Mapping the single-cell landscape of acral melanoma and analysis of the molecular regulatory network of the tumor microenvironments
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Evaluation Summary:
He and collaborators analyse eight samples from six patients with acral melanoma through single-cell RNA sequencing. They describe the tumour microenvironment in these tumours, including descriptions of interactions among distinct cell types and potential biomarkers. The study is thoroughly done. In its final form, this study will help to inform our knowledge of the immune infiltration on the poorly studied acral melanoma subtype, the most common type of the disease in several countries in Africa, Asia and Latin America.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Acral melanoma (AM) exhibits a high incidence in Asian patients with melanoma, and it is not well treated with immunotherapy. However, little attention has been paid to the characteristics of the immune microenvironment in AM. Therefore, in this study, we collected clinical samples from Chinese patients with AM and conducted single-cell RNA sequencing to analyze the heterogeneity of its tumor microenvironments (TMEs) and the molecular regulatory network. Our analysis revealed that genes, such as TWIST1 , EREG , TNFRSF9 , and CTGF could drive the deregulation of various TME components. The molecular interaction relationships between TME cells, such as MIF-CD44 and TNFSF9-TNFRSF9, might be an attractive target for developing novel immunotherapeutic agents.
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Author Response
Reviewer #1 (Public Review):
In this study, He and collaborators analyse eight samples from six patients with acral melanoma through single-cell RNA sequencing. They describe the tumour microenvironment in these tumours, including descriptions of interactions among distinct cell types and potential biomarkers. I believe the work is thoroughly done, but I have identified a few concerns in their depiction and interpretation of their results.
Strengths:
- One of the few available single-cell studies of acral melanoma, including a non-European cohort of patients.
- Data will be very useful to study the immune landscape of these rare tumours.
- Data include adjacent tissue, primary tumours and a metastatic sample, covering all disease stages.
- Analyses seem to be carefully done.
Things to improve:
- Figures need much …
Author Response
Reviewer #1 (Public Review):
In this study, He and collaborators analyse eight samples from six patients with acral melanoma through single-cell RNA sequencing. They describe the tumour microenvironment in these tumours, including descriptions of interactions among distinct cell types and potential biomarkers. I believe the work is thoroughly done, but I have identified a few concerns in their depiction and interpretation of their results.
Strengths:
- One of the few available single-cell studies of acral melanoma, including a non-European cohort of patients.
- Data will be very useful to study the immune landscape of these rare tumours.
- Data include adjacent tissue, primary tumours and a metastatic sample, covering all disease stages.
- Analyses seem to be carefully done.
Things to improve:
- Figures need much more description to be understandable, in particular, axes should be clearly labeled and the colour code should be specified
Thank you for your generous comments and suggestions. We have improved the integrity of some figures and added some figure legends. I believe this will further improve the quality of our manuscript.
- In some places, I would recommend the authors soften their interpretation of their analyses (for example, when they suggest targeting TNFRSF9+ T cells as a novel therapy), as these are nearly all bioinformatic in a small number of samples
As for the conclusions of TNFRSF9, we indeed provided a possibility that TNFRSF9 may serve as a novel therapy. We made some changes to soften the statement. In addition, we have added instructions and explanations in the Discussion section.
- I don't think the experiments add much to the literature, as these test already known oncogenes on a common, non-acral melanoma cell line. Thanks for your comments regarding the experiments included in our study. We have pointed out this deficiency in the Discussion section, and made some experimental changes. For example, we have removed the TWIST1-related experiments from the main Results section and shown them only as non-focus work in the Supplementary Figure.
