Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion, and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway

Round 1

Reviewer 1 Report

The authors have satisfactorily addressed my previous comments.

Author Response

Thank you for your insightful review 

Reviewer 2 Report

The modifications made by the authors greatly improved the reading and the understanding of the manuscript. Nevertheless, there are still some errors that need to be fixed:

Abstract:

- “Kindlin-2-deletetion”. Spelling mistake.

- Please, carefully read the revised version, in order to remove all the words that need to be removed. For example, “progression”, in this sentence: “its function during mammary gland development and BC initiation andin BC progression”.

Materials and Methods

2.2: “E0771-drived tumors ». Spelling mistake.

2.5: “The mammary gland branches the terminal end…”. Please correct the sentence.

2.8: The procedures of 3D multi-tumorsphere formation and 3D-tumorsphere invasion assays were present in the cited references (30,31), but not that of 3D single-tumorsphere formation. Authors, please complete.

Results

Figure 2H, lower panel: The brightness of the photo is not strong enough to see the Kindlin-2 staining.

Legend Figure 2: Several spelling mistakes must be corrected.

Legend Figure 3: “2-week old mice” must be replaced by 2-week-old pregnant mice.

To complete my remark about paragraphe 2.8, a rapid explanation of single and multiple-sphere formation assays should be added in paragraph 3.4.

P11: “(E-Ctl)” and “(E-Ctrl)” are both used. Please, homogenize.

Supplementary Materials :

The supplementary materials are presented both in the file supplementary materials and in the file S1. One of the two must be deleted.

In the file “supplementary materials”, the uncropped full Western blot for Figure 6D,E are in Figure S7 and not S8. Please, correct.

 

Author Response

To: Reviewer 2                                                                                               January 21, 2022

 

Dear Reviewer,

We would like to thank you for extending us the opportunity to submit our revised manuscript “Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway” for publication in Cancers Journal. We appreciate your time and efforts to review the article and provide constructive feedback that “The modifications made by the authors greatly improved the reading and the understanding of the manuscript. Nevertheless, there are still some errors that need to be fixed”. We have now addressed all the additional editorial issues raised by this Reviewer. These corrections are tracked in the revised manuscript and are described  in the point-by-point response to the Reviewer below.

Minor points:

Typographic errors

“Kindlin-2-deletetion”. Spelling mistake. Fixed

Please, carefully read the revised version, in order to remove all the words that need to be removed. For example, “progression”, in this sentence: “its function during mammary gland development and BC initiation andin BC progression. Fixed

2.2: “E0771-drived tumors ». Spelling mistake. Fixed

2.5: “The mammary gland branches the terminal end…”. Please correct the sentence. Fixed

Legend Figure 2: Several spelling mistakes must be corrected. Fixed

Legend Figure 3: “2-week old mice” must be replaced by 2-week-old pregnant mice. Fixed

P11: “(E-Ctl)” and “(E-Ctrl)” are both used. Please, homogenize. Fixed

We have now  performed a thorough proofing of the manuscript and ensured that are no remaining typographical or grammatical errors.

 

Materials and Methods

2.8: The procedures of 3D multi-tumorsphere formation and 3D-tumorsphere invasion assays were present in the cited references (30,31), but not that of 3D single-tumorsphere formation. Authors, please complete.

To complete my remark about paragraphe 2.8, a rapid explanation of single and multiple-sphere formation assays should be added in paragraph 3.4.

 

Our Response: We have now included a description of the 3D tumorspheres procedures in paragraph 2.8.

 

Results

Figure 2H, lower panel: The brightness of the photo is not strong enough to see the Kindlin-2 staining.

 

Our Response: We have now adjusted the brightness to adequate levels to clearly show Kindlin-2 staining in the K8-expressing cells in the lower panel of Figure 2H.

 

Supplementary Materials

 

The supplementary materials are presented both in the file supplementary materials and in the file S1. One of the two must be deleted.

 

Our Response: The supplementary information files are now revised to avoid duplicity.

 

In the file “supplementary materials”, the uncropped full Western blot for Figure 6D,E are in Figure S7 and not S8. Please, correct. Corrected

 

We hope that this new data and revisions have addressed all concerns of the Reviewer and that our manuscript is now acceptable for publication in Cancers.

