Cell Type-Specific Transcriptome Profiling Reveals a Role for Thioredoxin During Tumor Initiation.

Abstract

Neutrophils in the tumor microenvironment exhibit altered functions. However, the changes in neutrophil behavior during tumor initiation remain unclear. Here we used Translating Ribosomal Affinity Purification (TRAP) and RNA sequencing to identify neutrophil, macrophage and transformed epithelial cell transcriptional changes induced by oncogenic RasG12V in larval zebrafish. We found that transformed epithelial cells and neutrophils, but not macrophages, had significant changes in gene expression in larval zebrafish. Interestingly, neutrophils had more significantly down-regulated genes, whereas gene expression was primarily upregulated in transformed epithelial cells. The antioxidant, thioredoxin (txn), a small thiol that regulates reduction-oxidation (redox) balance, was upregulated in transformed keratinocytes and neutrophils in response to oncogenic Ras. To determine the role of thioredoxin during tumor initiation, we generated a zebrafish thioredoxin mutant. We observed an increase in wound-induced reactive oxygen species signaling and neutrophil recruitment in thioredoxin-deficient zebrafish. Transformed keratinocytes also showed increased proliferation and reduced apoptosis in thioredoxin-deficient larvae. Using live imaging, we visualized neutrophil behavior near transformed cells and found increased neutrophil recruitment and altered motility dynamics. Finally, in the absence of neutrophils, transformed keratinocytes no longer exhibited increased proliferation in thioredoxin mutants. Taken together, our findings demonstrate that tumor initiation induces changes in neutrophil gene expression and behavior that can impact proliferation of transformed cells in the early tumor microenvironment.