Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules

Round 1

Reviewer 1 Report

Comment 1: The author noted that FX administration considerably abrogated tumor development in a PC-PDX mouse model, but only provided a estimated tumor size data figure without the tumor photo.

Comment 2: The author made a conclusion that the increased DCN and pro-oxidant signals and the inhibition of IGFBP2-, EpCAM-, and LCN2-related signals are key regulators of tumor suppression in PC-PDX mice. They should provide the results of drug signaling pathway validation studies in pancreatic cancer cells to support this conclusion.

Comment 3: GO and KEGG analysis results should be provided.

 Comment 4: An explanation should be given as to why the signaling pathway results were inconsistent between in vivo and in vitro studies.

 The language need revision by a native speaker

Author Response

Aug 29, 2023

Dear Dr. Constantin N. Baxevanis

Editor-in-Chief, Onco

Manuscript ID.: onco-2563316

Ms.Title: Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules

   Thank you for your email of Aug 18, 2023, regarding our manuscript, “Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules”, and the valuable comments of the reviewers. The changed parts in the manuscript file are marked in the red with Track Changes.

   Please find enclosed the revised manuscript in Word format (file name: onco-2563316 with track changes).

Comments from the editors and reviewers:

Reviewer 1:

Comment 1: The author noted that FX administration considerably abrogated tumor development in a PC-PDX mouse model, but only provided a estimated tumor size data figure without the tumor photo.

We express our deep thanks for your comments. We are very sorry, but we did not take pictures of the tumors. When collecting tumors from mice, we had to very very quickly take the tumor and cut it into several pieces because we were afraid of degrading the RNAs and proteins. (Although the transcriptome data was removed in the revised manuscript, in accordance with the Reviewer-2’s comments…..)

Comment 2: The author made a conclusion that the increased DCN and pro-oxidant signals and the inhibition of IGFBP2-, EpCAM-, and LCN2-related signals are key regulators of tumor suppression in PC-PDX mice. They should provide the results of drug signaling pathway validation studies in pancreatic cancer cells to support this conclusion.

Thank you for your constructive comments. From the Reviewer-1’s comments, we have added some references and modified the Discussion section (line 565-575)

Comment 3: GO and KEGG analysis results should be provided.

Thank you for your valuable comments. In accordance with the Reviewer-2’s comments, we have removed all date on the transcriptome profiles. In addition, GO and KEGG analysis from proteome profile could not conducted because the proteins detected by proteome analysis were few (upregulated proteins, 318; downregulated proteins, 887; total proteins,1205).

    We are sorry that we could not enter GO and KEGG results according to your comments.

Comment 4: An explanation should be given as to why the signaling pathway results were inconsistent between in vivo and in vitro studies.

      We express our deep thanks for your comments. It is very difficult to explain the difference of molecular profiles in each cell or mouse with Fx or FxOH treatment.  Previously, we have demonstrated that FxOH addition induced apoptotic cell death in mouse pancreatic cancer KMPC44 cells and hamster pancreatic cancer HaPC-5 cells with alterations of various molecules (Ref.27 and 28). In addition, Furthermore, Fx administration could inhibit the onset of pancreatic cancer in a pancreatic cancer mouse (C57BL/6J) model with allogenic and orthotopic transplantations of the KMPC44 cells (Ref.29). Comparing the results (PANC-1, BxPC-3, KMPC44, HaPC-5, and Fx-treated C57BL/6J-KMPC44 and PDX mice), some of the molecular expression profiles were similar, while others were dissimilar. We speculate that these differences in reactivity to Fx or FxOH may be due to differences in cancer cell properties.

In accordance with the Reviewer-1’s comment-4, we have added the idea to the Discussion section (Line 593-601).

Comments on the Quality of English Language: The language need revision by a native speaker.

Thank you for your constructive comments. In accordance with the Reviewer-1’s comments, we did thoroughly English proofreading again by a native speaker (by Editage).

Please confirm below 3 manuscripts

・ onco-2563316 with Track Changes

・ Supplementary Figures_onco-2563316

・ Supplementary Tables_onco-2563316_modified

We strongly appreciate again for the Reviewer-1’s comments, regarding on our manuscript.

     We feel that the revised manuscript is a suitable response to the comments, and is significantly improved over the initial submission. We trust that is now suitable for publication in Onco.

