“Warburg metabolism” is characterised by an increased uptake of glucose and its conversion to lactate by glycolysis under normoxia. It is an important feature of cells under physiological/pathological stress such as immune cell activation during inflammation or cancer metabolism; moreover glycolysis is a characteristic of undifferentiated stem cells. To date, metabolic reprogramming of cancer cells has been recognized as a target for therapeutic intervention. However, it is unclear as to when and how metabolic changes occur during pre-neoplastic cell progression. We hypothesized that Warburg metabolism or metabolic change is an early event during pre-neoplastic cell initiation and plays an important role in supporting pre-neoplastic cell (PNC) progression. Until recently, it has been difficult to investigate PNC initiation and progression in vivo with precise spatiotemporal information due to limitations of available animal models. My lab has developed a tamoxifen-inducible system whereby fluorescent labeled PNCs can be induced in skin tissue with precise temporal control and their interaction with host tissues followed with live imaging. Using this model we have been monitoring PNC and innate immune cell interactions in vivo in real time. Our preliminary study using the Seahorse Analyzer® showed impaired mitochondria respiration and enhanced glycolysis in PNCs.
In the current project we seek first to characterize metabolic changes within the PNC niche, second to identify pathways in PNCs that are important for these metabolic alterations and finally to determine how metabolic alternations contribute to PNC progression.
Aim 1 Characterize metabolic alteration within the PNC niche
We will perform single cell RNA-sequencing of FAC-isolated PNCs and compare their transcriptomes with those of normal skin cells to identify altered gene expression profiles including altered metabolic pathways. We will then perform an Imaging Mass Spectrometry analysis of metabolites in situ using whole zebrafish larvae carrying PNCs. The two sets of data will be integrated and analyzed systematically to identify altered metabolic pathways and metabolites in the PNC niche.
Aim 2 In vivo live imaging of metabolic alterations
We have developed several transgenic reporter fish for monitoring metabolic changes in vivo. We will combine these reporter fish with our inducible skin PNC model to live image metabolic changes within the PNC niche, and determine when the earliest metabolic change occurs and whether this might play a role in regulating inflammatory cell recruitment toward PNCs.
Aim 3 Manipulating metabolic pathways within PNCs using a lineage specific Cas9 system
Having characterized metabolic alterations in Aim 1 and having set up live imaging protocols for metabolic changes in Aim 2, we will use a newly established lineage specific Cas9 gene inactivation system to restore normal metabolism in PNCs and examine its impact on PNC growth.
Student training includes:
Wet lab: zebrafish genetic manipulation techniques, in vivo live imaging; RNA-sequence sample and library preparation; Imaging Mass Spectrometry sample preparation and handling.
Computational lab: Cutting-edge analytical tools for single cell RNA-seq data analysis and interpretation; 4D imaging data analysis and Imaging Mass Spectrometry data analysis.