CD44 Sorted Cells Have an Augmented Potential for Proliferation, Epithelial-Mesenchymal Transition, Stemness, and a Predominantly Inflammatory Cytokine and Angiogenic Secretome

Round 1

Reviewer 1 Report

The methodology of this paper is questionable.

• Whilst 5 tumour samples were used, the text makes it seem as if cells from all tumours were combined together before sorting. If this is the case, I do not think this paper is publishable.
• If the tumours had been sorted separately but the final results aggregated during analysis, these results must be separated out for each tumour. Clinical details for each of the tumours must also be provided in the manuscript. A larger number of samples is necessary for any kind of meaningful interpretation of the results. 

The language in this manuscript, whilst free of major errors, is convoluted and opaque. More clarity in writing is required. Also note that the very first word of the paper is not capitalised.

Author Response

Reviewer 1:

The methodology of this paper is questionable.

• Whilst 5 tumour samples were used, the text makes it seem as if cells from all tumours were combined together before sorting. If this is the case, I do not think this paper is publishable.

Response: Given additional details on experiments which were performed independently for each individual sample and the data was added together. Moreover, we have performed statistical analysis by using paired two-sample t-test to combine the data and calculate p values.

• If the tumours had been sorted separately but the final results aggregated during analysis, these results must be separated out for each tumour. Clinical details for each of the tumours must also be provided in the manuscript. A larger number of samples is necessary for any kind of meaningful interpretation of the results. 

Response: Since this is the first time report, we performed the experimentation on a limited number of samples (n = 5). We will implement these experiments in a greater number of samples in our future studies. To perform statistical analysis and to give statistical significance, we have combined the results which were performed independently for all the five samples. For now, we hope that these results will definitely help in advancing the current research in the field of cancer stem cells and oral oncology. We have age range, gender, and history of tobacco abuse in the materials and methods section.

The language in this manuscript, whilst free of major errors, is convoluted and opaque. More clarity in writing is required. Also note that the very first word of the paper is not capitalised.

Response: the first letter has been capitalised Head and neck cancers (HNCs), 

Reviewer 2:

General comments:

The present study aimed to assess the association of CD44 with stemness-related, EMT-related genes and the secretome of the CSCs. Using 5 OSCC patients, they found that the sorted CD44+ CSCs expressed higher stemness and EMT-related genes, proliferation rate, inflammatory cytokines, and angiogenic factors than heterogeneous tumor cell populations. Therefore, the strategy to screen CSC by CD44+ for OSCC is promising.

Major comments:

1. Abstract: The authors mentioned too detailed descriptions for methods (line 14-20) but too few sentences for results. Moreover, the results may be written in more detail.

Response: The necessary changes have been done and highlighted

            The cancer stem cells were isolated by CD44+ selection using magnetic cell-sorting. The expression of CD44, proliferation rate, gene expression of EMT-related transcription factors, stemness markers, cytokine levels and angiogenic factors in both cell population was assessed. The sorted CD44+ cells showed significantly higher proliferation rate than heterogenous population. The CD 44 expression was >90% in the sorted cells which was higher than the heterogenous cells. The CD44+ CSCs cells demonstrated a significant increased levels of EMT-related genes TWIST1 and CDH2 (N-cadherin) , CSC-related genes CD44 and CD133 (PROM1), stemness-related genes OCT4, SOX2, inflammatory cytokines IL-1ß, IL-12, IL-18 and TNF-α and angiogenic factors Angiopoietin-1, Angiopoietin-2, bFGF and VEGF while decreased levels of epithelial gene CDH1 (E-cadherin) in comparison with mixed cell population. The genetic and secretome profiling of the CD44+ CSCs could serve as diagnostic and prognostic tools in the treatment of oral cancers.

1. Abstract: EMT should be provided in full name accompanied with the abbreviation.

Response: The same has been done and highlighted Epithelial Mesenchymal Transition

1. The author mentioned that “The data obtained were designated as the means ± standard deviations of the three independent experimental values.”. However, 5 OSCC were recruited. Therefore, it isn't very clear. For each figure, the repeated experiment number should be mentioned in each figure legend. How to deal with these 5 patient samples? Are they mixed for all experiments?

Response: We have added a line mentioning “Experiments were performed in triplicates for all five samples independently” in all figure legends. The samples were not mixed for any of the experiment and we have made additional changes in the materials and methods section for the clarification.

