Live Imaging and Gene Expression Analysis in Zebrafish Identifies a Link between Neutrophils and Epithelial to Mesenchymal Transition

Abstract

Chronic inflammation is associated with epithelial to mesenchymal transition (EMT) and cancer progression however the relationship between inflammation and EMT remains unclear. Here, we have exploited zebrafish to visualize and quantify the earliest events during epithelial cell transformation induced by oncogenic HRasV12. Live imaging revealed that expression of HRasV12 in the epidermis results in EMT and chronic neutrophil and macrophage infiltration. We have developed an in vivo system to probe and quantify gene expression changes specifically in transformed cells from chimeric zebrafish expressing oncogenic HRasV12 using translating ribosomal affinity purification (TRAP). We found that the expression of genes associated with EMT, including slug, vimentin and mmp9, are enriched in HRasV12 transformed epithelial cells and that this enrichment requires the presence of neutrophils. An early signal induced by HRasV12 in epithelial cells is the expression of il-8 (cxcl8) and we found that the chemokine receptor, Cxcr2, mediates neutrophil but not macrophage recruitment to the transformed cells. Surprisingly, we also found a cell autonomous role for Cxcr2 signaling in transformed cells for both neutrophil recruitment and EMT related gene expression associated with Ras transformation. Taken together, these findings implicate both autocrine and paracrine signaling through Cxcr2 in the regulation of inflammation and gene expression in transformed epithelial cells.

EMT, CSCs, and drug resistance: the mechanistic link and clinical implications

“…Intriguingly, such pro-tumour effects of these cells are also attributable, in part, to their capacity for activating EMT programmes within neighbouring cancer cells 121,122 . Hence, various secreted proteins and host cell types present in the inflammatory tumour microenvironment contribute, perhaps in a coordinated fashion, to the induction of EMT in carcinoma cells.…”

Imaging innate immune responses at tumour initiation: new insights from fish and flies

“…Also, because of their genetic tractability, engineering of appropriate lines and combinatorial crossing of these various lines is considerably easier than for mouse models. In larval zebrafish studies, if any one of a variety of cell lineages (melanoblasts, goblet cells or keratinocytes) is forced to express transgenic constitutively active human mutant HRAS G12V , then these mutant cells will be rapidly 'sensed' in an otherwise wild-type host epithelium 4,5 . Zebrafish larvae have a similar repertoire of innate immune cell lineages to mammals, including neutrophils and macrophages 6 , and both of these can be drawn to, and interact with, pre-neoplastic cells while they are still only single cells, or at the latest when they have divided only once or twice 4 .…”

“…These periods of attention may co incide with the duration of stochastic pulses of attractant (as discussed below). Just as in a wound repair scenario, macrophages arrive second but it may not be necessary for pre-neoplastic cells to have first been 'discovered' by neutrophils because blocking neutrophil recruitment does not substantially suppress recruitment of macrophages to clones of pre-neoplastic keratinocytes 5 . Once a clone of pre-neoplastic goblet or melanoblastic cells reaches >20 cells then inflammation becomes 'chronic' and neutrophils appear to remain 4 (FIG.…”

“…Furthermore, expression profiling studies in various late-stage human cancers have revealed induction of chemokines and cytokines 26 , and these might similarly be upregulated in early-stage pre-neoplastic cells. Indeed, a recent study in zebrafish larvae shows that CXCL8 is expressed by HRAS G12V -expressing epidermal cells, and this must be a major component of the immune cell attractant signal because global knockdown of its receptor, CXC chemokine receptor type 2 (CXCR2), leads to a significant disruption of neutrophil recruitment to these pre-neoplastic cell clones 5 . A similar oncogenic lesion in cells from a different tissue origin might release distinct immune cell attractants.…”

Neutrophils, Wounds, and Cancer Progression

“…Expression of oncogenic RasG12V in zebrafish skin melanocytes or epidermal cells induces cell transformation and recruitment of neutrophils, which drive cell proliferation (Feng et al, 2010) and expression of EMT markers (Freisinger and Huttenlocher, 2014). In the current study, Antonio et al first demonstrate that chronic tissue damage drives cancer progression; repeatedly wounding adult zebrafish expressing RasG12V in melanocytes significantly increased tumor formation.…”