It is difficult for us to obtain AM cell lines. No commercial AM cell lines can be purchased in ATCC or ECACC. AM cell lines are more difficult to establish and there are few reports on methods for establishing primary acral melanoma cell cultures (PMID: 22578220, PMID: 17488338). Some Japanese and Chinese researchers have isolated the primary generation of AM cells (e.g., PMID: 17488338, PMID: 22578220, PMID: 34097822), but due to the customs policy and the COVID-19 epidemic, we could not receive them within a short period. Moreover, these studies also stated their limitations; namely, that the stability during serial passaging had not been evaluated. Therefore, it may be very time-consuming to obtain operable AM cell lines for functional assays. However, our research group would like to have the opportunity to separate and culture primary cells in subsequent studies, and improve relevant experiments according to your valuable suggestions. Man thanks again for your comments.
Reviewer #2 (Public Review):
The study presented by Zan He et al dissects the main interactions between malignant and stromal cells present in acral melanoma samples and in adjacent tissues using single cell RNA sequencing. The study describes factors that allow communication between the different cell types, with a special focus on macrophages, lymphocytes and fibroblasts, along with malignant cells. Factors playing a role in cell-cell communication are identified and suggested to be relevant prognostic makers and/or attractive therapeutic targets.
Historically, the study of acral melanomas has been neglected due to the low incidence among Europeandescents and this formed an important gap of knowledge in the field and hindered the development of effective therapies to control the disease. Therefore, studies that address this unmet need in melanoma research are very important and should be motivated. This includes singlecell sequencing studies that allow one to study the complexity of tumours, including microenvironment features that influence the development and effectiveness of certain types of treatment. The present study contributes information on how cells interact in the acral melanoma microenvironment and this could be a first step toward better understanding how these interactions influence acral melanoma development, progression, and therapy response.
However, there are a few points that should be carefully considered. The authors use 3 adjacent tissues (which in theory is composed of normal skin next to a cancer lesion), 4 primary tumor samples, and one lymph node metastasis as a model to study tumor progression. Adjacent tissue is not considered a stage of tumour progression and the sample size is too small to rule out sample-dependent effects. The study is descriptive in nature and could better contextualize the findings regarding what is known for other subtypes of melanomas or other tumours. This is especially important to help readers understand why it would be relevant to study cutaneous melanomas located in acral skin. It would be helpful to explain how different it is from nonacral cutaneous melanoma, and what this study adds compared to other single-cell studies from cutaneous acral and non-acral melanomas.
Thank you for your generous comments. It is not accurate to represent the adjacent tissue samples as ‘tumour progression’, and our study did not want to focus on the tumour developmental process. We have revised related description in the text. Tumour adjacent tissues (ATs) have always been the focus of research on TMEs. Some studies believe that there are a lot of mutations and clone amplification in normal tissues adjacent to cancer, which may be in a pre-cancerous state (PMID: 33004515), and many single-cell studies of tumours have also sampled and paired para-cancer tissues (e.g., PMID: 29988129; PMID: 35303421).
The problem of sample size limits the generality of the results, as we pointed out in the Discussion section. Most acral melanoma (AM) patients opt for surgical resection at an early stage to avoid the possibility of metastasis. Hence, we rarely encounter patients with lymph gland (LG) metastases. We only collected one metastatic sample, because it is very rare in clinic. However, the sample has a high quality, such as a high cell activity of single cell suspension after dissociation (95.30%), and a rich amount of tumour cells and other stroma cells. Therefore, we added its sequencing data into the overall analyses, hoping to contribute to the comprehensiveness of resources and research.
It is important to link this study with the findings regarding what is known for other subtypes of melanomas. We have already supplied the comparison of AMs with non-acral skin cutaneous melanomas (CMs), using the published data. Your comments and advices are entirely helpful to us, and we believe that the current manuscript is more comprehensive and complete.
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Evaluation Summary:
He and collaborators analyse eight samples from six patients with acral melanoma through single-cell RNA sequencing. They describe the tumour microenvironment in these tumours, including descriptions of interactions among distinct cell types and potential biomarkers. The study is thoroughly done. In its final form, this study will help to inform our knowledge of the immune infiltration on the poorly studied acral melanoma subtype, the most common type of the disease in several countries in Africa, Asia and Latin America.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
In this study, He and collaborators analyse eight samples from six patients with acral melanoma through single-cell RNA sequencing. They describe the tumour microenvironment in these tumours, including descriptions of interactions among distinct cell types and potential biomarkers. I believe the work is thoroughly done, but I have identified a few concerns in their depiction and interpretation of their results.
