Using single transcriptomic and genetic manipulation to investigate the cellular interaction in preneoplastic cell development niche

Background

Tumourigenesis is initiated by a single cell acquiring an oncogenic mutation, which drives preneoplastic cell (PNC) development. To date very little is known as to how PNCs interact with healthy neighbours and host innate immune cells to establish a promotive niche that allows tumour development. A better understanding of the mechanisms that favours PNC progression toward tumourigenesis could provide novel targets for cancer early detection and prevention. We have established an inducible human HRAS^G12V driven PNC initiation model in the basal layer of zebrafish larval skin in which fluorescently tagged PNCs and innate immune cells can be visualized in vivo in real time. In this model we have found dynamic changes in PNC morphology and rapid recruitment of both neutrophils and macrophages within 24 hours of oncogene induction. Importantly, recruited neutrophils were found to play a trophic role in promoting PNC growth. To dissect the heterogeneity of cells in the PNC developmental niche, we performed single cell RNAseq analysis for PNCs as well as recruited macrophage and neutrophils. Our initial analysis revealed rapid cell fate alteration of PNCs and profound gene expression changes in neutrophils within a few hours of skin PNC induction. Data from additional time points beyond 24 hours PNC induction were also collected, these will be incorporated into future analysis. Initial cell-cell interaction (CellPhoneDB) analysis has identified candidate molecules that might mediate PNC-neutrophil crosstalk. However, the mechanisms underpinning PNC cellular state transition and the interaction with neutrophils are not fully understood, and these can be investigated through further in-depth analysis of the current scRNAseq data set and genetic manipulation of PNCs and neutrophils in the zebrafish PNC initiation model. More importantly, we wish to integrate data from our zebrafish model with multiple scRNAseq data-sets from human cancer patients to identify key features of PNC that may represent higher tumourigenesis potential.

In the current project:

1, we will identify key candidate pathways that control PNC cellular state transition and elucidate how might subsets of neutrophils impact on PNC cell fate transition through computational analysis.

2, We will use genetic and pharmacological tools to manipulate PNC cellular state transition and neutrophil function toward PNC suppression or elimination.

3, Using identified key factors (aim1) released from PNCs and/or neutrophils to interrogate publicly available databases (TCGA) and to establish the relevance of candidate factors in cancer early detection.

4, Finaly we will examine candidate marker genes in patient samples to evaluate the prognosis potential.

Training outcomes:

Through this project, the student will be proficient in the analysis of single cell transcriptomics and bioinformatics analysis. This will include a deep knowledge of the R computer language and basic Linux skill. Further they will learn about different package of pseudo time (Slingshot, PAGA) and inclusion of velocity and integration of published data from human datasets to understand the impact of our finding in the human context.

Wet lab skills: zebrafish genetics and zebrafish GM cancer models; zebrafish adult and embryo handling; zebrafish genetic manipulation; zebrafish larval wholemount histology methods; in vivo live imaging and image data analysis. Immunohistochemistry; Image analysis.

Reference:

1. Laux DW, Kelly L, Bravo IR, Ramezani T, Feng Y. Live imaging the earliest host innate immune response to preneoplastic cells using a zebrafish inducible KalTA4-ER(T2)/UAS system. Methods Cell Biol. (2017);138:137-150.
2. van den Berg MCW, MacCarthy-Morrogh L, Carter D, Morris J, Ribeiro Bravo I, Feng Y*, Martin P. Proteolytic and Opportunistic Breaching of the Basement Membrane Zone by Immune Cells during Tumor Initiation. Cell Rep. (2019); 27(10):2837-2846. PMID: 31167131
3. Myllymäki, H., Kelly, L., Elliot, A.M., Carter, R.N., Morton, N.M., and Feng, Y. (2023). Preneoplastic cells switch to Warburg metabolism from their inception exposing multiple vulnerabilities for targeted elimination. bioRxiv, 2023.01.09.523333. 10.1101/2023.01.09.523333.
4. Elliot A.M., Ribeiro Bravo I., Astorga Johansson J., Hutton E., Myllymaki H., Beltran M., Dobey R., Henderson N., Ponting C., Feng Y*. Oncogenic RAS drives rapid onset cellular plasticity and elicits a tumour-promoting neutrophil response at the inception of preneoplastic development BioRxiv 2023.11.10.566547; doi: https://doi.org/10.1101/2023.11.10.566547