Thank you in advance for your kind consideration of this paper.

Sincerely yours,

Masaru Terasaki, Ph.D.

Reviewer 2 Report

In the manuscript by Terasaki et al., the authors the anticancer effects of Fx in pancreatic cancer (PC)-PDX mice.  They also tried to dissect the molecular mechanisms behind these inhibitory effects. 

Although the data presented in the manuscript is interesting, further experiments need to be done to strengthen the manuscript.  

I have a few major/minor suggestions for polishing the manuscript.

Major Comments:

1.     The results section should be written in a more simple, comprehensive and categorical manner.  It is very confusing to follow. 

2.     In Line 372, three upregulated proteins (≥1.5-fold), i.e., decorin (DCN) (2.6-fold), heme oxygenase 1 (HO1) (2.5-fold), and FAF1 (1.8-fold), were identified as the most notable tumor suppressors and stress-inducible proteins (Supplementary Table S4). The authors should explain why they chose these three proteins as the most notable targets among others.  Similarly Moreover, we noted that the 26 downregulated proteins, INSR (N.D., not detected compared with that of control mice), BAG1 (N.D.), ITGB3 (N.D.), IGFBP3 (−4.0-fold), ITGA1 (−3.6-fold), LAMA5 (−3.2-fold), NECTIN2 (−3.1-fold), ECM1 (−3.0-fold), HNF4A (−2.7-fold), NFKB1 (−2.4-fold), NFKB2 (−2.3-fold), CLU (−2.3-fold), insulin growth factor binding protein 2 (IGFBP2) (−2.3-fold), ITGA3 (−1.9-fold), COL17A1 (−1.9-fold), CD44 (−1.9-fold), CDC42EP5 (−1.8-fold), TIMP1 (−1.8-fold), lipocalin 2 (LCN2) (−1.8-fold), ADAM17 (−1.7-fold), MDK (−1.7-fold), EpCAM (−1.7-fold), DAG1 (−1.7-fold), and MUC5AC (−1.6-fold), played an important role in the suppression of cancer development (Supplementary Table S5).  The authors should also justify the statement ‘played an important role in the suppression of cancer development’.

3.     Section 3.4 should be written in an easier way.

4.     The authors pointed out, three downregulated genes (≤ −2.0-fold), DES (−12.5-fold), TESC (−2.4-fold), and RASSF9 (−2.1-fold) were detected as most notable contributory molecules suppressing cancer development in epithelial tumor tissues following Fx-high diet administration.  Why the authors detected them as notable contributory molecules.  Do they have roles in pancreatic cancer in previously published literature.  The authors should explain the same thing in comprehensive gene alterations targeting the stromal tumor tissues  of PC-PDX mice administered with Fx-high diet.

5.     The authors should re-validate the transcriptome data (Section 3.4) using RT-qPCR analysis. This will strengthen their claims.

6.     In Figure 4 and Figure 5, the authors should put the representative western blot images in the main figure.  Also, the authors should comment on whether this quantification is done with three independent biological replicates.

7.     Also, the non-published supplementary figure cannot be deciphered properly.  No labelling is done.  The quantification in Figures 4 and 5 was done on the basis of this western blot images and hence it should be labelled properly. 

8.     The authors should comment on why they are measuring the protein expression of the expression of pAKT(Thr308), pSTAT3 (Ser727) pSmad2(Ser465/467), pFAK(Tyr397) pPaxillin(Tyr31) pAKT(Ser473), TGF-β1, p-p38(Thr180/Tyr182), pJNK(Thr183/Tyr185) and Cyclin D1.  Is there any previously published dataset.  Please comment on the results section.

9.     From Figure 6A, it is very difficult to differentiate between two conditions in both PANC-1 and BxPC-3 cell lines.  Some of the images are highly over exposed.  The authors should change the figures with a better resolution image.

10.   In Figure 6C, the authors pointed out that there is an upregulation of protein expression in HO1 in PANC-1. HO1 has two bands and both bands have merged.  Hence, the authors should point out the exact band.  Also, the protein expression of active caspase-3 does not seem to be upregulated in PANC-1 from the figure. Also, p-Paxillin protein level cannot be deciphered from the figure as there are two bands. Upregulation of protein expression in HO1and p-Paxillin in BxPC-3 cells cannot be deciphered for similar reasons.

