Establishment and Characterization of NCC-MFS5-C1: A Novel Patient-Derived Cell Line of Myxofibrosarcoma

Round 1

Reviewer 1 Report

The authors have addressed my concerns well and made required edits in the revised documents. The revised manuscript reads well and I have no additional revision requests.

Author Response

We are grateful to Reviewer #1 for recognizing the value of this paper well.

Reviewer 2 Report

The authors have addressed most points of criticism, at least the more important ones.

Two additional points:

(1)  Line 84:  "<0.1 / 100,000"  (not "<0,1 / 100,000 / years")

(2)  The authors must include at a prominent spot in the manuscript the willingness to provide the cell line to outside investigators upon reasonable request.

Author Response

Reviewer #2
We truly appreciate the constructive comments by Reviewer #2.

Comment 1
Line 84:  "<0.1 / 100,000"  (not "<0,1 / 100,000 / years")

Response to Comment 1
Accordingly, we corrected the manuscript.

Comment 2
The authors must include at a prominent spot in the manuscript the willingness to provide the cell line to outside investigators upon reasonable request.

Response to Comment 2
Accordingly, we added the statement regarding to the cell line distribution in the declaration section.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The authors presented the establishment and characterization of a novel patient-derived cell line of myxofibrosarcoma (NCC-MFS5-C1), which was shown typical MFS genetic abnormalities.

Four other MFS cell lines (NCC-MFS1-C1 [27], NCC-MFS2-C1 [28], NCC-MFS3-C1 [29], and NCC-MFS4-C1 [30] have already been published by this working group. All these papers are very similar in structure.

The present work is methodically well structured and meets the criteria that should be applied for the characterisation of novel cell lines. STR and SNP analyses correspond to the usual methods for the characterisation of cell lines. The effect of bortezomib and romidepsin are very interesting and should be further investigated. The efficacy of bortezomib in particular is already known in other sarcoma entities.

The establishment of new cell lines is of great importance in sarcoma research. Equally important would be a sharing of these cell lines with the research community.

Some minor comments:

The relevant ethical vote for the collection of the patient sample should be provided.

Why were MG63 cells used as a comparison for the migration analyses with the xCELLigence system? It would be interesting to compare this with the other MFS cell lines. And the question of whether genetic differences are reflected in migration behaviour.

The number of repetitions in each case (N=?) should be indicated (all methods).

Author Response

Reviewer #1

We are grateful to Reviewer #1 for the critical and constructive comments.

Comment 1

The relevant ethical vote for the collection of the patient sample should be provided.

Response to Comment 1

Accordingly, we submit the informed consent paper (Informed consent paper.pdf) to the editorial office.

Comment 2

Why were MG63 cells used as a comparison for the migration analyses with the xCELLigence system? It would be interesting to compare this with the other MFS cell lines. And the question of whether genetic differences are reflected in migration behaviour.

Response to Comment 2

MG63 is derived from osteosarcoma, which is one of the most representative histological subtypes of sarcomas. Therefore, we used MG63 cell line as a control. We added this reason to the materials and methods section.

In our previous reports about the establishment of MFS, we assessed the invasiveness using another method. It is interesting to evaluate the invasiveness of all MFS cell lines we established using the xCELLigence system. However, this study is not focused on the invasiveness of MFS cell lines. Therefore, we will assess it in another study.

Genetic differences may affect the migration behavior. However, the genotypes of MFS, including copy number alterations, are considered to be highly diverse and have not been fully elucidated (ref 1, WHO Classification of Tumours of Soft Tissue and Bone. 5th edn). We are currently investigating the whole genome sequence of the MFS cell line and the original tumor in another project, and hope to report the relationship between genetic differences and malignant potential including migration behavior in the future.

Comment 3

The number of repetitions in each case (N=?) should be indicated (all methods).

Response to Comment 3

Accordingly, we added the number of repetitions.

Reviewer 2 Report

This manuscript describes generation of a new cell lines derived from a patient of MFS. This work adds a useful tool to study these rare sarcomas. The paper is written well and conclusions are well supported by the data. My only concern is that the authors did not compare this new cell line with previous cell lines that presumable were also developed by same group. For all the characterization studies like growth and proliferation assays, they are using another MG63 cell line. The rationale for using this is not well justified.

It will be good to show the growth characteristics compared with previous 4 cell lines.