 

Sincerely yours,

 

 

Khalid Sossey-Alaoui

 

 

Khalid Sossey-Alaoui

Associate Professor

Case Western Reserve University

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have edited the manuscript for clarity and addressed many of the reviewer comments. 

Author Response

Thank you for your insightful review 

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

Wang and colleagues have generated a floxed Kindlin-2 mouse and looked at how loss of Kindlin-2 in the mammary gland effects breast cancer tumor growth using the E0771 mouse mammary cancer cell line. They provide some intriguing data showing that lack of Kindlin-2 in the host mammary gland, as well as the tumor cells themselves, reduces primary tumor growth in the mammary gland but not in the lung. They then show that loss of Kindlin-2 reduces TGFbeta and EGF signaling in the mammary epithelial cells. This builds on previous published work by the group showing that loss of Kindlin- 2 in human MDA-MB-231 and murine 4T1 cells reduces tumor growth. The novel aspect of this study is the use of the floxed Kindlin-2 mouse to demonstrate that the normal host cells also rely on Kindlin-2 to drive tumor growth.  However, this aspect of the study is not well developed with only preliminary observations showing reductions in both TGFbeta and EGF signaling. No attempt has been made to link these to their previous studies showing that CSF1 dependent recruitment of macrophages - are similar mechanisms involved? What factors are secreted by the MECs in a Kindlin-2 dependent manner that could be responsible for the increased tumor growth? Further information on how Kindlin-2 loss in the MECs impacts on tumor growth should be provided.

 

Specific questions:

1. Figure 2: Why would you have decrease in vimentin and keratin 5 in the mammary glands and MEC in the Kindlin-2-/- K14Cre mice?
2. As both approaches are not using purified basal cell populations to demonstrate specific deletion in the K14 lineage either immunohistochemistry with basal markers and Kindlin-2 antibodies should be carried out, or sorting of basal cells from the MECs should be carried out prior to analysis. From the data presented it is not possible to determine how efficient the deletion is in the K14 positive mammary cells. Without this information it is difficult to conclude that mammary gland development is not altered in the Kindlin-2-/- K14Cre as reported in Figure 3.
3. Figure 4 – experiments with non-targeting controls should be included for comparison.
4. Figure 5: basal cells in the lung will express K14 so this should be noted in the discussion on the experimental lung metastasis assay
5. The authors have used the HML2 cell line to carry out confirmatory in vivo studies and stated that this provides evidence in a second syngeneic model. However, as it is derived from the E0771 cell line it is misleading to suggest that this is a different model and the wording should be changed to reflect this.
6. Figure 6D/E: provide quantification of the westerns

Author Response

Dear Reviewer,

We would like to thank you for extending us the opportunity to submit our revised manuscript “Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway” for publication in Cancers Journal. We appreciate your time and efforts to review the article and provide constructive feedback. We have now addressed most if not all the issues raised by this Reviewer. We have performed new experiments and analyses to strengthen the study. These changes are highlighted within the manuscript and are described  in the point-by-point response to the Reviewer below.

 

Reviewer 1:

Specific questions:

• Figure 2: Why would you have decrease in vimentin and keratin 5 in the mammary glands and MEC in the Kindlin-2-/- K14Cre mice?

Our Response: We agree with the Reviewer that this is a valid question that we failed to articulate adequately. our published studies have shown that of Kindlin-2 is involved in the regulation of  the epithelial to mesenchymal transition (EMT) process [24]. Accordingly, here we show that, in purified mammary epithelial cells of K2-/-  mice, in addition to loss of expression of Kindlin-2, both Vimentin and K5 Keratin, two EMT markers, were also reduced by ~50% in mammary glands of K2--/K14 (Fig. 2F).

The above sentence was included in the revised manuscript in page 6 to explain the loss of expression of both vimentin and K5 in the K2-deficient mammary glands and MECs.

 

• As both approaches are not using purified basal cell populations to demonstrate specific deletion in the K14 lineage either immunohistochemistry with basal markers and Kindlin-2 antibodies should be carried out, or sorting of basal cells from the MECs should be carried out prior to analysis. From the data presented it is not possible to determine how efficient the deletion is in the K14 positive mammary cells. Without this information it is difficult to conclude that mammary gland development is not altered in the Kindlin-2-/- K14Cre as reported in Figure 3.