Strengths:
1. One of the few available single-cell studies of acral melanoma, including a non-European cohort of patients.
2. Data will be very useful to study the immune landscape of these rare tumours.
3. Data include adjacent tissue, primary tumours and a metastatic sample, covering all disease stages.
4. Analyses seem to be carefully done.Things to improve:
1. Figures need much more description …
Reviewer #1 (Public Review):
In this study, He and collaborators analyse eight samples from six patients with acral melanoma through single-cell RNA sequencing. They describe the tumour microenvironment in these tumours, including descriptions of interactions among distinct cell types and potential biomarkers. I believe the work is thoroughly done, but I have identified a few concerns in their depiction and interpretation of their results.
Strengths:
1. One of the few available single-cell studies of acral melanoma, including a non-European cohort of patients.
2. Data will be very useful to study the immune landscape of these rare tumours.
3. Data include adjacent tissue, primary tumours and a metastatic sample, covering all disease stages.
4. Analyses seem to be carefully done.Things to improve:
1. Figures need much more description to be understandable, in particular, axes should be clearly labeled and the colour code should be specified
2. In some places, I would recommend the authors soften their interpretation of their analyses (for example, when they suggest targeting TNFRSF9+ T cells as a novel therapy), as these are nearly all bioinformatic in a small number of samples
3. I don't think the experiments add much to the literature, as these test already known oncogenes on a common, non-acral melanoma cell line. -
Reviewer #2 (Public Review):
The study presented by Zan He et al dissects the main interactions between malignant and stromal cells present in acral melanoma samples and in adjacent tissues using single cell RNA sequencing. The study describes factors that allow communication between the different cell types, with a special focus on macrophages, lymphocytes and fibroblasts, along with malignant cells. Factors playing a role in cell-cell communication are identified and suggested to be relevant prognostic makers and/or attractive therapeutic targets.
Historically, the study of acral melanomas has been neglected due to the low incidence among European-descents and this formed an important gap of knowledge in the field and hindered the development of effective therapies to control the disease. Therefore, studies that address this unmet …
Reviewer #2 (Public Review):
The study presented by Zan He et al dissects the main interactions between malignant and stromal cells present in acral melanoma samples and in adjacent tissues using single cell RNA sequencing. The study describes factors that allow communication between the different cell types, with a special focus on macrophages, lymphocytes and fibroblasts, along with malignant cells. Factors playing a role in cell-cell communication are identified and suggested to be relevant prognostic makers and/or attractive therapeutic targets.
Historically, the study of acral melanomas has been neglected due to the low incidence among European-descents and this formed an important gap of knowledge in the field and hindered the development of effective therapies to control the disease. Therefore, studies that address this unmet need in melanoma research are very important and should be motivated. This includes single-cell sequencing studies that allow one to study the complexity of tumours, including microenvironment features that influence the development and effectiveness of certain types of treatment. The present study contributes information on how cells interact in the acral melanoma microenvironment and this could be a first step toward better understanding how these interactions influence acral melanoma development, progression, and therapy response.
However, there are a few points that should be carefully considered. The authors use 3 adjacent tissues (which in theory is composed of normal skin next to a cancer lesion), 4 primary tumor samples, and one lymph node metastasis as a model to study tumor progression. Adjacent tissue is not considered a stage of tumour progression and the sample size is too small to rule out sample-dependent effects. The study is descriptive in nature and could better contextualize the findings regarding what is known for other subtypes of melanomas or other tumours. This is especially important to help readers understand why it would be relevant to study cutaneous melanomas located in acral skin. It would be helpful to explain how different it is from non-acral cutaneous melanoma, and what this study adds compared to other single-cell studies from cutaneous acral and non-acral melanomas.
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