Single cell gene expression profiling of prenoplastic cells and myeloid cells during tumour initiation wihin a larval zebrafish model of skin oncogenis driven by inducible HRASG12V

Tumour initiation begins with a single oncogenic mutation within a single cell, leading to hyperproliferation and clonal expansion. These mutant preneoplastic cells may undergo various fates such as: death, dormancy, benign growth or malignant growth. Whilst further genetic mutations have been linked to progression of preneoplastic cells, even genetically identical preneoplastic cells may undergo different fates. To better understand the phenotypic heterogeneity between genetically identical preneoplastic cells during tumour initiation we have performed single-cell RNA sequencing on preneoplastic cells derived from an inducible larval zebrafish model of skin cancer. Furthermore, inflammation has been identified as a potent driving force in oncogenesis. Macrophages have especially been shown to promote cancer cell growth and progression, whilst neutrophils have the potential to perform either pro- or anti-tumour functions. To study the first responses of myeloid cells following initial oncogenic transformation we also profiled sorted myeloid cells at the single cell level. Preneoplastic cells and myeloid cells were sorted by FACS from transgenic zebrafish larvae, in which the human oncogene HRASG12V is expressed specifically within epidermal keratinocytes following induction with 4OH-Tamoxifen. Induction was carried out at 3dpf. To determine how these preneoplastic cells develop over time within their first day of oncogenic transformation we profiled cells at both 8 and 24 hours post induction. As a control, we also sorted keratinocytes and myeloid cells from time-matched larvae which do not express HRASG12V. Preneoplastic cells and keratinocytes were identified by mCherry fluorescence, whilst myeloid cells were identified by EGFP fluorescence from the following larvae: Tg(krtt1c19e:KALTA44-ERT2; UAS:mCherry-HRASG12V;mpeg1.1:EGFP) and Tg(krtt1c19e:KALTA44-ERT2; UAS:mCherry-CAAX;mpeg1.1:EGFP).