Our Response: At the time of the initial submission, we did not have all the reagents to perform additional experiments to confirm the findings described in Figure 3. While the manuscript was under review, we initiated immunostaining experiments of MECs isolated form mammary glands of both WT and K2-deficient mice and stained the MECs. The experiment and the results are described in the revised Figure 3 in page and 7 of the revised manuscript as follow:  We used immunofluorescence analyses to confirm that Kindlin-2 was specifically deleted in the K14 mammary epithelial lineage (Fig. 2G). In mammary epithelial cells isolated from wildtype mice, Kindlin-2 can be found in cells expressing the basal marker (K14) (Fig. 2G, upper panel) as well as the cells expressing the luminal marker (K8)(Fig. 2H, up-per panel). However, in the mammary epithelial cells isolated from the  mammary glands of K2--/K14 mice, Kindlin-2 can only be found in cells expressing K8 (Fig. 2H; lower panel), but not K14-expressing MECs (Fig. 2G, lower panel).

We also included a description of the immunostaining assays in the Material and Methods section in page 4.

• Figure 4 – experiments with non-targeting controls should be included for comparison.

Our Response: We apologize for this editorial oversight from our side. In fact, all our KO experiments included a scrambled sgRNA as a control. We have now revised our figures throughout the manuscript to describe our control cells as “Control-sg”.

• Figure 5: basal cells in the lung will express K14 so this should be noted in the discussion on the experimental lung metastasis assay.

Our Response: We agree with the Reviewer on the fact that K14 is indeed expressed in the lungs as well as in other tissues such as the esophagus, tongue, thymus and  even the skin. Indeed, our analyses showed that crossing the K2 floxed mice with the Cre-K14 mice resulted in targeting K2 in these tissues, but this targeting was  very minimal and had no effect on the normal development of mice bearing the transgenic K2 deletion. On the other hand, since K14 is also expressed in the lungs, this possibly may have influenced lung metastasis. However, our data in Figure 5 showed no difference in lung metastasis between WT and K2-deficient mice when injected with the same cells (Parental or K2-KO E0771 cells). We have now addressed this point in the discussion (page 14).

 

• The authors have used the HML2 cell line to carry out confirmatory in vivo studies and stated that this provides evidence in a second syngeneic model. However, as it is derived from the E0771 cell line it is misleading to suggest that this is a different model and the wording should be changed to reflect this.

 

Our Response: We have now revised our statement to reflect the fact that HML2 cells are indeed derived from E0771 cells. The following statement was included I our revised manuscript in page 10: . Even though HML2 cells were derived from the E0771 cells, and do not represent a true independent replicate, they still provide a second confirmatory cell model for our tumor growth assay.

 

• Figure 6D/E: provide quantification of the westerns

We have now revised Figure 6 to include the quantification and statistical analyses of the WBs.

 

We hope that this new data and revisions have addressed all concerns of the Reviewer and that our manuscript is now acceptable for publication in Cancers.

 

 

Sincerely yours,

 

 

 

Khalid Sossey-Alaoui

 

 

 

Khalid Sossey-Alaoui

Associate Professor

Case Western Reserve University

Author Response File: Author Response.pdf

Reviewer 2 Report

In the present study, Wang and collaborators assessed the consequences of Kindlin-2 (K2) loss in the mammary gland on the aggressiveness of breast carcinoma. For this, they generated mice, in which flox sequences were inserted around the K2 gene. The resulting mice were then crossed with a Keratin14-Cre mouse, in order to obtain a mouse model in which K2 gene was invalidated in the basal compartment of the mammary gland. They first checked for the consequences of Kindlin-2 knock-out (K2-KO) on mammary gland development and did not observe any significant modification.

Then, by using the CRISPR/Cas9 technology, they derived a K2-KO cell line from the mouse E0771 breast cancer cell line. Loss of K2 resulted in decreased migration and invasion capacities of K2-KO cells compared to the parental cells.

By combining both cell lines and mice with or without K2 expression, they investigated the effect of K2 loss i) on tumor growth in vivo, after inoculation of tumor cells in the mammary fat pad and ii) on appearance of lung metastases, after inoculation of tumor cells in the tail vein. Their results confirmed the role of K2 on tumor growth and metastasis development. More interestingly, they indicated that K2 expression in the basal compartment of the mammary gland favors tumor growth.

