# Transcriptional mechanisms and melanoma

> **NIH NIH P01** · MASSACHUSETTS GENERAL HOSPITAL · 2024 · $698,985

## Abstract

ABSTRACT
The melanoma niche facilitates growth, differentiation, and drug resistance of cancer cells. Although many of the
cell types, including immune, endothelial, and stromal cells, have been characterized, the interactions of the
many signals that directly regulates these niche cells have not been evaluated in vivo. The niche cells also could
expand or alter their differentiation in response to the tumor cells or extracellular signals, and such changes
could trigger clonal expansion. We have utilized the zebrafish to study melanoma. Recently we developed
zebrafish reporters for the signaling pathways TGF
β
, Wnt, and Type I Interferons (IFN). Surprisingly, we see
certain regions of tumors that express the reporters and the Wnt and IFN reporters co-localize. Using a new
system to image tumors continuously for up to 24 hours, we noted small indentations of the surface of
melanomas that we termed “craters”. These regions contain CD8+ T cells that are both activated and exhausted
with dendritic cells. These immune hubs function as a conduit for T cells to enter tumors. We have found craters
also in human melanoma samples. In zebrafish melanomas, we specifically found Wnt positive macrophages
localized to craters. We plan to probe the function of the Wnt positive macrophages and to examine the role of
Wnt in the craters. Macrophages also phagocytose TGFβ-signaled and some Wnt-signaling melanoma cells. We
plan to probe the “eat-me” signals that are involved. We recently have examined clonality using a variety of
cellular barcoding approaches in zebrafish and human tumors. In the zebrafish, we have a Brainbow approach
that uses colors to examine clonality and a CRISPR based DNA barcode system called GESTALT. Using single
cell analysis, we found that the cancer associated fibroblasts secrete high IL-8 levels. Clonal analyses of tumors
overexpressing IL-8 revealed that IL8 mediates endothelial cell clonal expansion which is associated with a
decrease of CD8+T cell infiltration. We have used mitochondrial barcoding to examine single cell clonality, gene
expression and chromatin accessibility in human melanomas. We plan to compare zebrafish and human tumors,
examining spatial transcriptomics and multiplex imaging, coupled with clonality. The clonality of the blood and
immune cells in melanoma will also be assessed, independent of the TCR sequencing. Our studies should shed
light on how multiple signals in cancer niche intersect and function and will relate these effects to clonal growth.
This work with have impact on the choice of primary therapies or when drug resistance occurs.

## Key facts

- **NIH application ID:** 10933666
- **Project number:** 2P01CA163222-11
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** LEONARD Ira ZON
- **Activity code:** P01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $698,985
- **Award type:** 2
- **Project period:** 2013-03-12 → 2029-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10933666

## Citation

> US National Institutes of Health, RePORTER application 10933666, Transcriptional mechanisms and melanoma (2P01CA163222-11). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10933666. Licensed CC0.

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