11.  The discussion section should be more concise.

12.  The authors should include a model figure in order to elucidate the mechanism of Fx mediated pancreatic cancer treatment.  They should take all the data together and devised a model behind this inhibition to make the manuscript more attractive.

Minor Comments

1.     In the Simple Summary Section, the authors should introduce the full form of DCN, IGFB2, EpCAM, LCN2, HO1 etc. as they have not introduced this term before.  Also follow this pattern everywhere in the manuscript.

2.     Line 64 should be placed after Line 57 to maintain continuity.  Then the authors should start from Line 58.

3.     In line 293 the dilution of anti-mouse and anti-rabbit secondary antibodies should be mentioned.

Author Response

Aug 29, 2023

Dear Dr. Constantin N. Baxevanis

Editor-in-Chief, Onco

Manuscript ID.: onco-2563316

Ms.Title: Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules

   Thank you for your email of Aug 18, 2023, regarding our manuscript, “Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules”, and the valuable comments of the reviewers. The changed parts in the manuscript file are marked in the red with Track Changes.

   Please find enclosed the revised manuscript in Word format (file name: onco-2563316 with track changes).

Comments from the editors and reviewers:

Reviewer 2: In the manuscript by Terasaki et al., the authors the anticancer effects of Fx in pancreatic cancer (PC)-PDX mice. They also tried to dissect the molecular mechanisms behind these inhibitory effects.

Although the data presented in the manuscript is interesting, further experiments need to be done to strengthen the manuscript. 

I have a few major/minor suggestions for polishing the manuscript.

Major Comments:

1. The results section should be written in a more simple, comprehensive and categorical manner. It is very confusing to follow.

We express our deep thanks for your comments. We thought these section (3.1, 3.2) were fine as they were.

In accordance with the Reviewer-2’s comments, we have removed all descriptions and data on the transcriptome analyses using human and mouse microarray (remove: section 3.4 in original manuscript). With this modification, we have removed on the transcriptome analysis section (remove: section 2.5 in original manuscript) in the Materials and Methods section.

We hope this modification clears up your confusion.

2. In Line 372, three upregulated proteins (≥1.5-fold), i.e., decorin (DCN) (2.6-fold), heme oxygenase 1 (HO1) (2.5-fold), and FAF1 (1.8-fold), were identified as the most notable tumor suppressors and stress-inducible proteins (Supplementary Table S4). The authors should explain why they chose these three proteins as the most notable targets among others. Similarly Moreover, we noted that the 26 downregulated proteins, INSR (N.D., not detected compared with that of control mice), BAG1 (N.D.), ITGB3 (N.D.), IGFBP3 (−4.0-fold), ITGA1 (−3.6-fold), LAMA5 (−3.2-fold), NECTIN2 (−3.1-fold), ECM1 (−3.0-fold), HNF4A (−2.7-fold), NFKB1 (−2.4-fold), NFKB2 (−2.3-fold), CLU (−2.3-fold), insulin growth factor binding protein 2 (IGFBP2) (−2.3-fold), ITGA3 (−1.9-fold), COL17A1 (−1.9-fold), CD44 (−1.9-fold), CDC42EP5 (−1.8-fold), TIMP1 (−1.8-fold), lipocalin 2 (LCN2) (−1.8-fold), ADAM17 (−1.7-fold), MDK (−1.7-fold), EpCAM (−1.7-fold), DAG1 (−1.7-fold), and MUC5AC (−1.6-fold), played an important role in the suppression of cancer development (Supplementary Table S5). The authors should also justify the statement ‘played an important role in the suppression of cancer development’.

Thank you for your valuable comments. In accordance with the Reviewer-2’s comment-2, we have considerably modified the descriptions (line 347-364).

3. Section 3.4 should be written in an easier way.

　In accordance with the Reviewer-2’s comment-1, we have removed all descriptions and data on the transcriptome analyses using human and mouse microarray (section 3.4 in the original manuscript).

We hope this modification clears up your confusion.

4. The authors pointed out, three downregulated genes (≤ −2.0-fold), DES (−12.5-fold), TESC (−2.4-fold), and RASSF9 (−2.1-fold) were detected as most notable contributory molecules suppressing cancer development in epithelial tumor tissues following Fx-high diet administration. Why the authors detected them as notable contributory molecules. Do they have roles in pancreatic cancer in previously published literature?  The authors should explain the same thing in comprehensive gene alterations targeting the stromal tumor tissues of PC-PDX mice administered with Fx-high diet.

