Metabolic Reprogramming of Tumor Microenvironment to Maximize Immunotherapy for Pediatric Cancers

Goals A critical barrier for immunotherapy is the hostile metabolic microenvironment within solid tumors, where the highly metabolic-demanding tumor cells compromise the function of T cells by competing for glucose. In addition, tumor cells also release immune suppressive metabolite, adenosine into the extracellular space. While recent advances have helped determine the metabolic reprogramming of T cell activation and its regulatory signaling mechanism, the detailed landscape of metabolic programs in active T cells and the impact of the tumor’s metabolic microenvironment on the development and anti-tumor immune function of cytotoxic T effector (Teff) cells remains elusive. Our studies have shown that glucose restriction significantly dampened the growth and tumor-killing activities of Teff cells. Our recent metabolite-reconstitution screen identified inosine as a potent metabolite that could replace glucose in promoting Teff cells growth and tumor-killing activities. Using inosine as the alterative metabolic substrate indicates a layer of metabolic plasticity on T cells and further implicates the potential of inosine in relieving tumor-imposed metabolic restrictions on T cells in vivo. Intriguingly, adenosine is the metabolic precursor of inosine and adenosine deaminase (ADA) catalyzes their conversion by the substitution of the amino group of the base adenine for a keto group. While our data suggest that inosine promotes T cell growth and effector functions, stressed or damaged tumor cells release ATP and its product, adenosine, into the extracellular space to suppress Teff cells and Natural Killer (NK) cells, shaping the efficacy and magnitude of anti-tumor immune response. Hence, we hypothesize that reprogramming of adenosine-inosine metabolic axis can maximize anti-tumor immune response in treating pediatric solid tumors. To test our hypothesis, we propose to decipher inosine catabolic pathways and to assess the impact of key metabolic steps on Teff cells. Here we will apply the Stable Isotope Resolved Metabolomics (SIRM) approach coupled with NMR and high-resolution mass spectrometry to generate an atom-resolved metabolic map of Teff and NK cells to define uptake and utilization pathways of inosine and adenosine in supporting cell growth and activity. Experimental Design The metabolism of Teff and NK cells will be analyzed in the absence and presence of glucose using different tracers, namely 13C inosine, 13C adenosine, 13C glucose and 13C,15N glutamine in triplicate, i.e. 2 cell types, 4 tracers in triplicate = 24 cell samples. Both polar and non-polar metabolites will be analyzed : 48 analytical samples. Media samples will be taken at 5 timed intervals and flash frozen for analysis of the extracellular metabolites, particularly for nutrient uptake and waste product excretion. 120 analytical samples Cells will be grown at OHSU, and metabolites will be extracted according to standard protocols to provide polar metabolites and non–polar metabolites (lipids) simultaneously, as well as the proteins of the cells to be used for normalization. Media samples will be deproteinized according to standard protocols. Glucose, inosine, adenosine and glutamine carbon and nitrogen atoms will be traced into the various downstream metabolites, and the isotopomer and isotopologue distributions quantified. These provide direct information about the relative importance of different networks of central metabolism, in terms of cell proliferation and survival. Methodology Polar metabolites will be identified and quantified by NMR and IC-MS. Uptake of nutrients from medium (amino acids, glucose), excretion of products (e.g. lactate, alanine) with enrichment factors are determined from 1D 1H NMR and 1D HSQC NMR experiments and IC-MS from the time course experiments. This gives the overall nutrient flux for biomass and energy production. Data will be collected and reduced at UKY CESB. 2D NMR experiments of polar cell extracts will be carried out at CESB, and the data reduced by CESB staff under the supervision of A.N Lane and T. Fan. The polar cell extracts provide information about the following pathways: Glycolysis (and glycogenolysis)/lactic fermentation Pentose phosphate pathway Hexosamine pathway Krebs cycle Anaplerosis nucleotide biosynthesis Glutathione redox biochemistry Glutamine metabolism. Cellular lipids will be analyzed by direct infusion FT-MS at CESB. Each run provide accurate mass of resolved peaks corresponding to all of the major classes of lipids, including intact TAGs/DAGs; phosphatidyglycerides (choline, ethanolamine, serine, inositols), ceramides, sphingolipids and ether lipids and steroids. Typically >500 lipid species and their isotopologue distributions are determined for each sample. Data reduction comprising peak identification and isotopologue distribution analysis will be carried out at UKY CESB. NMR analyses of the lipids will also be carried out to cross verify global isotopic incorporation in groups of lipids. Biochemical interpretation in terms of cancer biology and metabolic networks will be carried out as collaboration between the OHSU (Wang) and UKY (Fan, Lane) groups.