Investigating the role of endoplasmic reticulum stress in neutrophils during tumour initiation

Abstract

Neutrophils are pivotal innate immune cells conventionally recognised for their rapid response to infection sites. In recent years, immune suppressive and tumourpromoting neutrophils were found enriched in the tumour microenvironment, suggesting their role in driving the development of cancer. However, the role of neutrophils during tumour initiation is less clear. Studies in zebrafish tumour initiation models suggest that neutrophils might play a trophic role in promoting early tumour cell proliferation. However, the underlying mechanism remains to be determined.

To elucidate the mechanisms associated with this trophic function during tumour initiation, the Feng lab employed single-cell RNA sequencing analysis to capture all neutrophils in preneoplastic cell-carrying larvae to compare that with neutrophils from control larvae. This revealed a list of upregulated genes linked to endoplasmic reticulum (ER) stress. I hypothesize a potential connection in mechanisms between ER stress and the trophic function of neutrophils associated with PNCs.

In this project, I established an ER stress reporter zebrafish line Tg(5XATF6RE:eGFP) to visualise ER stress. I confirmed the functionality of the reporter fish, as previously described by Clark et al. (2020). However, utilising a time window of 24 hours post-induction (hpi) induced by 4-hydroxytamoxifen (4-OHT), I didn’t visualise PNCs and PNC-associated neutrophils and proceeded with timelapse confocal live imaging from 24 to 36 hpi to monitor ER stress in these cells. Surprisingly, my results indicated subtle intensity changes indicative of ER stress in PNC-associated neutrophils, as confirmed by FACS analysis.

I also generated a neutrophil-specific CAS9 expression transgenic zebrafish Tg(lyzC: CAS9), which paved the way for future neutrophil-specific modulation of ER Stress to study its functionality.

In summary, although I did not detect elevated ER stress in neutrophils in the PNC development model using the Tg(5XATF6RE:eGFP), I did not have enough time to employ alternative methods to evaluate ER stress in neutrophils. Therefore, whether ER stress is involved in modulating neutrophil function in the PNC development model remains to be determined. However, I have generated tools to facilitate future studies into ER stress and functional studies in neutrophils in zebrafish models.