　In accordance with the Reviewer-2’s comment-1, we have removed all descriptions and data on the transcriptome analyses using human and mouse microarray (section 3.4 in the original manuscript). With this modification, we may no longer have to correct your comment-4 and we thought the result sections were changed simpler.

5. The authors should re-validate the transcriptome data (Section 3.4) using RT-qPCR analysis. This will strengthen their claims.

　In accordance with the Reviewer-2’s comment-1, we have removed all descriptions and data on the transcriptome analyses using human and mouse microarray (section 3.4 in the original manuscript). With this modification, we may not need the revalidation of transcriptome profiles using real-time PCR.

6. In Figure 4 and Figure 5, the authors should put the representative western blot images in the main figure. Also, the authors should comment on whether this quantification is done with three independent biological replicates.

Thank you for your constructive comments.

In accordance with the Reviewer-2’s comment-1, we removed Figure 2 and 3 regarding transcriptome analyses on the original manuscript.

With the modification, Figure 4 and 5 changed to Figure 2 and 3, respectively.

      In accordance with the Reviewer-2’s comment-6, we have attached the representative western blot images in Figure 2 and 3, and modified the figure legends (line 387-423).

    Figure 2 and 3 are the results of an animal experiment. We think it would be difficult to repeat this animal experiment two more times. Is it really necessary to repeat animal experiments three times? We think we have secured the necessary n number in the animal experiment.

7. Also, the non-published supplementary figure cannot be deciphered properly. No labelling is done. The quantification in Figures 4 and 5 was done on the basis of this western blot images and hence it should be labelled properly.

     Thank you for your valuable comments. In accordance with the Reviewer-2’s comment-7, we have attached the representative western blot images in Figure 2 and 3, and modified the figure legends (line 387-423).

8. The authors should comment on why they are measuring the protein expression of the expression of pAKT(Thr308), pSTAT3 (Ser727) pSmad2(Ser465/467), pFAK(Tyr397) pPaxillin(Tyr31) pAKT(Ser473), TGF-β1, p-p38(Thr180/Tyr182), pJNK(Thr183/Tyr185) and Cyclin D1. Is there any previously published dataset? Please comment on the results section.

Thank you for your constructive comments. These molecules are the central molecules controlling each signal. In accordance with the Reviewer-2’s comment-8, we have modified the sentence of the result section (line 375-376).

9. From Figure 6A, it is very difficult to differentiate between two conditions in both PANC-1 and BxPC-3 cell lines. Some of the images are highly over exposed. The authors should change the figures with a better resolution image.

 It was changed from Figure 6 to 4.

We express our deep thanks for your comment-9. In accordance with the Reviewer-2’s comments, we have replaced the higher resolution of Figure 4A. Some cells were becoming round form and floating, therefore, it could not be avoided to be over exposed by phase-contrast microscopy. Even when observed with the naked eye, some of these cells are overexposed.

10. In Figure 6C, the authors pointed out that there is an upregulation of protein expression in HO1 in PANC-1. HO1 has two bands and both bands have merged. Hence, the authors should point out the exact band. Also, the protein expression of active caspase-3 does not seem to be upregulated in PANC-1 from the figure. Also, p-Paxillin protein level cannot be deciphered from the figure as there are two bands. Upregulation of protein expression in HO1and p-Paxillin in BxPC-3 cells cannot be deciphered for similar reasons.

It was changed from Figure 6C to 4C.

Thank you for your constructive comment-10. The bands of HO1 and pPaxillin are likely to be detected as multiple bands, as well as IGFBP2, pERK1/2(Thr²⁰²/Tyr²⁰⁴), and pJNK(Thr¹⁸³/Tyr¹⁸⁵). We have so far investigated the expression of these molecules in various cells (colon, breast, and pancreatic cancers). As a result, it was found that the number of bands depends on the cell line. We think that this phenomenon is probably due to the difference in the degree of adducts such as sugar chains. In some cases, we have confirmed that those multiple bands were correct using positive control of western blot. Hope you don’t worry.

