Chemokine Homeostasis in Healthy Volunteers and during Pancreatic and Colorectal Tumor Growth in Murine Models

Round 1

Reviewer 1 Report

 

Review: CIMB-1927714

Chemokine homeostasis in norm and tumor growth 

The authors of this manuscript tried to compare the levels of chemokines in healthy humans, mice, and mice with tumor growth. The take-home message from them is to not rely on the chemokines as the prognostic factors for the cancers tested in this manuscript. This manuscript has several inconsistencies. 

1.      The word “norm” in this paper does not seem appropriate, as there is no “standard” measurement for a set of biological values. The same is proven in this manuscript. Please change it to “healthy.”

2.      The abstract should be rewritten in a grammatical and scientifically accurate manner.

3.      Table 1: Headings in the table must be adequately rearranged.

4.      Page 4: Please correct Lines 67 - 69.

5.      Methods: You mentioned blood samples included different sex. Mention the differences in sex for the measured values of the same chemokines. How about the sex of mice? And differences in chemokine levels?

6.      Methods 2.3: Correct multiple to Multiple.

7.      Line 131; eliminate the word “one.”

8.      You have mentioned individual differences in the levels of chemokines even in healthy individuals, including mice. Can you identify the factors that cause those differences, family history, sex, race, etc.? In mice, it can be a species difference. Can you cite papers that can explain relevant differences?

9.      3.3 Tumor models: Since both species of mice were used, show the survival results of both the species for the same cancer and the histopathology results.

10.  3.4:  Dynamics of blood chemokines in pancreatic and colorectal tumor models:  Please revisit the graph and write the results correctly. Also, mention clearly the species, their cancer treatments and appropriate results. 

11.  Line 200:  Revisit the word “To day.” 

12.  Fig 3: Correct the group name to CT26 consistently with the text. Also, label the images for their respective cancer types and staining for a clear understanding.

13.  Fig 4: Label the graphs with the appropriate species and the tumor type. The current representation is confusing. What is BL int or BA int group? If int means intact mice, did you use the mice as their own controls? If so, please correct lines 190 - 191. 

14.  The results section appears to be incomplete without proper evidence. Please add supporting evidence at least in supplemental figures.

15.  Modify the discussion accordingly. 

 

 

Author Response

We are thankful to our reviewers and tried to meet all the remarks below  

The authors of this manuscript tried to compare the levels of chemokines in healthy humans, mice, and mice with tumor growth. The take-home message from them is to not rely on the chemokines as the prognostic factors for the cancers tested in this manuscript. This manuscript has several inconsistencies. 

1. The word “norm” in this paper does not seem appropriate, as there is no “standard” measurement for a set of biological values. The same is proven in this manuscript. Please change it to “healthy.”

We have changed it for

Chemokine homeostasis in healthy donors and during pancreatic and colorectal tumor growth in murine models

 

 

2. The abstract should be rewritten in a grammatical and scientifically accurate manner.

 

We have tried to improve it

 

Chemokines are involved in humoral regulation of body homeostasis. Changes in blood level of chemokines were found in cancer, atherosclerosis, diabetes, and other systemic diseases. It is essential to distinguish the effects of co-morbid pathologies and cancer on the level of chemokines in blood. The aimed to analyze by multiplex cytometry the levels of chemokines in blood of healthy young donors as well as of intact mice and mice with CT26 colon and Pan02 pancreatic tumors. Two types of factors were identified both in human and murine plasma: homeostatic chemokines found in high concentrations (>100 pg/mL) and inducible ones which can be undetectable or determined at very low level (0-100 pg/mL). There was a high variability in the chemokine levels between the donors. To analyze chemokine levels during tumor growth, C57BL/6 and BALB/c were inoculated with Pan02 or CT26 tumor cells accordingly. The tumors significantly differed in the growth and the mortality of mice. However, the blood chemokine levels did not change in tumor bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer.

 

3. Table 1: Headings in the table must be adequately rearranged.

Table 1. Role of chemokines in cancer

 

4. Page 4: Please correct Lines 67 - 69.

We have corrected.

 

5. Methods: You mentioned blood samples included different sex. Mention the differences in sex for the measured values of the same chemokines. How about the sex of mice? And differences in chemokine levels?

We have added the information on the differences in men and women (Fig. S1). We used only females in animal experiments. Some data on the difference in chemokines in males and females is shown by Ray et al (doi: 10.1038/s41416-020-0913-8). They have estimated only 3 chemokines from our panel and found no differences.  

