Live imaging and gene expression analysis in zebrafish identifies a link between neutrophils and epithelial to mesenchymal transition.

Christina M Freisinger,Anna Huttenlocher,Olivier De Wever

doi:10.1371/journal.pone.0112183

Christina M Freisinger, Anna Huttenlocher + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0112183

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Journal: PloS one	Publication Date: Nov 5, 2014
Citations: 54	License type: CC BY 4.0

Affiliation: University of Wisconsin–Madison

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.

Highlights

Epithelial to mesenchymal transition (EMT) is essential for normal embryonic development, and occurs during wound healing and the invasion of transformed cells during cancer progression [1,2]
We further show that both Cxcr2 in transformed cells and the presence of neutrophils, but not macrophages, in the microenvironment are required for expression of EMT-related genes
Microinjection of the krt4 morpholino oligonucleotide (MO) inhibited GFP expression in krt4:GFP transgenic larvae (Figure 1D) and reduced transgene expression in 24 hpf embryos injected with Tol2 flanked:krt4: RFP-HRasV12 (Figure 1E)

Summary

Introduction

Epithelial to mesenchymal transition (EMT) is essential for normal embryonic development, and occurs during wound healing and the invasion of transformed cells during cancer progression [1,2]. Chronic inflammation has been linked to EMT and cancer invasion [10,11,12], it is not clear how innate immune inflammation is associated with EMT and participates in cancer progression. Understanding how transformed cells induce chronic innate immune inflammation in tissues and if this inflammation plays a role in EMT progression has been limited due to the difficulty visualizing the early tissue microenvironment around small clusters of transformed epithelial cells in live animals. Chronic inflammation of the epidermis in zebrafish has been associated with the activation of EMT programs and developmental defects in epidermal homeostasis, further supporting the use of zebrafish to study the relationship between inflammation and EMT [10,20]

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