From a mechanistic point of view, they showed that loss of Kindlin-2 in the mammary epithelial cells was accompanied by a decrease of both TGF-β and EGF signaling.

 

The manuscript presents interesting results. However, it is confusing, difficult to read and contains approximations, over interpretations and many errors.

 

Major points:

 

- Page 6: “Vimentin and K5 Keratin were also reduced by ~50% in mammary glands of K2--/K14. These results are consistent with the specific deletion of Kindlin-2 in the basal cell lineage, but not the luminal cell lineage in the mammary glands, as both Vimentin and K5 Keratin are specific to the basal subtype.” Could the authors explain the relationship between the decreased level of K2 and that of vimentin and K5? Moreover, why the decrease of the last two does not seem to have an effect on the development of the mammary gland (Figure 3)?

- Figure 2 and 6: Looking at the images and numbers in Figure 2F and 6 A,B,C, it appears that for each protein, the level in WT cells/mice is taken as a reference, disregarding actin level. It is essential to describe the quantification method used to have a true idea of the relative expression.

- Page 9-10: The description of results of Figure 5 is very confusing. This paragraph should be rewritten to be clearer.

- Page 10, Figure 5: How is it possible to have the red curve below the blue (Fig 5D), since all the tumors obtained with the red conditions are larger than those with the blue condition (Fig 5C)?

- Page 12: In the discussion, the authors explain that “previous studies consistently showed that loss of Kindlin-2 function does not lead to total elimination of invasion and metastasis, possibly because both shRNA knockdown and CRISPR/Cas9-mediated knockout used in these studies did not result in complete loss of Kindlin-2 expression in the cancer cells.” Yet, in a previous article, the team reported a very efficient inhibition of K2 in the 4T1 mouse breast cancer cell line, as well as the almost complete elimination of lung metastases after inoculation of these K2-KO cells in syngeneic mice (Sossey-Alaoui et al., Sci. Reports, 2018). These previous published data invalidate the sentence mentioned above. Could the authors clarify this point and modify the discussion?

- Page 11 and Figure 6: here, the authors showed a relationship between K2 and the TGF-β and EGF signaling pathways in mammary epithelial cells. They already reported a similar relationship in breast cancer cells (Sossey-Alaoui et al., Cancer Res., 2017). Taken together, these results highlight an important link between K2 and TGF-β and EGF, both in physiological and pathological conditions. It would have been very beneficial for the present manuscript to have an explanation of the molecular mechanism connecting these pathways, as reported with DNMT1 and E-Cadherin (Wang et al., Int. J Biochem. Cell Biol., 2018).

 

Minor points:

 

- General: The nomenclature about the control and K2-KO cells and mice must be homogenized throughout the manuscript, in order to allow a better understanding of the text and the figures.

- General: The number of all figures and tables should be checked.

 

Materials and methods:

- Please, use the power of 3 rather than K to write 1000 (number of cells)

- Page 3, 2.2: HML2 cells origin and characteristics are described page 10 only. They should also be mentioned in the material and methods part.

- Page 3, 2.3: Replace “Figure S1” by Supplementary File S1 and “Supplementary Table 1” by Supplementary Table 2.

- Page 3: “(EGFR inhibitor ZD1839 and TGF-β receptor inhibitor SB431542…” Replace the bracket before EGFR by a full stop.

- Page 3, 2.4, 2.5: Please indicate which software was used to quantify the number of end buds and branches in Figure 3.

- Page 4: “dissociation solution containing 9 parts of Gentle Collagenase/ Hyaluronidase and 1 part of complete EpiCult-B Medium”. This is the contrary (1/9). Please correct.

 

Results:

- General: The molecular weight of proteins revealed by western blot must be indicated.

- Fig 3A and Figure 4E and G: Magnification?

- Page 10, Figure 5: The insert with the tumor samples shown in Figure 5C is not useful on the graph Figure 5D and should be removed.

- Page 10, legend Figure 5F: “Quantification of lung metastasis after tail vein injection of control E0771 cells or their K2-KO derivatives into the mammary fat pads”. Tail vein injection or fat pad injection? Please clarify.

- Page 10, legend Figure 5: ** corresponds to p<0.005 in the legend, but to p<0.01 in the text page 9. Moreover, all the statistical differences in Figure 5D are noticed by **, whereas one of the difference is written p<0.05 in the text. Please, check and clarify.