 

6.       Methods 2.3: Correct multiple to Multiple.

We have corrected

7. Line 131; eliminate the word “one.”

Done, thanks

 

8. You have mentioned individual differences in the levels of chemokines even in healthy individuals, including mice. Can you identify the factors that cause those differences, family history, sex, race, etc.? In mice, it can be a species difference. Can you cite papers that can explain relevant differences?

We do not know the reason for this difference, we just state the fact. We added some information and ideas, more studies are needed.

 Mice are inbred animals, even between the same strain of mice here was a difference, however 5 times less than in human, this can be explained by their genetic similarity.

However, there are no papers devoted to these differences. Moreover, there are no papers clearly showing such a variability in chemokine levels.

 

9. 3.3 Tumor models: Since both species of mice were used, show the survival results of both the species for the same cancer and the histopathology results.

It is not clear. We have shown both the survival and histology for both CT26 and Pan02 models in Fig. 3. If you mean CT26 in C57BL/6 mice and Pan02 in BALB/c mice it is impossible as CT26 have BALB/c origin, they will not grow in C57BL/6 mice and vice versa.

 

10. 3.4:  Dynamics of blood chemokines in pancreatic and colorectal tumor models:  Please revisit the graph and write the results correctly. Also, mention clearly the species, their cancer treatments and appropriate results. 

There was no treatment, other comments we have tried to meet.

 

11. Line 200:  Revisit the word “To day.” 

Done, thanks

12. Fig 3: Correct the group name to CT26 consistently with the text. Also, label the images for their respective cancer types and staining for a clear understanding.

We have done it

 

13. Fig 4: Label the graphs with the appropriate species and the tumor type. The current representation is confusing. What is BL int or BA int group? If int means intact mice, did you use the mice as their own controls? If so, please correct lines 190 - 191. 

We have changed it.

14. The results section appears to be incomplete without proper evidence. Please add supporting evidence at least in supplemental figures.

We have done multiple studies including blood cytokine level, matrix proteinases, expression

of hypoxia genes in tumors, immune cell subpopulations in organs and tumors. Unfortunately, all our data show the absence of a systemic response to tumor growth with only minor changes in the exception of some hypoxia gene expression in tumors. Our major interest was to find any parameters which can reflect tumor growth. Finally, we decided to publish negative data to warn scientists to be careful to develop anti-chemokine/receptor therapy.

 

15. Modify the discussion accordingly. 

We have done it.

 

Thank you, now we know a little bit more.

Reviewer 2 Report

The authors have investigated the level of chemokines in the blood of healthy young donors and in mice with colon and pancreatic tumors using multiplex cytometry. In addition, used histological methods to chartacterize the resulting tumor growth. The introduction is very concise with relevant citations to review articles to aid the reader. However, I recommend that the introduction reports the current normal level or range of chemokines in the plasma of healthy people and/or mice that has been reported to date in the literature. This is later described in the discussion section but it’s unclear what your data provides without defining norm, high or low levels to the reader at the start. However, I note that high value is defined in the abstract. 

 

The authors have clearly shown that there was a high variability in the levels of all chemokines in the 24 different healthy young individuals (28 years) and identified some chemokine correlations, but I’m not convinced with the R² value reported (<0.65). However, differences in diet, smoking status, weight, height, ethic group could account for this. Data on this was not included and perhaps could have a mention in a demographic table to show the ranges. 

 

Next, they investigated the chemokine levels in the blood of two mice strains and showed differences in chemokine levels in blood plasma of naïve C56BL/6 and BALB/c mice with correlation between CXCL5 and 149 CCL22 levels in blood plasma from C57BL/6 (c) and BALB/c (d) mice. This data was much more convincing with the inbred mice and controlled conditions as expected. 

 

Next, they monitored chemokine changes during tumor growth and demonstrated a difference in mice survival time, tumor characteristics and Chemokine levels at 4 time points (0, 12, 28, and 211 42 post inoculation). This data is very clear and convincing. 

 

I enjoyed reading the discussion as it highlighted the issues and what it means to the scientific community. Thus, caution is required when using chemokines as biomarkers diseases such as cancer. 

 

The following recommendations to improve the manuscript are outline below:

 

Line 28 – Remove the first word “The”

The Table 1 summarizes the available data….

 

Line 32 – The role of CXCL1 and CXCR2 in osteosarcoma metastasis was shown.