1. Figure 1 (proliferation curve) is based on 48 and 72 h experiments. However, the experiments for other figures were not mentioned. Please add this information.

Response: We have implemented the changes in all the materials and methods and added timepoints for all the experiments.

1. Why the sorted cells in culture-expanded cells had higher gene expressions than the sorted cells in freshly isolated cells? Please provide the discussion for this finding.

Response: This is for the first time we have obtained these results. In our future studies we will try to find out by utilizing more experimental modalities and a greater number of samples.

Minor comments:

1. The PCR program should be provided in section 2.5.

Response: Provided PCR program in the materials and methods section (Table 1).

1. “in vitro” should be typed in italic font.

Response: The same has been done and highlighted throughout the manuscript.

1. Figure 2 and Figure 4: For consistency, I suggest the author change the column color from black to white for the sorted cells. In that way, all sorted cell data will be shown in the white column, and unsorted data will be shown in black.

Response: We appreciate your suggestion. We have implemented the change in figure 2 and figure 4.

Reviewer 3:

In this manuscript, author characterized sorted cd44+ cells in term of proliferation, EMT, stemness and secretome. There are some spelling errors, extra blanks. author need rechecking text.

1. more detail information is needed for materials and methods 2.5 part, ie, qPCR machine and cycling conditions, etc

Response: Provided detailed information on RT-qPCR in materials and methods section.

1. The conclusion of 3.3 part is not very clearly. how many passages have cells been cultured/expanded? figure 3 is also similar with figure2D and 2E, while the relative expression to ACTB are different scale in figure 3 and figure2D-E. Could author comment on this?

Response: We have mentioned passage number. It was a calculation mistake while splitting the figure 3 into 2 parts and the scales changed while calculating gene expressions of SOX2 and OCT4 relative to ACTB. But, we removed figure 2D and 2E which were SOX2 and OCT4 gene expression analyses as they are already included in figure 3 so there is no meaning including them repeatedly.

1. in each figure, author should clarify how many patients the data are derived from.

Response: We have added a line mentioning “Experiments were performed in triplicates for all five samples independently” in all figure legends.

Questions from reviewer: how long can author culture the enriched cd44+ cells?

Response: Though we have used passage 4 CD44+ cells in this study, we were able to expand and sustain these cells in culture upto passage 23 from two samples. At late passages from passage 9, these cells started to change their morphology and very late passages, these cells started losing their surface adhesion characteristic.

Author Response File: Author Response.docx

Reviewer 2 Report

General comments:

The present study aimed to assess the association of CD44 with stemness-related, EMT-related genes and the secretome of the CSCs. Using 5 OSCC patients, they found that the sorted CD44+ CSCs expressed higher stemness and EMT-related genes, proliferation rate, inflammatory cytokines, and angiogenic factors than heterogeneous tumor cell populations. Therefore, the strategy to screen CSC by CD44+ for OSCC is promising.

Major comments:

1. Abstract: The authors mentioned too detailed descriptions for methods (line 14-20) but too few sentences for results. Moreover, the results may be written in more detail.
2. Abstract: EMT should be provided in full name accompanied with the abbreviation.
3. The author mentioned that “The data obtained were designated as the means ± standard deviations of the three independent experimental values.”. However, 5 OSCC were recruited. Therefore, it isn't very clear. For each figure, the repeated experiment number should be mentioned in each figure legend. How to deal with these 5 patient samples? Are they mixed for all experiments?
4. Figure 1 (proliferation curve) is based on 48 and 72 h experiments. However, the experiments for other figures were not mentioned. Please add this information.
5. Why the sorted cells in culture-expanded cells had higher gene expressions than the sorted cells in freshly isolated cells? Please provide the discussion for this finding.

Minor comments:

1. The PCR program should be provided in section 2.5.
2. “in vitro” should be typed in italic font.
3. Figure 2 and Figure 4: For consistency, I suggest the author change the column color from black to white for the sorted cells. In that way, all sorted cell data will be shown in the white column, and unsorted data will be shown in black.

Author Response

Reviewer 1:

The methodology of this paper is questionable.

• Whilst 5 tumour samples were used, the text makes it seem as if cells from all tumours were combined together before sorting. If this is the case, I do not think this paper is publishable.

Response: Given additional details on experiments which were performed independently for each individual sample and the data was added together. Moreover, we have performed statistical analysis by using paired two-sample t-test to combine the data and calculate p values.