Although the bands of the active form of caspase-3 in PANC-1 may be hard to see, there is a clear difference between the control cells and the FxOH 20 μM-treated cells. However, the western blot bands may be low resolution. Then, we have changed Figure 6C to higher resolution one.

11. The discussion section should be more concise.

Thank you for your constructive comments. In accordance with the Reviewer-2’s comment-11, we have removed the descriptions regarding transcriptome analyses and have modified the Discussion section (line 469-629).

12. The authors should include a model figure in order to elucidate the mechanism of Fx mediated pancreatic cancer treatment. They should take all the data together and devised a model behind this inhibition to make the manuscript more attractive.

Thank you for your valuable comments. In accordance with the Reviewer-2’s comment-12, we have made the summarized figure (Figure 5) and modified the Conclusion section (Line 630-651).

Minor Comments:

1. In the Simple Summary Section, the authors should introduce the full form of DCN, IGFB2, EpCAM, LCN2, HO1 etc. as they have not introduced this term before. Also follow this pattern everywhere in the manuscript.

Thank you for your valuable comments. In accordance with the Reviewer-2’s comments, we have modified the title (line 29-31 and 44).

2. Line 64 should be placed after Line 57 to maintain continuity. Then the authors should start from Line 58.

Thank you for your constructive comments. In accordance with the Reviewer-2’s comments, we have moved these two paragraphs (line 61-74).

3. In line 293 the dilution of anti-mouse and anti-rabbit secondary antibodies should be mentioned.

Thank you for your constructive comments. In accordance with the Reviewer-2’s comments, we have added the dilution information (line 273).

Please confirm below 3 manuscripts

・ onco-2563316 with Track Changes

・ Supplementary Figures_onco-2563316

・ Supplementary Tables_onco-2563316_modified

We strongly appreciate again for the Reviewer-2’s comments, regarding on our manuscript.

     We feel that the revised manuscript is a suitable response to the comments, and is significantly improved over the initial submission. We trust that is now suitable for publication in Onco.

Thank you in advance for your kind consideration of this paper.

Sincerely yours,

Masaru Terasaki, Ph.D.

Round 2

Reviewer 1 Report

Comment 1: Genomic data have been removed from the text, but the abstract has not been modified accordingly.

 Comment 2: Results of western blot validation and cellular results are not described in the Abstract.

 Comment 3: In Figure 2, 3 and 4, the destination bands and β-actin should be put together in the western blot results.

 Comment 4: What are the central molecules controlling the upstream and downstream signaling counterparts of DCN, HO1, IGFBP2, EpCAM, and LCN2, respectively?

 Comment 5: The molecules upstream and downstream of the target showed in Figure 3 do not represent the signalling pathway.

 Comment 6: Fx-administered PC-PDX mice showed increased expression of non-glycanated DCN in figure 2, but figure 5 showed the opposite result.

 The language need revision by a native speaker.

Author Response

Sep 20, 2023

Dear Dr. Constantin N. Baxevanis

Editor-in-Chief, Onco

Manuscript ID.: onco-2563316

Ms.Title: Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules

   Thank you for your email of Sep 14, 2023, regarding our manuscript, “Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules”, and the valuable comments of the reviewers. The changed parts in the manuscript file are marked in the red with Track Changes.

   Please find enclosed the revised manuscript in Word format (file name: onco-2563316 with track changes_v2).

Comments from the editors and reviewers:

Reviewer 1:

Comment 1: Genomic data have been removed from the text, but the abstract has not been modified accordingly.

Thank you for your valuable comments. In accordance with the Reviewer-1’s comment-1, we have removed “gene and” from the Abstract section (Line 43).

Comment 2: Results of western blot validation and cellular results are not described in the Abstract.

Thank you for your constructive comments. From the Reviewer-1’s comment-2, we have added some sentences regarding in vitro experiments in the Abstract section (line 30, 33-34, 46-47, and 49-52).

Comment 3: In Figure 2, 3 and 4, the destination bands and β-actin should be put together in the western blot results.

We express our deep thanks for your comments. As you pointed out, we also had a lot of trouble at first trying to place the β-actin bands under each molecule on the same membrane, in first revised manuscripts (we tried to β-actin placement as your comments once). However, the molecular bands in Figure 2 are arranged in the order detected by proteome analysis. In addition, the molecular bands in Figure 3 are grouped together for each signal pathway. Because β-actin was not analyzed in accordance with the order described above, placing β-actin between these molecules makes it difficult to see the order of molecules to be analyzed (Figure 2) and the pathway groups (Figure 3). In order to improve these, β-actin was placed together at the bottom left.