Statement is ambiguous; what was the role CXCL1 and CXCR2?

 

Line 50 – 

Improve formatting of Table 1 as column headings are not interpretable. 

 

Line 83 – word “ultiplex” is missing a letter

Include an “M” in the title: 2.3. Multiplex analysis of chemokines

 

Line 88

How much sample volume was used? 

 

Line 93

Include the pH and concentration of the PBS used.

 

Line 99

Include a reference on the methodology used for preparing the paraffin specimens for sectioning.

 

Line 136 – Figure 1

Remove the colour (blue and green). Just use black symbols. 

Even though, the data clearly shows the high and low chemokine levels, it might be more meaningful using a Box and whisker plot. 

 

Line 137

Only 23 green dots present on Figure 1d. Not 24.

 

Line 138 – Typo? Pirson’s 

 

Should it be Pierson’s coefficients or Pearson’s correlation.

Also, the R² values are low to support correlation. Any comment on this? Interestingly, the R² values are much higher for the mice data. 

 

Line 148

Include the number of mice analyzed in the figure legend. 

 

Line 193 – Figure 3 formatting

 

Make sure the X-axis (days) is the same length for each graph (a, b) so easier to compare.

Include a scale bar on the Figures 3e, f, g, h.

 

Line 223 – Those that found in blood

Insert a word – were.

Those that were found in blood

 

Lines 228-240 – relevance?

I’m not sure what the relevance of this data from other diseases since you are investigating norm and cancer. How does your data compare to other cancer studies? Can you include more information relevant to cancer, even if it’s not multiplex rather than be broader at the disease front? Is there anything regarding RNAseq data of chemokine signalling genes? What about knockout chemokine mouse studies?

Author Response

 

The following recommendations to improve the manuscript are outline below:

 

Line 28 – Remove the first word “The”

The Table 1 summarizes the available data….

Thank you, done

 

Line 32 – The role of CXCL1 and CXCR2 in osteosarcoma metastasis was shown.

Statement is ambiguous; what was the role CXCL1 and CXCR2?

CXCL1 binds CXCR2, that is a axis.

 

Line 50 – 

Improve formatting of Table 1 as column headings are not interpretable. 

We have modified it  

Line 83 – word “ultiplex” is missing a letter

Include an “M” in the title: 2.3. Multiplex analysis of chemokines

Sorry, done 

Line 88

How much sample volume was used? 

For the analysis 25 ul is used, in 96 plates 

Line 93

Include the pH and concentration of the PBS used.

 Done. All components were from the panel provided including a dilution buffer. Should be about 7-7.2

Line 99

Include a reference on the methodology used for preparing the paraffin specimens for sectioning.

Slides were prepared by a commercial firm, it is prepared automatically.

 

Line 136 – Figure 1

Remove the colour (blue and green). Just use black symbols. 

Even though, the data clearly shows the high and low chemokine levels, it might be more meaningful using a Box and whisker plot. 

 We have removed colours. However, we do not have the soft for Box and whisker plot soft.

Line 137

Only 23 green dots present on Figure 1d. Not 24. Some are overlayered

 

Line 138 – Typo? Pirson’s 

 

Should it be Pierson’s coefficients or Pearson’s correlation.

Also, the R² values are low to support correlation. Any comment on this? Interestingly, the R² values are much higher for the mice data. 

 

Sorry for Pearson’s. R² values are low but the table says it is significant. We think that a better distribution for mice results from their genetic similarity.

 

Line 148

Include the number of mice analyzed in the figure legend. 

 Done

Line 193 – Figure 3 formatting

 

Make sure the X-axis (days) is the same length for each graph (a, b) so easier to compare.

Include a scale bar on the Figures 3e, f, g, h.

 The length is the same, 20, 40, and 60 days

Line 223 – Those that found in blood

Insert a word – were.

Those that were found in blood

Done

 

Lines 228-240 – relevance?

I’m not sure what the relevance of this data from other diseases since you are investigating norm and cancer. How does your data compare to other cancer studies? Can you include more information relevant to cancer, even if it’s not multiplex rather than be broader at the disease front? Is there anything regarding RNAseq data of chemokine signalling genes? What about knockout chemokine mouse studies?

 

Data on knockout chemokine mouse studies were discussed a little. Signalling in cancer is very complex, just see Table 1, impossible to discuss it wider here. There multiple papers describing it. Sorry for not given more. Our message was to alert the researcher studding chemokines and developing related therapies.