• If the tumours had been sorted separately but the final results aggregated during analysis, these results must be separated out for each tumour. Clinical details for each of the tumours must also be provided in the manuscript. A larger number of samples is necessary for any kind of meaningful interpretation of the results. 

Response: Since this is the first time report, we performed the experimentation on a limited number of samples (n = 5). We will implement these experiments in a greater number of samples in our future studies. To perform statistical analysis and to give statistical significance, we have combined the results which were performed independently for all the five samples. For now, we hope that these results will definitely help in advancing the current research in the field of cancer stem cells and oral oncology. We have age range, gender, and history of tobacco abuse in the materials and methods section.

The language in this manuscript, whilst free of major errors, is convoluted and opaque. More clarity in writing is required. Also note that the very first word of the paper is not capitalised.

Response: the first letter has been capitalised Head and neck cancers (HNCs), 

Reviewer 2:

General comments:

The present study aimed to assess the association of CD44 with stemness-related, EMT-related genes and the secretome of the CSCs. Using 5 OSCC patients, they found that the sorted CD44+ CSCs expressed higher stemness and EMT-related genes, proliferation rate, inflammatory cytokines, and angiogenic factors than heterogeneous tumor cell populations. Therefore, the strategy to screen CSC by CD44+ for OSCC is promising.

Major comments:

1. Abstract: The authors mentioned too detailed descriptions for methods (line 14-20) but too few sentences for results. Moreover, the results may be written in more detail.

Response: The necessary changes have been done and highlighted

            The cancer stem cells were isolated by CD44+ selection using magnetic cell-sorting. The expression of CD44, proliferation rate, gene expression of EMT-related transcription factors, stemness markers, cytokine levels and angiogenic factors in both cell population was assessed. The sorted CD44+ cells showed significantly higher proliferation rate than heterogenous population. The CD 44 expression was >90% in the sorted cells which was higher than the heterogenous cells. The CD44+ CSCs cells demonstrated a significant increased levels of EMT-related genes TWIST1 and CDH2 (N-cadherin) , CSC-related genes CD44 and CD133 (PROM1), stemness-related genes OCT4, SOX2, inflammatory cytokines IL-1ß, IL-12, IL-18 and TNF-α and angiogenic factors Angiopoietin-1, Angiopoietin-2, bFGF and VEGF while decreased levels of epithelial gene CDH1 (E-cadherin) in comparison with mixed cell population. The genetic and secretome profiling of the CD44+ CSCs could serve as diagnostic and prognostic tools in the treatment of oral cancers.

1. Abstract: EMT should be provided in full name accompanied with the abbreviation.

Response: The same has been done and highlighted Epithelial Mesenchymal Transition

1. The author mentioned that “The data obtained were designated as the means ± standard deviations of the three independent experimental values.”. However, 5 OSCC were recruited. Therefore, it isn't very clear. For each figure, the repeated experiment number should be mentioned in each figure legend. How to deal with these 5 patient samples? Are they mixed for all experiments?

Response: We have added a line mentioning “Experiments were performed in triplicates for all five samples independently” in all figure legends. The samples were not mixed for any of the experiment and we have made additional changes in the materials and methods section for the clarification.

1. Figure 1 (proliferation curve) is based on 48 and 72 h experiments. However, the experiments for other figures were not mentioned. Please add this information.

Response: We have implemented the changes in all the materials and methods and added timepoints for all the experiments.

1. Why the sorted cells in culture-expanded cells had higher gene expressions than the sorted cells in freshly isolated cells? Please provide the discussion for this finding.

Response: This is for the first time we have obtained these results. In our future studies we will try to find out by utilizing more experimental modalities and a greater number of samples.

Minor comments:

1. The PCR program should be provided in section 2.5.

Response: Provided PCR program in the materials and methods section (Table 1).

1. “in vitro” should be typed in italic font.

Response: The same has been done and highlighted throughout the manuscript.

1. Figure 2 and Figure 4: For consistency, I suggest the author change the column color from black to white for the sorted cells. In that way, all sorted cell data will be shown in the white column, and unsorted data will be shown in black.

Response: We appreciate your suggestion. We have implemented the change in figure 2 and figure 4.

Reviewer 3:

In this manuscript, author characterized sorted cd44+ cells in term of proliferation, EMT, stemness and secretome. There are some spelling errors, extra blanks. author need rechecking text.

