cell growth and metabolism, mTOR can promote anabolic processes and inhibit catabolic processes (autophagy). Activated Akt can stimulate mTOR through a series of signal transduction processes, and ultimately inhibit autophagy. Our study suggests that the Akt/mTOR pathway and its downstream molecules were closely involved in the induction of autophagy by T-DM1 in SGC7901 cells. Furthermore, autophagy inhibition can significantly enhance T-DM1-mediated cytotoxicity and apoptosis. The modified contents were as follows. “…Together, these results suggest that the Akt/mTOR pathway is restrained in the induction of autophagy by T-DM1 in SGC7901 cells.…” The figures and results sections include excessive repetition, particularly in the presentation of apoptotic and autophagy-related assays. Streamlining this information would improve clarity and enhance the manuscript's readability. Thanks very much for your valuable advice. Based on your suggestion, I simplified Figure 2. I placed the statistics from the FCM and Figure 2c into the supplementary data to improve the usability of the article. The Figure 2. are modified and replaced appropriately. Supplementary Figure S4. are added appropriately. The modified contents were as follows. “…Figure 2 T-DM1 dose-dependently induced apoptosis in SGC7901 cells. (A) Apoptotic SGC7901 cells were stained with Annexin V-FITC/PI and observed after being incubated with T-DM1 for the indicated time (48 h) and were then examined by FCM. (B) Immunoblot analysis was used to measure the expression of apoptotic proteins (cleaved PARP, cleaved caspase 9, and cleaved caspase 3) after SGC7901 cells were treated with T-DM1 for 48 hours.…” “…Supplementary Figure S4. (A) Apoptotic cells examined by FCM for the indicated time (48 h) were counted and analyzed, with results shown in bar charts (n = 3; mean ± S.D.; **P < 0.001). (B-D) Densitometric values of the expression of apoptotic proteins (cleaved PARP, cleaved caspase 9, and cleaved caspase 3) after SGC7901 cells treated with T-DM1 for 48 hours were calculated using ImageJ software and normalized to the value of the corresponding vehicle bands. The values of the vehicle group were adjusted to 1.0 and are shown as the mean ± S.D. Student’s t-test was used to analyze the data. P < 0.05 and *P < 0.001.…” The manuscript lacks detailed descriptions of the statistical analyses performed, including criteria for significance and the sample sizes used for each experiment. This omission undermines the reproducibility and robustness of the findings. Thank you for your advice. GraphPad Prism 8 (San Diego, CA) was used to analyze the data. The results were analyzed by Student’s t-test (n = 3 independent experiments) and are presented as the mean ± S.D. Differences were believed statistically significant when P < 0.05. According to your comment, we have added related data in our revised manuscript. The modified contents were as follows. Figure 1 T-DM1 exhibited limited cytotoxicity and was taken up by SGC7901 cells. (A) CCK-8 assays were used to evaluate the survival rates of SGC7901 cells treated with T-DM1 in a concentration-dependent manner for 72 hours (mean ± S.D.; P < 0.001, n = 3). The results were analyzed by Student’s t-test (n = 3 independent experiments). The sample size and mean ± S.D. in Figure 1A are shown in the table. Figure 3 Autophagosomes formation and autophagic flux were induced by T-DM1 in SGC7901 cells. (A) Immunoblot analysis showed the expression of autophagic proteins (SQSTM1 and LC3-Ⅱ) after SGC7901 cells were incubated with T-DM1 for 48 hours. The densitometric values were estimated by ImageJ software and normalized to the vehicle values. The data in the vehicle group were adjusted to 1.0, and data from three independent experiments are shown showed as the mean ± S.D. (Student’s t-test; P < 0.05; **P < 0.001, n = 3). The results were analyzed by Student’s t-test (n = 3 independent experiments) and are presented as the mean ± S.D. Differences were believed statistically significant when P < 0.05. Figure 4 Autophagy inhibition induced by T-DM1 enhanced the cytotoxicity of SGC7901 cells. (E) Autophagy inhibition with 3-MA or LY294002 reduced the viability of SGC7901 cells, as determined by relevant kits (mean ± S.D.; P < 0.05 versus vehicle, n = 3). (F) The percentages of apoptotic SGC7901 cells were determined by FCM after incubation with 10 μg/ml T-DM1 and autophagy inhibitors (3-MA or LY294002). The results are presented in the side bar charts (**P < 0.001, n = 3). Figure 5 The Akt/mTOR pathway and downstream components were involved in autophagy induced by T-DM1. (B) The densitometric values of SGC7901 cell samples were evaluated using ImageJ software and normalized to the corresponding values of the vehicle group. The values of the vehicle group were adjusted to 1.0 (mean ± S.D.; P < 0.05; P < 0.01 versus vehicle, n = 3). The results were analyzed by Student’s t-test (n = 3 independent experiments) and are presented as the mean ± S.D. Differences were believed statistically significant