A chronic signaling TGFb zebrafish reporter identifies immune response in melanoma

  1. Haley R. Noonan
  2. Julia Barbano
  3. Michael Xifaras
  4. Chloé S. Baron
  5. Song Yang
  6. Katherine Koczirka
  7. Alicia M. McConnell
  8. Leonard I. Zon

  • Curated by eLife

    eLife logo

    eLife assessment

    This study presents a valuable enhancer reporter of TGFb signaling in melanoma that has a conserved function in both human cell lines and zebrafish. The reporter data is solid and provides interesting insights into TGFb targets in melanoma. However, the model that macrophages preferentially phagocytose certain subsets of melanoma cells is still incomplete, and more data will be needed before this process is clearly understood.

Reviewed by

Read the full article See related articles

Listed in

Log in to save this article

Abstract

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein ( TIE:EGFP ). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single cell RNA- sequencing revealed that TIE:EGFP + melanoma cells down-regulated interferon response, while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2, a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in melanoma precursor zones and early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages are recruited to TIE:EGFP + regions and preferentially phagocytose TIE:EGFP + cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors.

Article activity feed

  1. eLife

    eLife assessment

    This study presents a valuable enhancer reporter of TGFb signaling in melanoma that has a conserved function in both human cell lines and zebrafish. The reporter data is solid and provides interesting insights into TGFb targets in melanoma. However, the model that macrophages preferentially phagocytose certain subsets of melanoma cells is still incomplete, and more data will be needed before this process is clearly understood.

  2. eLife

    Reviewer #1 (Public Review):

    This study by Noonan et al. explores the role of TGFb signaling in melanoma. TGFb signaling in melanoma and in the tumor microenvironment is complex, acting as both a tumor suppressor and tumor promoter, as well as an immune suppressor. The authors identified a human TGFb-responsive genomic regulatory element that is activated in TGFb-treated melanoma cell lines. This human genomic regulatory element also functions in a zebrafish melanoma model (TIE:EGFP) in specific regions of advanced melanoma. The enhancer region is bound by SMAD2/3, JunB, and ATF3. The proposed model that TIE:EGFP+ melanoma cells are preferentially phagocytosed by macrophages suggests there is some signal specific to this subset of melanoma cells. How this subset of melanoma cells is phagocytosed by macrophages is still poorly understood and will require further investigation. In addition, the authors found that SATB2 overexpression drives the early onset of the TIE:EGFP reporter in melanoma. This novel zebrafish TGFb reporter line has provided unique insights into the dynamic in vivo interactions between melanoma cells and the microenvironment, as well as immune cells. This study will be of interest to researchers looking for novel signaling mechanisms of melanoma progression.

  3. eLife

    Reviewer #2 (Public Review):

    This study combines molecular analysis of human melanoma cells with in vivo functional experiments in zebrafish. ChIP-seq analysis of A375 melanoma cells stimulated with TGFB revealed a TGFB enhancer. The human enhancer was a clone and a zebrafish transgenic line driving GFP by this enhancer (TIE:EGFP) revealed that TIE:EGFP was only expressed in late melanomas. TIE:EGFP+ cells showed downregulated IFN response but upregulation of novel chronic GHB target genes. AP-1 transcription factor is required for the activation of this enhancer. Expression of the chromatin remodeller SATB2 promoted activation of TIE:EGFP in early melanomas. Finally, in vivo imaging, flow cytometry and scRNA seq showed that macrophages preferentially phagocytosed TIE:EGFP+ melanoma cells.

    The identification of this novel TGFB enhancer is important since most studies focused on acute TGFB effects in melanoma. However, the present study identified a set of chronic TGFB target genes that may be relevant in melonoma and probably other tumors. Therefore, this study paves the way for future studies aiming at revealing the importance of this enhancer in different tumor histotypes and the novel identified chronic TGFB target genes.

    Most conclusions are supported by the data, with the exception of the ones related to macrophages that are not fully convincing.

  4. eLife

    Reviewer #3 (Public Review):

    Noonan et al. developed a clever reporter of TGFbeta signaling using human A375 melanoma cells to identify a TGFbeta-induced enhancer and generated a zebrafish transgenic line to monitor TGFbeta activation during the development of melanoma. They found that few discrete cells in advanced melanoma express the TIE:EGFP reporter, and used single-cell sequencing to identify differences in gene expression between these TGFbeta-responsive melanoma cells and the remaining population. They found that these cells downregulate interferon signaling and upregulate a gene signature compatible with chronic TGFbeta signaling that favours metastasis and requires AP-1 binding to regulatory elements of the target genes. Then they overexpressed SATB2, a known inducer of TGFbeta activation, in whole melanoma to increase the amount of TIE:EGFP positive cells for better characterization. Among the TIE:EGFP positive cells they retrieved a population of macrophages (Marco positive in single-cell analysis) and interpreted this observation as due to the phagocytic activity of macrophages that preferentially phagocytose TIE:EGFP positive melanoma cells. Since melanoma cells expressing TGFbeta upregulate a chronic TGFbeta signature that favours metastasis, downregulate interferon signaling, and are preferentially phagocytosed by macrophages that, as a consequence, turn on M2 markers (immunosuppressive), the authors conclude that this work highlights the need for the identification of a chronic TGFbeta biomarker signature to predict patient response to TGFbeta inhibitors.

    The conclusions of this paper on melanoma cells are mostly well supported by data, while the data concerning macrophages and their interpretation need strengthening with better images and additional data analysis.

  5. Version published to 10.1101/2022.09.29.510035v1 on bioRxiv