1. more detail information is needed for materials and methods 2.5 part, ie, qPCR machine and cycling conditions, etc

Response: Provided detailed information on RT-qPCR in materials and methods section.

1. The conclusion of 3.3 part is not very clearly. how many passages have cells been cultured/expanded? figure 3 is also similar with figure2D and 2E, while the relative expression to ACTB are different scale in figure 3 and figure2D-E. Could author comment on this?

Response: We have mentioned passage number. It was a calculation mistake while splitting the figure 3 into 2 parts and the scales changed while calculating gene expressions of SOX2 and OCT4 relative to ACTB. But, we removed figure 2D and 2E which were SOX2 and OCT4 gene expression analyses as they are already included in figure 3 so there is no meaning including them repeatedly.

1. in each figure, author should clarify how many patients the data are derived from.

Response: We have added a line mentioning “Experiments were performed in triplicates for all five samples independently” in all figure legends.

Questions from reviewer: how long can author culture the enriched cd44+ cells?

Response: Though we have used passage 4 CD44+ cells in this study, we were able to expand and sustain these cells in culture upto passage 23 from two samples. At late passages from passage 9, these cells started to change their morphology and very late passages, these cells started losing their surface adhesion characteristic.

Author Response File: Author Response.docx

Reviewer 3 Report

In this manuscript, author characterized sorted cd44+ cells in term of proliferation, EMT, stemness and secretome. There are some spelling errors, extra blanks. author need rechecking text.

1. more detail information is needed for materials and methods 2.5 part, ie, qPCR machine and cycling conditions, etc
2. The conclusion of 3.3 part is not very clearly. how many passages have cells been cultured/expanded? figure 3 is also similar with figure2D and 2E, while the relative expression to ACTB are different scale in figure 3 and figure2D-E. Could author comment on this?
3. in each figure, author should clarify how many patients the data are derived from.

Questions from reviewer: how long can author culture the enriched cd44+ cells?

Author Response

Reviewer 1:

The methodology of this paper is questionable.

• Whilst 5 tumour samples were used, the text makes it seem as if cells from all tumours were combined together before sorting. If this is the case, I do not think this paper is publishable.

Response: Given additional details on experiments which were performed independently for each individual sample and the data was added together. Moreover, we have performed statistical analysis by using paired two-sample t-test to combine the data and calculate p values.

• If the tumours had been sorted separately but the final results aggregated during analysis, these results must be separated out for each tumour. Clinical details for each of the tumours must also be provided in the manuscript. A larger number of samples is necessary for any kind of meaningful interpretation of the results. 

Response: Since this is the first time report, we performed the experimentation on a limited number of samples (n = 5). We will implement these experiments in a greater number of samples in our future studies. To perform statistical analysis and to give statistical significance, we have combined the results which were performed independently for all the five samples. For now, we hope that these results will definitely help in advancing the current research in the field of cancer stem cells and oral oncology. We have age range, gender, and history of tobacco abuse in the materials and methods section.

The language in this manuscript, whilst free of major errors, is convoluted and opaque. More clarity in writing is required. Also note that the very first word of the paper is not capitalised.

Response: the first letter has been capitalised Head and neck cancers (HNCs), 

Reviewer 2:

General comments:

The present study aimed to assess the association of CD44 with stemness-related, EMT-related genes and the secretome of the CSCs. Using 5 OSCC patients, they found that the sorted CD44+ CSCs expressed higher stemness and EMT-related genes, proliferation rate, inflammatory cytokines, and angiogenic factors than heterogeneous tumor cell populations. Therefore, the strategy to screen CSC by CD44+ for OSCC is promising.

Major comments:

1. Abstract: The authors mentioned too detailed descriptions for methods (line 14-20) but too few sentences for results. Moreover, the results may be written in more detail.

Response: The necessary changes have been done and highlighted

            The cancer stem cells were isolated by CD44+ selection using magnetic cell-sorting. The expression of CD44, proliferation rate, gene expression of EMT-related transcription factors, stemness markers, cytokine levels and angiogenic factors in both cell population was assessed. The sorted CD44+ cells showed significantly higher proliferation rate than heterogenous population. The CD 44 expression was >90% in the sorted cells which was higher than the heterogenous cells. The CD44+ CSCs cells demonstrated a significant increased levels of EMT-related genes TWIST1 and CDH2 (N-cadherin) , CSC-related genes CD44 and CD133 (PROM1), stemness-related genes OCT4, SOX2, inflammatory cytokines IL-1ß, IL-12, IL-18 and TNF-α and angiogenic factors Angiopoietin-1, Angiopoietin-2, bFGF and VEGF while decreased levels of epithelial gene CDH1 (E-cadherin) in comparison with mixed cell population. The genetic and secretome profiling of the CD44+ CSCs could serve as diagnostic and prognostic tools in the treatment of oral cancers.

