Oncogenic RAS drives rapid onset cellular plasticity and elicits a tumour-promoting neutrophil response at the inception of preneoplastic development

To date, very little is known regarding the earliest cellular and molecular events within the initial preneoplastic cell (PNC) niche that might determine the outcome of PNC development. Here, we combine in vivo live imaging and single cell transcriptomics to investigate the immediate cellular and molecular changes that occur in PNCs and responding innate immune cells within the first 24 hours following tumour initiation. We do so using an inducible transgenic zebrafish model of tumour initiation, in which oncogenic HRAS is conditionally expressed within the zebrafish epidermis. We found that, when expressed within basal keratinocytes, oncogenic HRAS was sufficient to drive several cancer hallmarks in nascent PNCs and their niche. Within 8 hours of oncogenic HRAS induction, basal keratinocytes began to dedifferentiate, followed by a bifurcation in PNC fate towards either a cellular state characterised by partial Epithelial-Mesenchymal-Transition (pEMT) and cancer stem cell features or a more differentiated cell state resembling suprabasal keratinocytes. By directly integrating data from PNCs and human SCC patients we show that both of these PNC states likely persist with established tumours, whilst the pEMT state corresponded with a more aggressive phenotype. Strikingly, markers of the PNC subpopulation that underwent pEMT correlated with poor prognosis in multiple human cancer types in the TCGA database. In addition, the pEMT PNC subpopulation expressed higher levels of cytokines that modulate neutrophil development and function, suggesting a major role for these cells in governing pro-tumour neutrophil responses. Indeed, we found that granulopoiesis was altered within PNC-bearing larvae, with a large proportion of neutrophils switching to an Arginase 2 positive phenotype that functioned to promote PNC proliferation. Similar to tumour associated neutrophils in the mammalian system, the cxcr1/2-Il8 axis is involved in mediating the PNC-promoting function of neutrophils in our model. Thus, oncogenic RAS, when activated in the permissive cellular environment is sufficient to drive enabling cancer hallmarks, such as cellular plasticity and inflammation from the inception of the preneoplastic stage of cancer development. Furthermore, these same features may persist to drive malignant progression. ### Competing Interest Statement The authors have declared no competing interest.