4Dplus_Metaflux · Multidimensional in vivo metabolic flux analyses: Resolving immune cells based on in vivo metabolic phenotypes

The development of better immunotherapies is being hammered by our lack of understanding about immune cell metabolism in vivo at the sites of disease. We know that immune cell metabolism is pivotal in controlling immune function. For instance, disrupting Natural Killer cell metabolism prevents them from killing cancer cells. Cellular immunotherapies for diseases like solid cancer are underperforming and one central reason for this is the metabolic pressure exerted on immune cells in solid tumours. The core problem is that we cannot currently measure the metabolism of immune cells at the site of disease. As a result, the biopharma industry is ill equipped to design immmunotherapies that are effective at these sites in the face of the metabolic stress.My ERC-CoG project (DC_Nutrient) has developed a new type of nutrient uptake assay with single cell resolution that can be used to probe metabolism in vivo. This is highly informative because the uptake of nutrients is the first limiting step for cellular metabolism. The core advance in our approach is to use bioorthogonal chemistry to attach a fluorophore to the nutrient after it has been transported into the cell. This bypasses all the pitfalls and failings of previous attempts to develop such assays using fluorophore tags. This PoC will advance this technology to simultaneously measure the uptake of 3 separate nutrients into immune cells at the site of disease, all with single cell resolution. Combined with an in vivo measurement of protein translation this will provide 4 dimensions of metabolic flux analysis for each cell analysed by multiparmetric flow cytometry. This innovative idea will provide a licensable assay technology that will be of high value to the biopharma industry as it will enable preclinical mouse studies to gain a detailed understanding of the metabolic changes occurring in immune cells at the site of disease. This technology will drive innovation towards metabolically enhanced immunotherapies.