1. Abstract: EMT should be provided in full name accompanied with the abbreviation.

Response: The same has been done and highlighted Epithelial Mesenchymal Transition

1. The author mentioned that “The data obtained were designated as the means ± standard deviations of the three independent experimental values.”. However, 5 OSCC were recruited. Therefore, it isn't very clear. For each figure, the repeated experiment number should be mentioned in each figure legend. How to deal with these 5 patient samples? Are they mixed for all experiments?

Response: We have added a line mentioning “Experiments were performed in triplicates for all five samples independently” in all figure legends. The samples were not mixed for any of the experiment and we have made additional changes in the materials and methods section for the clarification.

1. Figure 1 (proliferation curve) is based on 48 and 72 h experiments. However, the experiments for other figures were not mentioned. Please add this information.

Response: We have implemented the changes in all the materials and methods and added timepoints for all the experiments.

1. Why the sorted cells in culture-expanded cells had higher gene expressions than the sorted cells in freshly isolated cells? Please provide the discussion for this finding.

Response: This is for the first time we have obtained these results. In our future studies we will try to find out by utilizing more experimental modalities and a greater number of samples.

Minor comments:

1. The PCR program should be provided in section 2.5.

Response: Provided PCR program in the materials and methods section (Table 1).

1. “in vitro” should be typed in italic font.

Response: The same has been done and highlighted throughout the manuscript.

1. Figure 2 and Figure 4: For consistency, I suggest the author change the column color from black to white for the sorted cells. In that way, all sorted cell data will be shown in the white column, and unsorted data will be shown in black.

Response: We appreciate your suggestion. We have implemented the change in figure 2 and figure 4.

Reviewer 3:

In this manuscript, author characterized sorted cd44+ cells in term of proliferation, EMT, stemness and secretome. There are some spelling errors, extra blanks. author need rechecking text.

1. more detail information is needed for materials and methods 2.5 part, ie, qPCR machine and cycling conditions, etc

Response: Provided detailed information on RT-qPCR in materials and methods section.

1. The conclusion of 3.3 part is not very clearly. how many passages have cells been cultured/expanded? figure 3 is also similar with figure2D and 2E, while the relative expression to ACTB are different scale in figure 3 and figure2D-E. Could author comment on this?

Response: We have mentioned passage number. It was a calculation mistake while splitting the figure 3 into 2 parts and the scales changed while calculating gene expressions of SOX2 and OCT4 relative to ACTB. But, we removed figure 2D and 2E which were SOX2 and OCT4 gene expression analyses as they are already included in figure 3 so there is no meaning including them repeatedly.

1. in each figure, author should clarify how many patients the data are derived from.

Response: We have added a line mentioning “Experiments were performed in triplicates for all five samples independently” in all figure legends.

Questions from reviewer: how long can author culture the enriched cd44+ cells?

Response: Though we have used passage 4 CD44+ cells in this study, we were able to expand and sustain these cells in culture upto passage 23 from two samples. At late passages from passage 9, these cells started to change their morphology and very late passages, these cells started losing their surface adhesion characteristic.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Language remains convoluted. More direct phrasing of points raised in the introduction and discussion would be of benefit.

Whilst the text has now clarified that the 5 samples were indeed analysed separately, the changes made in the paper are not sufficient for me to change my decision. The expression levels for each gene in each sample needs to be represented on the plots for a more complete representation of data. Adding points to the boxplots is the common way of doing this (see https://0-bmcplantbiol-biomedcentral-com.brum.beds.ac.uk/articles/10.1186/s12870-019-1988-3/figures/3 for an example). 

Other

Please acknowledge in an acknowledgments section individuals who supported this work as well as funding sources - all experimental research has to receive funding/support from someone/some organisation and it is not reasonable to state that there is no funding source. (This would not apply were it a review article or a purely theoretical paper)

If any other person made substantial intellectual contributions to the work, please include them as a co-author.

