Restraint of melanoma progression by cells in the local skin environment

Reviewer #1 (Public review):

Summary:

Ma et al. show that melanoma cells induce an EMT-like state in nearby keratinocytes and that when this state is induced experimentally by Twist-overexpression the resulting alteration in keratinocytes is inhibitory for melanoma invasion. These conclusions are based on experiments in vivo with zebrafish and, in vitro, with human cells. The work is carefully done and provides new insights into the interactions between melanoma cells and their environment.

Strengths:

The use of both zebrafish and human cells adds confidence that findings are relevant to human melanomas while also further demonstrating the utility of the zebrafish system for discovering important new features of melanoma biology that could ultimately have clinical impacts. The work also combines a nice suite of approaches including different models for induced melanomagenesis in zebrafish, single-cell RNA-sequencing, and more. Some of the final observations are intriguing as well, especially the possibility of EMT-induced melanocyte-keratinocyte interactions via Jam3 expression; it will be interesting to see if this is indeed a mechanism for restraining melanoma invasion. The paper is clearly written and the inferences are appropriate for the results obtained. Overall the work makes a solid contribution to our understanding of important, but too often neglected, roles of the tumor microenvironment in promoting or inhibiting tumor progression and outcome.

Weaknesses:

No critical weaknesses were noted.

Reviewer #2 (Public review):

Summary:

The manuscript by Ma et. al. utilizes a zebrafish melanoma model, single-cell RNA sequencing (scRNA-seq), a mammalian in vitro co-culture system, and quantitative PCR (Q-PCR) gene expression analysis to investigate the role keratinocytes might play within the melanoma microenvironment. Convincing evidence is presented from scRNA-seq analysis showing that a small cluster of melanoma-associated keratinocytes upregulates the master EMT regulator, transcription factor, Twist1a. To investigate how Twist-expressing keratinocytes might influence melanoma development, the authors use an in vivo zebrafish model to induce melanoma initiation while overexpressing Twist in keratinocytes through somatic transgene expression. This approach reveals that Twist overexpression in keratinocytes suppresses invasive melanoma growth. Using a complementary in vitro human cell line co-culture model, the authors demonstrate reduced migration of melanoma cells into the keratinocyte monolayer when keratinocytes overexpress Twist. Further scRNA-seq analysis of zebrafish melanoma tissues reveals that in the presence of Twist-expressing keratinocytes, subpopulations of melanoma cells show altered gene expression, with one unique melanoma cell cluster appearing more terminally differentiated. Finally, the authors use computational methods to predict putative receptor-ligand pairs that might mediate the interaction between Twist-expressing keratinocytes and melanoma cells.

Strengths:

The scRNA-seq approach reveals a small proportion of keratinocytes undergoing EMT within melanoma tissue. The use of a zebrafish somatic transgenic model to study melanoma initiation and progression provides an opportunity to manipulate host cells within the melanoma microenvironment and evaluate their impact on tumour progression. Solid data demonstrate that Twist-expressing keratinocytes can constrain melanoma invasive development in vivo and reduce melanoma cell migration in vitro, establishing that Twist-overexpressing keratinocytes can suppress at least one aspect of tumour progression.

Weaknesses:

While the scRNA-seq analysis of melanoma tissue and RT-PCR analysis of EMT gene expression in isolated keratinocytes provide evidence that a subpopulation of host keratinocytes upregulates Twist and other EMT marker genes and potentially undergoes EMT, the in vivo evidence for keratinocyte EMT within the melanoma microenvironment is based on cell morphology in a single image without detailed characterization and quantification. No EMT marker gene expression was examined in melanoma tissue sections to determine the proportion and localization of Twist+ve keratinocytes within the melanoma microenvironment.

The scRNA-seq UMAP suggests the proportion of EMT keratinocytes within the melanoma microenvironment is very small, raising questions about their precise location and significance within the tumour microenvironment. Although both in vivo and in vitro evidence demonstrates that Twist-expressing keratinocytes can suppress melanoma progression, the conditions modelled by the authors involve over-expression of Twist in all keratinocytes, which do not naturally occur within the melanoma microenvironment and, therefore, might not be relevant to naturally occurring melanoma progression. The author did not test whether blocking EMT through down-regulation of Twist in keratinocytes may influence melanoma development, which would establish the role of Twist expression keratinocytes in the melanoma microenvironment.

To address the potential mechanism by which Twist-expressing keratinocytes suppress melanoma progression, a second scRNA-seq analysis was conducted. However, this analysis is not adequately presented to provide strong evidence for proposed mechanisms for how Twist-expressing keratinocytes suppress melanoma cell invasion. CellChat analysis was used to attempt to identify receptor-ligand pairs that might mediate keratinocyte-melanoma cell interaction, but the interactions between tumour-associated keratinocytes (TAK) and melanoma cells were not included in the analysis. Furthermore, although genetic reporters were used to label both keratinocytes and melanoma cells, no images showing the detailed distribution and positional information of these cells within melanoma tissue are presented in the report. None of the gene expression changes detected through Q-PCR or scRNA-seq were validated using immunostaining or in situ hybridization.

Overall, the data presented in this report draw attention to a less-studied host cell type within the tumour microenvironment, the keratinocytes, which, similar to well-studied immune cells and fibroblasts, could play important roles in either promoting or constraining melanoma development.

Counterintuitively, the authors show that Twist-expressing EMT keratinocytes can constrain melanoma progression. While the detailed mechanisms remain to be uncovered, this is an interesting observation.

Reviewer #3 (Public review):

Summary:

In this study the authors use the zebrafish model and in vitro co-cultures with human cell lines, to study how keratinocytes modulate the early stages of melanoma development/migration. The authors demonstrate that keratinocytes undergo an EMT-like transformation in the presence of melanoma cells which leads to a reduction in melanoma cell migration. This EMT transformation occurs via Twist; and resulted in an improvement in OS in zebrafish melanoma models. Authors suggest that the limitation of melanoma cell migration by Twist-overexpressing keratinocytes was through altered cell-cell interactions (Jam3b) that caused a physical blockage of melanoma cell migration.

Strengths:

The authors describe a new cross-talk between melanoma and its major initial microenvironment: the keratinocytes and how instructed by melanoma cells keratinocytes undergo an EMT transformation, which then controls melanoma migration.

Overall, the paper is very well written, and the results are clearly organized and presented.

Weaknesses:

(1) To really show their last point it would be important to CRISPR KO Jam3b in melanoma with twist OE keratinocytes, in vivo or in vitro.

(2) The use of patient biopsies from early-stage melanomas vs healthy tissue to assess if there is a similar alteration of morphology of adjacent keratinocytes and an increase in vimentin in human samples would strengthen the author's findings.

(3) The cell-cell junctions and borders between cells (melanoma/ keratinocytes) should be characterized better, with cellular and sub-cellular resolution. Since melanocytes can "touch" with their dendrites ~40 keratinocytes - can authors expand and explain better their model? Can this explain that in some images we cannot observe a direct interface between the cells?