Otherwise it is a good detailed study.

Author Response

Reviewer #2

We truly appreciate the constructive comments by Reviewer #2.

Comment

My only concern is that the authors did not compare this new cell line with previous cell lines that presumable were also developed by same group. For all the characterization studies like growth and proliferation assays, they are using another MG63 cell line. The rationale for using this is not well justified.

Response to Comment

According to your advice, we added Supplementary Table which compares the MFS cell lines we established.

Regarding to MG63, this cell line is derived from osteosarcoma, which is one of the most representative histological subtypes of sarcomas. Therefore, we think it is reasonable to use MG63 cell line as a control. We added this reason to the materials and methods section.

Reviewer 3 Report

The authors described the establishment and the characterization of a new cell line derived from a patient with myxofibrosarcoma (MFS). This cell line is the fifth cell line in a series of MFS cell lines that these authors have published in 2019-2021 in five different papers. One wonders whether these five cell lines could not have been published all together in one paper.

Specific Points of Criticism and Suggestions for Alterations:

1. Line 37: For the non-specialist in MFS a number could be given for the overall incidence (per 100.000) in parenthesis.
2. Line 52: What means „poor prognosis“? What is generally the 5-year survival rate (range)? This could be indicated in parenthesis at the end of this sentence.
3. Lines 67-68: While the authors indicate that only 2 MFS-derived cell lines are publicly available, they themselves have not deposited their previous 4 MFS cell lines in a public non-profit cell lines bank. The authors are urgently encouraged to do so and also to provide the cell lines to outside researchers upon reasonable request.
4. Line 78: Was it only „one tumor specimen“ (as suggested on line 102) - or „tumor specimens“?
5. Lines 84-89: First, the authors write: „right upper extremity amputation“ (lines 84-85). Later it is written: „surgical tumor resection“ (line 89). Which one is correct?
6. Table 1: This table is very important for the authentication but for the "normal" reader not very interesting. Hence Table 1 could go into the supplements.
7. Lines 206-210: In this paragraph it is not Figure 4, but Figure 3 that should be cited (Figure 3a-e).
8. Line 220: Better use „significantly“ instead of „sufficiently“.
9. Line 229: After „in cluster C“ Figure 4b should be cited.
10. Line 230: „BCR-ABL1“ instead of BCR-ABL“.
11. Figure 4:

11a.  In Figure 4a the colors in the dendrogram groups are red for A (high effect group), blue for B (medium effect group) and yellow for C (low effect group). However in the scale for „Cell viability“ (to the left of the dendrogram), blue stands for low viability (= results from high cytotoxicity of the drug), yellow for medium viability  and red for high viability (= ineffective cytotoxicity). Maybe the colors of the 3 dendrogram groups can be changed appropriately in order to avoid confusion.

11b.  Bottom of figure:  The official designation is no longer BCR-ABL, but BCR-ABL1 (see www.genenames.org), also in line 230.

11c.  Use "Other inhibitors" instead of "Others inhibitor".

12. Lines 271-277:

12a.  A table comparing the CDKN2A, CDKN2B and TP53 mutation status among the 5 MFS cell lines of this group, possible including data from other MFS cell lines if available, would be useful

12b.  In the Introduction (line 46) gene RB1 is also mentioned as being commonly mutated in MFS. Was the RB1 status tested here? Are any data on other MFS cell lines available (for the table)?

13. Lines 283-287:  Here a comparison table of the 5 MFS cell lines would be useful, e.g. doubling time, heterotransplantability, and other features.

Author Response

Reviewer #3

We really appreciate the critical and constructive comments by Reviewer #3.

Overall Comment

The authors described the establishment and the characterization of a new cell line derived from a patient with myxofibrosarcoma (MFS). This cell line is the fifth cell line in a series of MFS cell lines that these authors have published in 2019-2021 in five different papers. One wonders whether these five cell lines could not have been published all together in one paper.

Response to Overall Comment

When we first started to establish MFS cell lines, there were less than 10 MFS cell lines in existence. Even now, the number of MFS cell lines is only 12 as we described in the introduction section. Therefore, in order to promote the research on MFS, we report the cell line as soon as it is established.

Comment 1

Line 37: For the non-specialist in MFS a number could be given for the overall incidence (per 100.000) in parenthesis.

Response to Comment 1

Accordingly, we added the incidence of MFS (<0,1/100,000/years).