- Page 10: HML2 cells cannot be considered as a “second syngeneic model”, since they directly derive from the first syngeneic model, i.e. the E0771 cells. Please, correct.

- Page 11: “We also found both EGF and TGFβ signaling to be significantly lower in the in the tumors derived from E0771 (Fig. 6D) and HML2 (Fig. 6E) when injected in K2-/- mice as compared to their wild-type counterparts”. First, did you perform any statistical analysis showing a significant decrease of EGF and TGFβ downstream targets? Second, one of the two “in the” must be deleted.

- Page 11, legend Figure 6: (E&F) must be replaced by (D&E). 6F does not exist.

- Page 11, legend Figure 6: “Detailed information about the Western blotting can be found at Figure S4–S6”. Western blot of Figure 6 correspond to Figures S6-S8, not S4-S6.

- Figure S2, with the exception of Figure S2D, is included in Figure 5. Please, regroup Figures 5 and S2D and delete Figure S2 (present both in S1 file and supplementary data).

Author Response

Dear Reviewer,

We would like to thank you for extending us the opportunity to submit our revised manuscript “Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway” for publication in Cancers Journal. We appreciate your time and efforts to review the article and provide constructive feedback. We have now addressed most if not all the issues raised by this Reviewer. We have performed new experiments and analyses to strengthen the study. These changes are highlighted within the manuscript and are described  in the point-by-point response to the Reviewer below.

Major points:

• Page 6: “Vimentin and K5 Keratin were also reduced by ~50% in mammary glands of K2--/K14. These results are consistent with the specific deletion of Kindlin-2 in the basal cell lineage, but not the luminal cell lineage in the mammary glands, as both Vimentin and K5 Keratin are specific to the basal subtype.” Could the authors explain the relationship between the decreased level of K2 and that of vimentin and K5? Moreover, why the decrease of the last two does not seem to have an effect on the development of the mammary gland (Figure 3)?

Our Response: We agree with the Reviewer that this is a valid question that we failed to articulate adequately. our published studies have shown that of Kindlin-2 is involved in the regulation of  the epithelial to mesenchymal transition (EMT) process [24]. Accordingly, here we show that, in purified mammary epithelial cells of K2-/-  mice, in addition to loss of expression of Kindlin-2, both Vimentin and K5 Keratin, two EMT markers, were also reduced by ~50% in mammary glands of K2--/K14 (Fig. 2F).

The above paragraph was included in the revised manuscript in page 6 to explain the loss of expression of both vimentin and K5 in the K2-deficient mammary glands and MECs.

 

• Figure 2 and 6: Looking at the images and numbers in Figure 2F and 6 A,B,C, it appears that for each protein, the level in WT cells/mice is taken as a reference, disregarding actin level. It is essential to describe the quantification method used to have a true idea of the relative expression.

Our Response: All the signal quantifications were normalized to the β-Actin signal before deriving the fold-change from the control lane. We have now included this statement in the captions of Figure 2 and 6.

• Page 9-10: The description of results of Figure 5 is very confusing. This paragraph should be rewritten to be clearer.

Our Response: We have now revised the description of the data presented in Figure 5 in a way that we hope is much clear and concise. Please refer to page 10 of the revised manuscript.

• Page 10, Figure 5: How is it possible to have the red curve below the blue (Fig 5D), since all the tumors obtained with the red conditions are larger than those with the blue condition (Fig 5C)?.

Our Response: Please accept or apologies for this oversight and honest mistake. The red bar in the picture of the tumors (Fig. 5C) represent the E-K2-KO/WT B6 group and should indeed be below the blue one that represent the E-Ctl/K2^-/- B6 group. We have now corrected this mistake to conform with the data showed in the Fig. 5D.

 

• Page 12: In the discussion, the authors explain that “previous studies consistently showed that loss of Kindlin-2 function does not lead to total elimination of invasion and metastasis, possibly because both shRNA knockdown and CRISPR/Cas9-mediated knockout used in these studies did not result in complete loss of Kindlin-2 expression in the cancer cells.” Yet, in a previous article, the team reported a very efficient inhibition of K2 in the 4T1 mouse breast cancer cell line, as well as the almost complete elimination of lung metastases after inoculation of these K2-KO cells in syngeneic mice (Sossey-Alaoui et al., Sci. Reports, 2018). These previous published data invalidate the sentence mentioned above. Could the authors clarify this point and modify the discussion?

