The zebrafish as a model for T cell recruitment to melanoma
Aya Ludin1, John Beausang2, Derek Croote2, Song Yang3, Yi Zhou3, Stephan R. Quake2 and Leonard I. Zon1,3,4*
1Department of Stem Cell and Regenerative Biology, Harvard stem cell institute, Harvard University, Cambridge MA; 2Department of Bioengineering and Howard Hughes Medical Institute, Stanford University, Stanford CA; 3Stem Cell Program and Division of Haematology/Oncology, Boston Children's Hospital and Dana-Farber Cancer Institute Boston MA; 4Howard Hughes Medical Institute, Boston MA
Immunotherapy shows great promise in melanoma treatment. However, mechanisms of T cell recruitment to tumors are not fully known. The zebrafish model allows high throughput screens, genomic manipulation and in vivo imaging in relative ease. Melanoma is well modeled in zebrafish. To image T cell recruitment into zebrafish melanoma, we labelled tumors with mCherry driven by the ubiquitin promoter and placed them in a dorsal location on the skin of an adult zebrafish expressing the T cell specific lck promoter driving GFP. 3-5 weeks later, the tumor was imaged using a 2-photon confocal microscope. This revealed that T cells occupy a position on the rim of the tumor. These T cells and T cell derived from zebrafish lymphoid organs were characterized using single cell and bulk RNA Seq and ATAC seq. We examined T cell markers, signaling genes, chemokines, cytokines and transcription factors expression and found high similarity between T cell populations of zebrafish and mammals. The fish thymus harbors immature, developing thymocytes while the kidney marrow presents active T cells with characteristics similar to mammalian cells. Single cell RNA Seq of tumor derived T cells revealed CD8 and CD4 populations which presented mature, active profile of migrating cells rather than tissue resident. Interestingly, granzyme genes expression was low in the CD8 population, and IL-16 expression was high among the CD4 population, both implicated in cancer microenvironment. Our findings highlight the zebrafish as a reliable tool with powerful characteristics in the field of cancer immunology to further screen for factors that will improve immunotherapy outcome.
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