Author Response

Reviewer 1:

1) Language remains convoluted. More direct phrasing of points raised in the introduction and discussion would be of benefit.

Response: As advised, the manuscript is subjected to a professional language editing service to reduce complex sentences.

2) Whilst the text has now clarified that the 5 samples were indeed analyzed separately, the changes made in the paper are not sufficient for me to change my decision. The expression levels for each gene in each sample need to be represented on the plots for a more complete representation of data. Adding points to the boxplots is the common way of doing this (see https://0-bmcplantbiol-biomedcentral-com.brum.beds.ac.uk/articles/10.1186/s12870-019-1988-3/figures/3 for an example). 

Response: Implemented the changes in all the graphs. The values for all 5 samples in triplicates are represented in the graphs.

3) Please acknowledge in an acknowledgments section individual who supported this work as well as funding sources - all experimental research has to receive funding/support from someone/some organisation and it is not reasonable to state that there is no funding source. (This would not apply were it a review article or a purely theoretical paper). If any other person made substantial intellectual contributions to the work, please include them as a co-author.

Response: The study was performed by me Dr.Shankargouda Patil, BDS, MDS (Oral & Maxillofacial Pathology), Ph.D., FRCPath, MFDS, RCPS (Glasgow), FICD, and FPFA. It was self-funded, meaning I paid for the study. This is not the first study I had performed on my own without any funding source as shown in the example below:

Shankargouda Patil. Metformin treatment decreases the expression of cancer stem cell marker CD44 and stemness related gene expression in primary oral cancer cells. Arch Oral Biol. 2020 May;113:104710. doi: 10.1016/j.archoralbio.2020.104710.

Reviewer 3:

It will be better if you can give more information (some staining pictures/phase contrast pictures) on cell culture, how sorted cells behave during culture. Otherwise, the manuscript is acceptable.

Response: We have included photomicrographs of the cells at different passages in figure 1.

At this stage we were unable to explain the changes occurring in CD44 cells during continuous culture, but we are working to find out why this is happening and what significant information this data will yield to know the cancer stem cells extensively at cellular and molecular level.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Author,

It will be better if you can give more information (some staining pictures/phase contrast pictures) on cell culture, how sorted cells behave during culture. Other wise the manuscript is acceptable.

best regards,

Yafeng

Author Response

Reviewer 1:

1) Language remains convoluted. More direct phrasing of points raised in the introduction and discussion would be of benefit.

Response: As advised, the manuscript is subjected to a professional language editing service to reduce complex sentences.

2) Whilst the text has now clarified that the 5 samples were indeed analyzed separately, the changes made in the paper are not sufficient for me to change my decision. The expression levels for each gene in each sample need to be represented on the plots for a more complete representation of data. Adding points to the boxplots is the common way of doing this (see https://0-bmcplantbiol-biomedcentral-com.brum.beds.ac.uk/articles/10.1186/s12870-019-1988-3/figures/3 for an example). 

Response: Implemented the changes in all the graphs. The values for all 5 samples in triplicates are represented in the graphs.

3) Please acknowledge in an acknowledgments section individual who supported this work as well as funding sources - all experimental research has to receive funding/support from someone/some organisation and it is not reasonable to state that there is no funding source. (This would not apply were it a review article or a purely theoretical paper). If any other person made substantial intellectual contributions to the work, please include them as a co-author.

Response: The study was performed by me Dr.Shankargouda Patil, BDS, MDS (Oral & Maxillofacial Pathology), Ph.D., FRCPath, MFDS, RCPS (Glasgow), FICD, and FPFA. It was self-funded, meaning I paid for the study. This is not the first study I had performed on my own without any funding source as shown in the example below:

Shankargouda Patil. Metformin treatment decreases the expression of cancer stem cell marker CD44 and stemness related gene expression in primary oral cancer cells. Arch Oral Biol. 2020 May;113:104710. doi: 10.1016/j.archoralbio.2020.104710.

Reviewer 3:

It will be better if you can give more information (some staining pictures/phase contrast pictures) on cell culture, how sorted cells behave during culture. Otherwise, the manuscript is acceptable.

Response: We have included photomicrographs of the cells at different passages in figure 1.

At this stage we were unable to explain the changes occurring in CD44 cells during continuous culture, but we are working to find out why this is happening and what significant information this data will yield to know the cancer stem cells extensively at cellular and molecular level.

Author Response File: Author Response.docx