when P < 0.05. The results of Supplementary Material were analyzed by Student’s t-test (n = 3 independent experiments) and are presented as the mean ± S.D. Differences were believed statistically significant when P < 0.05. The study does not adequately compare the efficacy of the proposed combination therapy with existing treatment options for HER2-low gastric cancer, limiting its clinical relevance and applicability. Thank you for your advice. In our study, the effect of combination therapy was significantly better than that of single therapy for HER2-low gastric cancer in vitro experiments (Figure 4 E and F). Clinical relevance and applicability need to be further studied. Phrases such as "significantly enhanced cytotoxicity" are used without providing clear quantitative benchmarks, leaving the practical significance of the findings unclear. Thanks very much for your valuable advice. Based on your suggestion, I would be more careful to use "significantly enhanced cytotoxicity". The discussion fails to place the findings within the broader landscape of HER2-low gastric cancer research. Conflicting evidence or alternative interpretations are not sufficiently addressed, leaving gaps in the contextual analysis. Thank you for your suggestion. The modified contents were as follows. “…GC, a prevalent malignant tumor, is receiving increasing attention because of its poor prognosis and high incidence [2]. HER2-targeted therapies are commonly used in clinical settings, but their application in HER2-low GC patients remains limited [4]. While HER2-targeted therapies have shown effectiveness in HER2+ GC, patients with HER2-low GC exhibit poor responses to these treatments [18, 19, 20]. Classification based on clinical features cannot fully describe biological characteristics, as nearly half of the HER2 negative BC tumors currently defined exhibit some degree of IHC expression, which has recently been renamed as HER2 low expression [21]. T-DM1, which is the first antibody-drug conjugate (ADC) approved for clinical use, is designed to decompose in lysosomes and disrupt microtubule organization, thereby selectively targeting HER2+ malignant tumor cells [20, 22, 23 , 24]. However, the effectiveness of T-DM1 is largely reduced in HER2-low tumors, so T-DM1 cannot be applied in clinical practice in HER2-low tumors. In recent years, people began to gradually pay attention to HER2-low gastric cancer, hoping to improve the effectiveness of targeted therapy [25]. This limited efficacy underscores the urgent need to better understand the mechanisms by which T-DM1 can be optimized for treating HER2-low GC.…” “…ADCs represent a promising class of chemotherapy drugs that are engineered to deliver cytotoxic agents directly to target cells via monoclonal antibodies [36]. Ideally, target cells should remain highly sensitive to ADCs. However, our study showed that T-DM1 did not exhibit a superior therapeutic advantage against HER2-low GC cells [37]. Novel ADC drugs have been tested in clinical trials in HER2-low tumors, which suggests that fewer available HER2 receptors on cancer cells may be sufficient to achieve clinical benefits [38]. We observed the processing of T-DM1 from receptor recognition to intracellular lysosomes in SGC7901 cells, finding that fusion with lysosomes was strengthened by autophagy inhibition. Increased fusion of T-DM1 with lysosomes effectively increased the cytotoxic effect of T-DM1 on SGC7901 cells. Importantly, our findings, along with previous studies, suggest that autophagy interference in the dissociation of ADCs may be a universal phenomenon across different tumors [15, 39]. Therefore, regulating autophagy could potentially alter the sensitivity of ADC-based cancer treatment and could be a viable strategy to broaden the therapeutic range of ADCs.…” The authors assert that autophagy inhibition may have universal applications across various tumor types but provide no supporting evidence beyond the context of HER2-low gastric cancer cells. This weakens the broader impact of the conclusions. Thanks very much for your valuable suggestion. In a previous study, we found that autophagy occurs extensively during the treatment of tumors. The study of Zhang J, et al. showed that targeting the autophagy promoted antitumor effect of T-DM1 on HER2-positive Gastric Cancer (Zhang J, et al. Targeting the autophagy promoted antitumor effect of T-DM1 on HER2-positive gastric cancer. Cell Death Dis. 2021 Mar 17;12(4):288. doi: 10.1038/s41419-020-03349-1.). Another paper by Shen, W. et al. also revealed the same antitumor effect in non-small cell lung cancer (Shen, W. et al. A novel and promising therapeutic approach for NSCLC: recombinant human arginase alone or combined with autophagy inhibitor. Cell Death Dis. 2017 Mar 30;8(3): e2720. doi: 10.1038/cddis.2017.137.). We will add the corresponding references to reinforce the conclusion of the article. The modified contents were as follows. “…Importantly, our findings, along with previous studies, suggest that autophagy interference in the