 

Our Response: We apologize if our description of the facts were taken as an  attempt to mislead the readers about the effect of loss of Kindlin-2 on tumor growth and metastasis. In both this present study and our previously published studies, none of the techniques used resulted in a 100% knockout of Kindlin-2, as can be seen in our WBs that all showed a residual K2-expression. Therefore, we posited that the residual K2 expression in cancer cells may have been responsible for not eliminating metastasis completely. We have revised the discussion (page 14) to address this point .

 

• Page 11 and Figure 6: here, the authors showed a relationship between K2 and the TGF-β and EGF signaling pathways in mammary epithelial cells. They already reported a similar relationship in breast cancer cells (Sossey-Alaoui et al., Cancer Res., 2017). Taken together, these results highlight an important link between K2 and TGF-β and EGF, both in physiological and pathological conditions. It would have been very beneficial for the present manuscript to have an explanation of the molecular mechanism connecting these pathways, as reported with DNMT1 and E-Cadherin (Wang et al., Int. J Biochem. Cell Biol., 2018).

Our Response: We thank the Reviewer for this comment. Our data showed that loss Kindlin-2 in the  mammary glands resulted in inhibition of tumor growth in the mice lacking K2 in the MECs. We also showed that the molecular mechanism driving this effect is associated with the TGF-β/EGF signaling in similar manner that we reported for loss of K2 in the cancer cells (Cancer Research 2017). In that study, we provided a through discussion on the link between  K2 and TGF-β/EGF signaling to regulated BC progression and metastasis. In the discussion of the present study, to avoid duplicity, we provided a brief discussion about this link and referred to our Cancer Research manuscript for further detailed information. We hope that this explanation will satisfy the Reviewer.

 

Minor Points

• General: The nomenclature about the control and K2-KO cells and mice must be homogenized throughout the manuscript, in order to allow a better understanding of the text and the figures.

Our Response: We have now revised the manuscript and figures throughout to have a homogeneous description of the K2-KO cells and mice.

 

• General: The number of all figures and tables should be checked.

Our Response: We have now revised the manuscript to make sure the numbers of Figures and Tables are correct.

Materials and methods:

• Please, use the power of 3 rather than K to write 1000 (number of cells): This was corrected

 

• Page 3, 2.2: HML2 cells origin and characteristics are described page 10 only. They should also be mentioned in the material and methods part.

Our Response: We have now included a description of HML2 cells in the M7M section in page 2.

 

• -Page 3, 2.3: Replace “Figure S1” by Supplementary File S1 and “Supplementary Table 1” by Supplementary Table 2.

Our Response: The nomenclature and titles of figures and table of the manuscript was now revised according to the Editors’ instructions.

 

• Page 3: “(EGFR inhibitor ZD1839 and TGF-β receptor inhibitor SB431542…” Replace the bracket before EGFR by a full stop. This was corrected

 

• Page 3, 2.4, 2.5: Please indicate which software was used to quantify the number of end buds and branches in Figure 3.

 

Our Response: We used ImageJ software for our image analyses. This is now indicated in captions of Figure 3.

 

• Page 4: “dissociation solution containing 9 parts of Gentle Collagenase/ Hyaluronidase and 1 part of complete EpiCult-B Medium”. This is the contrary (1/9). Please correct. This typo was corrected.

 

Results:

 

• General: The molecular weight of proteins revealed by western blot must be indicated.

Our Response: The molecular weights of protein bands are now shown in all WBs.

 

• Fig 3A and Figure 4E and G: Magnification?

Our Response: A scale bar is now shown in FIG. 3A to reflect the magnification.

 

• Page 10, legend Figure 5F: “Quantification of lung metastasis after tail vein injection of control E0771 cells or their K2-KO derivatives into the mammary fat pads”. Tail vein injection or fat pad injection? Please clarify.

Our Response: We have now corrected this typo and removed  the “into the mammary fat pads” words form this sentence.

 

• Page 10, legend Figure 5: ** corresponds to p < 0.001 in the legend, but to p < 0.01 in the text page 9. Moreover, all the statistical differences in Figure 5D are noticed by **, whereas one of the difference is written p<0.05 in the text. Please, check and clarify.