 

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Re-review

Chemokine homeostasis in healthy donors and during pancreatic and colorectal tumor growth in murine models

 

Although the authors have requested changes in the manuscript, some edits are required. 

 

1. “Healthy donors” is not an apt word here. Change it to “Healthy Volunteers.”

2. Please correct the “factors” to either groups or classes.

3. Mention that the tumor models being used are “Syngeneic models.”

4. Line 146 - 147: Where was the correlation graph shown?

5. Please verify the statements in Lines 149 - 161 with the corresponding figures.

6. Fig S2 should have a value on Day 0 as well. 

7. Line 164 - 170: Given the variability and massive differences in the ranges, I'm not sure if it is a valid statement.

8. Fig. 4: Put the legends for all graphs. 

9. Line 246: change “accordingly” to “respectively.”

 

Author Response

We again thank our reviewers for reading and checking our paper.

1. “Healthy donors” is not an apt word here. Change it to “Healthy Volunteers.”

Done

2. Please correct the “factors” to either groups or classes.

Done

3. Mention that the tumor models being used are “Syngeneic models.”

Done

4. Line 146 - 147: Where was the correlation graph shown?

They are shown in Fig S1 and mentioned in the text.

5. Please verify the statements in Lines 149 - 161 with the corresponding figures.

Sorry, supplementary figures were incorrectly placed, we changed the order.

6. Fig S2 should have a value on Day 0 as well. 

Fig 2 gives data for intact mice, there is no Day 0 for these data.

7. Line 164 - 170: Given the variability and massive differences in the ranges, I'm not sure if it is a valid statement.

The variability is observed between people not for a single person. The results on Fig.  S3 show that individual concentrations do not change significantly. We have done only for three people such timing data just because wanted to see individual changes. May be more data will help to understand it better. We ask to keep it in the supplement. I have added some soft comment on it in the text.

8. 4: Put the legends for all graphs. 

Done, thank you for finding it.

 

9. Line 246: change “accordingly” to “respectively.”

      Done

Thank you again!

Author Response File: Author Response.docx

Round 3

Reviewer 1 Report

Below are my comments on the revised manuscript

1. The title is misleading, as healthy mice are included in the study. Please change the title.

2. Abstract: Line 22: Correct the word “donors.” Also, include a statement on variability in intact mice. Line 26: “Blood chemokines levels are”

3. Line 78: Change “groups” to “chemokines.”

4. Line 115: Add models after Syngeneic. Or “Subcutaneous syngeneic tumor models were employed.”

5. Fig S2: State what S1 and S2 are in the text and the figure legend.

6. Line 164: Only two but not three volunteers' blood was collected.

7. Fig 3 legend: Remove syngeneic.

Author Response

Thanks again for new comments

 

1. The title is misleading, as healthy mice are included in the study. Please change the title.

The mice used were transplanted with tumors, so they are not “healthy”. Indeed we have not treated them as the aim was to study the effect of tumor growth on chemokine level. 

 

2. Abstract: Line 22: Correct the word “donors.” Also, include a statement on variability in intact mice.

Cahnged for “There was a high variability in the chemokine levels both in healthy human and mice”.

3. Line 26: “Blood chemokines levels are”

 

 However, the blood chemokine levels did not change in tumor bearing mice until the very late stages. Taken collectively, blood chemokine level is highly variable and reflects in situ homeostasis. Care should be taken when considering chemokines as prognostic parameters or therapeutic targets in cancer.

 

May be we have changed it earlier

 

4. Line 78: Change “groups” to “chemokines.”

Done

….identify the most significant chemokine involved in tumor formation and response to it

 

5. Line 115: Add models after Syngeneic. Or “Subcutaneous syngeneic tumor models were employed.”

Done

 

6. Fig S2: State what S1 and S2 are in the text and the figure legend.

Page 147 – Fig. S1, Difference between men and women in chemokines. Page 155 – Fig. S2, Reproducibility of chemokine estimation in different days.

 

7. Line 164: Only two but not three volunteers' blood was collected.

We have data for 3 people, for simplicity showed only for two persons. I do not think we have to add these data too. Please see these data in the doc file. We have calculated the ratios of the data from month to month. It appeared that average ratios are 1±0.5. If you insist we can add these data.

 

 

8. Fig 3 legend: Remove syngeneic.

Done

 

Author Response File: Author Response.docx