dissociation of ADCs may be a universal phenomenon across different tumors [15, 36]. Therefore, regulating autophagy could potentially alter the sensitivity of ADC-based cancer treatment. To date, it remains unclear whether modulating autophagy could be a viable strategy to broaden the therapeutic range of ADCs. …” Referee #2 (Remarks to the Author): In the manuscript entitled: “Enhanced cytotoxicity of T-MD1 in HER2-low carcinomas via autophagy inhibition”, Zhang et al. showed that T-DM1 induces autophagy and combination of T-DM1 with autophagy inhibitors increases T-DM1 efficacy in HER2-low gastric cancer. A potential expansion of the use of T-DM1 and may be other HER2-targeted therapies to tumors with low HER2 expression is significant since the HER2-low patient subpopulation usually exhibits an aggressive disease progression. Despite that, there are several major points that need to be addressed as listed below: Major points: 1- All the IF images need quantification. Thanks very much for your valuable suggestion. All the IF images had been quantified. This can be seen in Supplementary Figure S1, Supplementary Figure S2. (B-D) and Supplementary Figure S5. The modified contents were as follows. “…The brightness values of HER2 red dye staining were estimated using ImageJ software and the densitometric values were normalized to the corresponding values of the vehicle group. The values of the vehicle group were set as 1.0 (n = 3 independent experiments; mean ± S.D.; P < 0.01 and *P < 0.001). …” “…(C) The brightness values of the Alexa Fluor™ 647 were evaluated using ImageJ software, and the values were normalized to the corresponding values of the vehicle group. (D) …” “…Supplementary Figure S5. The brightness values of the Alexa Fluor™ 647 visualized by red fluorescence, Cyto-ID visualized by fluorescence and LysoTracker exhibited yellow fluorescence were evaluated using ImageJ software, and the values were normalized to the corresponding values of the vehicle group. …” 2- In Fig. 1B, it was claimed that endocytosis happens at 6 hrs, however, it is not visible from the image provided. Thank you very much for your constructive comments. we claimed that endocytosis happening at 6 hrs was based on the uniform distribution of the labeling T-DM1 with red fluorescence on the cells. This conclusion really lacks more accurate experimental support, so we have made corresponding modifications to the paper. The modified contents were as follows. “…Laser confocal microscopy was used to track the uptake of T-DM1 at different time points, revealing that T-DM1 bound to the HER2 receptor at 1 hour and internalized at 12 hours post-treatment (Figure 1B). …” 3- Is there any change in T-DM1 internalization upon combination of T-DM1 with autophagy inhibitors. Thank you for your asking. We speculated that T-DM1 internalization upon combination of T-DM1 with autophagy inhibitors should be enhanced, thus enhancing cytotoxicity (Figure 6 and Supplementary Figure S5). 4- It is not clear how the apoptotic cells were quantified. Were they the Annexin V+ and PI- cells corresponding to early apoptosis or Annexin V+ PI+ cells corresponding to late apoptosis? Thank you very much for your constructive comments. We quantitatively counted early apoptosis (the Annexin V+ and PI- cells). The modified contents were as follows. “…(F) The percentages of early apoptotic SGC7901 cells (the Annexin V+ and PI- cells) were determined by FCM after incubation with 10 μg/ml T-DM1 and autophagy inhibitors (3-MA or LY294002). The results are presented in the side bar charts (P < 0.001, n = 3).…” “…Apoptotic cells (the Annexin V+ and PI- cells) examined by FCM for the indicated time (48 h) were counted and analyzed, with results shown in bar charts (n = 3; mean ± S.D.; *P < 0.001).…” 5- Is DM1 treatment also inducing autophagy induction upon mTOR inhibition? Thank you for your asking. Our current experimental results can only prove than the Akt/mTOR pathway is restrained in the induction of auto Attachments Attachment Submitted filename: Response to Reviewers.docx* https://doi.org/10.1371/journal.pone.0322029.r003
16 Mar 2025 Decision Letter - Ruo Wang, Editor <p>Enhanced cytotoxicity of T-DM1 in HER2-low carcinomas via autophagy inhibition PONE-D-24-51611R1 Dear Dr. Xin, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Ruo Wang Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: (No Response) ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: (No Response) ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors answers all comments and they improve the manuscript. The manuscript is accepted for publication ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy . Reviewer #1: No ******** https://doi.org/10.1371/journal.pone.0322029.r004
Formally Accepted
Acceptance Letter - Ruo Wang, Editor PONE-D-24-51611R1 PLOS ONE Dear Dr. Xin, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Ruo Wang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0322029.r005

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