Comment 2

Line 52: What means „poor prognosis“? What is generally the 5-year survival rate (range)? This could be indicated in parenthesis at the end of this sentence.

Response to Comment 2

Accordingly, we added the 5-year survival rate.

Comment 3

Lines 67-68: While the authors indicate that only 2 MFS-derived cell lines are publicly available, they themselves have not deposited their previous 4 MFS cell lines in a public non-profit cell lines bank. The authors are urgently encouraged to do so and also to provide the cell lines to outside researchers upon reasonable request.

Response to Comment 3

Due to the regulations of our institution, it is difficult to deposit our cell lines to the public cell banks immediately. However, we have already distributed established cell lines free of charge upon request. We are also applying for registration of established cell lines as “NCC sarcoma cell line panel” in Cellosaurus cell line database. We believe that this attempt will make our cell lines more accessible to researchers in the world.

Comment 4

Line 78: Was it only „one tumor specimen“ (as suggested on line 102) - or „tumor specimens“?

Response to Comment 4

We used “one” tumor specimen, and corrected the manuscript.

Comment 5

Lines 84-89: First, the authors write: „right upper extremity amputation“ (lines 84-85). Later it is written: „surgical tumor resection“ (line 89). Which one is correct?

Response to Comment 5

The patient underwent “right upper extremity amputation”. We removed the sentence “surgical tumor resection”.

Comment 6

Table 1: This table is very important for the authentication but for the "normal" reader not very interesting. Hence Table 1 could go into the supplements.

Response to Comment 6

As you mentioned, the STR status is quite important. And other reports about cell line establishments usually include the STR status as a main table. Therefore, we think it is acceptable to demonstrate STR status in the main table.

Comment 7

Lines 206-210: In this paragraph it is not Figure 4, but Figure 3 that should be cited (Figure 3a-e).

Response to Comment 7

Accordingly, we corrected the figure number.

Comment 8

Line 220: Better use „significantly“ instead of „sufficiently“.

Response to Comment 8

Accordingly, we corrected the expression.

Comment 9

Line 229: After „in cluster C“ Figure 4b should be cited.

Response to Comment 9

Accordingly, we corrected the manuscript.

Comment 10

Line 230: „BCR-ABL1“ instead of BCR-ABL“.

Response to Comment 10

Accordingly, we corrected the manuscript.

Comment 11

Figure 4:

11a.  In Figure 4a the colors in the dendrogram groups are red for A (high effect group), blue for B (medium effect group) and yellow for C (low effect group). However in the scale for „Cell viability“ (to the left of the dendrogram), blue stands for low viability (= results from high cytotoxicity of the drug), yellow for medium viability  and red for high viability (= ineffective cytotoxicity). Maybe the colors of the 3 dendrogram groups can be changed appropriately in order to avoid confusion.

11b.  Bottom of figure:  The official designation is no longer BCR-ABL, but BCR-ABL1 (see www.genenames.org), also in line 230.

11c.  Use "Other inhibitors" instead of "Others inhibitor".

Response to Comment 11

As for the color coding of the clusters, we think it is obvious that this is just to show the clusters clearly and not the viability of the cells. We think this is a matter of preference. Therefore, we did not modify the color spectrum.

Otherwise, we corrected the Figure 4 and manuscript as the reviewer’s suggestion.

Comment 12

Lines 271-277:

12a.  A table comparing the CDKN2A, CDKN2B and TP53 mutation status among the 5 MFS cell lines of this group, possible including data from other MFS cell lines if available, would be useful

12b.  In the Introduction (line 46) gene RB1 is also mentioned as being commonly mutated in MFS. Was the RB1 status tested here? Are any data on other MFS cell lines available (for the table)?

Response to Comment 12

According to your advice, we added Supplementary Table which compared the MFS cell lines we had established.

NCC-MFS5-C1 cell line did not harbor the copy number alteration of RB1 (Supplementary Table 3).

As for the mutation status, we did not assess it because this is not included in the diagnostic criteria for MFS. We also described other genetic data in Supplementary Table.

Comment 13

13. Lines 283-287: Here a comparison table of the 5 MFS cell lines would be useful, e.g. doubling time, heterotransplantability, and other features.

Response to Comment 13

According to your advice, we added Supplementary Table which compares the MFS cell lines we established including the characteristics of cell line.