Our Response: We have now revised the manuscript to reflect the concordance between the p values and their significance: * means p < 0.01, ** means p< 0.001.

 

• Page 10: HML2 cells cannot be considered as a “second syngeneic model”, since they directly derive from the first syngeneic model, i.e. the E0771 cells. Please, correct.

Our Response: we have now revised this statement to reflect the true origin of HML2 cells and their contribution as a secondary confirmatory cell line. Please refer to page 10 of the revised manuscript:  Even though HML2 cells were derived from the E0771 cells, and do not represent a true independent replicate, they still provide a second confirmatory cell model for our tumor growth assay.

 

• Page 11: “We also found both EGF and TGFβ signaling to be significantly lower in the in the tumors derived from E0771 (Fig. 6D) and HML2 (Fig. 6E) when injected in K2-/- mice as compared to their wild-type counterparts”. First, did you perform any statistical analysis showing a significant decrease of EGF and TGFβ downstream targets? Second, one of the two “in the” must be deleted.

Our Response: We have now revised Figure 6 and provided statistical analyses for the difference in expression levels of pSMAD and pERK between WT and K2-KO tumors. The statistical analyses are now presented in the new graphs in revised Figure 6.

The duplicated “in the” words were also deleted.

 

• Page 11, legend Figure 6: (E&F) must be replaced by (D&E). 6F does not exist. Figure 6 has now been revised.

 

• Page 11, legend Figure 6: “Detailed information about the Western blotting can be found at Figure S4–S6”. Western blot of Figure 6 correspond to Figures S6-S8, not S4-S6.

 

Our Response: We have now revised the supplementary tables and figure according the Editors’ instructions.

 

We hope that this new data and revisions have addressed all concerns of the Reviewer and that our manuscript is now acceptable for publication in Cancers.

 

 

Sincerely yours,

 

 

 

Khalid Sossey-Alaoui

 

 

 

Khalid Sossey-Alaoui

Associate Professor

Case Western Reserve University

Author Response File: Author Response.pdf

Reviewer 3 Report

The manuscript explores a logical in vivo extension the authors' previous in vitro studies. The findings are important because they reveal the critical contribution of Kindlin 2 to the mammary TME for the initiation and progression of tumors upon injection in the fat pad. The role of TGFbeta in this process is interesting and the mouse model may be useful for future exploration of TGF beta contribution to these processes.

Note that the legend for figure 6 has an error (there is no panel F in the figure).

The authors should address the finding that the levels of P-SMAD3 and P-ERK don't always correlate in the tumors from either the wt and KO mice (Figure 6 D and E). 

The addition of data from additional tumors could be summarized to show the prevalence of P-SMAD or P-Erk in tumors from control vs KO mice.

 

 

Author Response

Dear Reviewer,

We would like to thank you for extending us the opportunity to submit our revised manuscript “Targeted Deletion of Kindlin-2 in Mouse Mammary Glands Inhibits Tumor Growth, Invasion and Metastasis Downstream of a TGF-β/EGF Oncogenic Signaling Pathway” for publication in Cancers Journal. We appreciate your time and efforts to review the article and provide constructive feedback. We have now addressed most if not all the issues raised by this Reviewer. We have performed new experiments and analyses to strengthen the study. These changes are highlighted within the manuscript and are described  in the point-by-point response to the Reviewer below.

Minor points:

• Note that the legend for figure 6 has an error (there is no panel F in the figure).

Our Response: We have now revised Figure 6 to make sure that all the panels are described.

• The authors should address the finding that the levels of P-SMAD3 and P-ERK don't always correlate in the tumors from either the wt and KO mice (Figure 6 D and E).
• The addition of data from additional tumors could be summarized to show the prevalence of P-SMAD or P-Erk in tumors from control vs KO mice.

Our Response to both points 2 and 3: We have now revised Figure 6 and provided statistical analyses for the difference in expression levels of pSMAD and pERK in tumors between WT and K2-KO mice. The statistical analyses are now presented in the new graphs in revised Figure 6.

We hope that this new data and revisions have addressed all concerns of the Reviewer and that our manuscript is now acceptable for publication in Cancers.

 

Sincerely yours,

 

Khalid Sossey-Alaoui

 

 

Khalid Sossey-Alaoui

Associate Professor

Case Western Reserve University

Author Response File: Author Response.pdf