              Thank you very much for your very helpful comments. However, in Figure 2 and 3, we think it would be easier to see the profile of each molecule if β-actin was placed as is. What do you think on it? If you really think it’s better to change it, we will modify it. However, we think the bands and bars will become very small and difficult to see….

Comment 4: What are the central molecules controlling the upstream and downstream signaling counterparts of DCN, HO1, IGFBP2, EpCAM, and LCN2, respectively?

Thank you for your constructive comments. From the Reviewer-1’s comments, we have modified the sentences in the Results section (line 382-385).

Comment 5: The molecules upstream and downstream of the target showed in Figure 3 do not represent the signaling pathway.

Thank you for your insightful comments. As you pointed out, there are many pivotal molecules involved in Adhesion, PI3K/AKT, STAT, TGF-β, MAPK, WNT, Cell cycle, ECM, TME, EMT, and Apoptosis. Therefore, the reader may be confused as to why these molecules were selected. We selected representative 26 molecules belonging to 11 upstream and downstream signals closely related to all HO1, IGFBP2, EpCAM, and LCN2 (Line 382-385).

If more molecules are analyzed, new target molecules may be discovered. However, we believe that the results of this study yielded sufficiently interesting results for anticancer effects of Fx in the PDX mice. On the other hands, we think it will be difficult to conduct additional experiments due to the amount of remaining sample….

Comment 6: Fx-administered PC-PDX mice showed increased expression of non-glycanated DCN in figure 2, but figure 5 showed the opposite result.

Thank you for your comments. We created Figure 5 incorrectly. In accordance with the Reviewer-1’s comment-6, we have modified it (Line 654).

Please confirm below 3 manuscripts

・ onco-2563316 with Track Changes_v2

・ Supplementary Figures_onco-2563316

・ Supplementary Tables_onco-2563316_modified

We strongly appreciate again for the Reviewer-1’s comments, regarding on our manuscript.

     We feel that the revised manuscript is a suitable response to the comments, and is significantly improved over the initial submission. We trust that is now suitable for publication in Onco.

Thank you in advance for your kind consideration of this paper.

Sincerely yours,

Masaru Terasaki, Ph.D.

Reviewer 2 Report

In the manuscript by Terasaki et al., the authors the anticancer effects of Fx in pancreatic cancer (PC)-PDX mice.  They also tried to dissect the molecular mechanisms behind these inhibitory effects. 

The revised manuscript is nicely written and well explained.  The authors have addressed most of the previous comments. 

I have a few minor comments for the manuscript.

Minor Comments

1.     In Line 349, the authors say that 'the current functional knowledge regarding cancer, three upregulated proteins…. proteins.'  The authors have cited some research regarding these proteins in the discussion section.  They should also cite the same articles here.  The authors should also rephrase the sentence “the current functional knowledge regarding cancer” to based on some available studies (insert citations)”.  The same thing should be done in Line 355.

2.     The most notable proteins in LC-MS/MS analysis in Supplementary figures 4 and 5 used in the study should be included in the form of a table in the main figures (say Figure 2 and 3).

3.     There are some proteins which have significant fold changes in LC-MS/MS data sets compared to the proteins they studied.  Did the authors check their levels as well? 

4.     In Figure 4, the authors should include an image quantification of the notable upregulated and downregulated proteins just they did in Figures 2 and 3.

5.     Line 372-373 Fx-high diet in Group 1 significantly decreased the expression of IGFBP2 (0.6-fold) and EpCAM (0.7-fold) and tended to decrease LCN2 expression (0.6-fold).  What did the authors mean by tending to decrease?  Are they not significant? LCN and IGFBP2 both decreased by 0.6-fold.  So why LCN2 expression is not significant?  The authors should comment on this.

Author Response

Sep 20, 2023

Dear Dr. Constantin N. Baxevanis

Editor-in-Chief, Onco

Manuscript ID.: onco-2563316

Ms.Title: Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules

   Thank you for your email of Sep 14, 2023, regarding our manuscript, “Anticancer Effects of Fucoxanthin in a PDX Model of Advanced Stage Pancreatic Cancer with Alteration of Several Multifunctional Molecules”, and the valuable comments of the reviewers. The changed parts in the manuscript file are marked in the red with Track Changes.

   Please find enclosed the revised manuscript in Word format (file name: onco-2563316 with track changes_v2).

Comments from the editors and reviewers:

Reviewer 2: In the manuscript by Terasaki et al., the authors the anticancer effects of Fx in pancreatic cancer (PC)-PDX mice.  They also tried to dissect the molecular mechanisms behind these inhibitory effects.

The revised manuscript is nicely written and well explained.  The authors have addressed most of the previous comments.

I have a few minor comments for the manuscript.

Comment 1: In Line 349, the authors say that 'the current functional knowledge regarding cancer, three upregulated proteins…. proteins.'  The authors have cited some research regarding these proteins in the discussion section.  They should also cite the same articles here.  The authors should also rephrase the sentence “the current functional knowledge regarding cancer” to based on some available studies (insert citations)”.  The same thing should be done in Line 355.

We express our deep thanks for your comments. In accordance with the Reviewer-2’s comment-1, we have modified the sentence and added some references (line 356-357).

Comment 2: The most notable proteins in LC-MS/MS analysis in Supplementary figures 4 and 5 used in the study should be included in the form of a table in the main figures (say Figure 2 and 3).

Thank you for your valuable comments. We thought your point was very constructive. However, there is one concern. If the table of the most notable proteins from Supplementary Figure 4 and 5 is placed in Figure 2, we think the western blot bands (Figure 2A) and bars (Figure 2B) in Figure 2 will become very small and difficult to see. We described the detailed results of the most notable proteins in Supplementary Table S4 and S5 to the Results section 3.3 (Line 357-370). Would it be better to insert it as a table?

Comment 3: There are some proteins which have significant fold changes in LC-MS/MS data sets compared to the proteins they studied.  Did the authors check their levels as well?

              Thank you very much for pointing this out. As you said, there are many other attractive molecules in the LC-MS/MS data set that have not yet been analyzed by WB. The answer to your question is that those molecules does not check it. If more molecules are analyzed, new target molecules may be discovered.

However, this time, we selected these molecules from the molecular functions and interactions regarding cancer. We believe that the results of this study yielded sufficiently interesting results for anticancer effects of Fx in the PDX mice. In accordance with the Reviewer-2’s comment-3, we have modified the sentence (line 355 and 361-362).

Comment 4: In Figure 4, the authors should include an image quantification of the notable upregulated and downregulated proteins just they did in Figures 2 and 3.

Thank you for your constructive comments. We repeated the same in vitro experiment twice in completely independent experimental systems and obtained similar results. The data is not as diverse as in animal experiments, and we are confident that these two repeated experiments are reliable for this study. Therefore, there is no need to add bar graphs like in animal experiments, and we think it is sufficient to quantify each band.

In accordance with the Reviewer-2’s comment-4, we have inserted on it in the Figure 4 legends (line 475-476).

Comment 5: Line 372-373 Fx-high diet in Group 1 significantly decreased the expression of IGFBP2 (0.6-fold) and EpCAM (0.7-fold) and tended to decrease LCN2 expression (0.6-fold).  What did the authors mean by tending to decrease?  Are they not significant? LCN and IGFBP2 both decreased by 0.6-fold.  So why LCN2 expression is not significant?  The authors should comment on this.

Thank you for your valuable comments. Through rigorous statistical calculations, it was determined that there was no significant difference in LCN2. Therefore, we described LCN expression as a trend. In accordance with the Reviewer-2’s comment-5, we have modified the sentences regarding LCN2 plus HO1 (line 377-378 and 379-380).

Please confirm below 3 manuscripts

・ onco-2563316 with Track Changes_v2

・ Supplementary Figures_onco-2563316

・ Supplementary Tables_onco-2563316_modified

We strongly appreciate again for the Reviewer-2’s comments, regarding on our manuscript.

     We feel that the revised manuscript is a suitable response to the comments, and is significantly improved over the initial submission. We trust that is now suitable for publication in Onco.

Thank you in advance for your kind consideration of this paper.

Sincerely yours,

Masaru Terasaki, Ph.D.