Abstract 373: Bioluminescent non-lytic metabolite and cytotoxicity assays for cancer and immunotherapy research
Authors: Leippe Donna M. , Karassina Natasha , Valley Michael P. , Cali James J. , Vidugiriene Jolanta 
Published: August 2018

Abstract
The growing interest in understanding metabolic adaptations that occur in cancer and immune cells in the tumor microenvironment and the advances made towards developing effective immunotherapies have increased the need for sensitive and efficient methods for studying cell metabolism and viability. We have developed bioluminescent assays for monitoring cell metabolism and cytotoxicity during cell growth and in response to environmental conditions and treatments. The plate-based assays employ coupled enzyme reactions and bioluminescent NADH detection chemistry to detect metabolites and enzymes. We used the assays in a non-lytic format, analyzing samples of cell culture medium and leaving the cells intact for additional measurements or multiplexing with other assays. In this way, changes occurring in a single well of cells were followed over time by collecting medium at multiple time points. We used assays for glucose, lactate, glutamine and glutamate detection to obtain information about the key metabolic pathways, glycolysis and glutaminolysis, and metabolically profile cancer cell lines. When we compared the metabolism of two ovarian cancer cell lines, OVCAR-3 and SKOV-3, the rates for glucose consumption and lactate secretion were similar, but the SKOV-3 cells consumed more glutamine and secreted 20-fold more glutamate. Glutamate secretion by the OVCAR-3 cells was low, only 5-fold higher than background controls. Analysis of the metabolites in cell lysates further supported the conclusion that the two cell lines differed in glutamine metabolism. The metabolite detection assays were also used to monitor metabolic pathway reprogramming in response to the environment. The induction of glycolysis is an early, critical step in the activation of T cells, and results in increased lactate production. We investigated the requirements for T-cell activation by measuring lactate levels over time and observed robust activation only in the presence of glutamine and anti-CD3/anti-CD28 costimulatory signals. In addition to metabolite levels, cell culture medium was used to assess cytotoxicity by following lactate dehydrogenase enzyme (LDH) release into cell culture medium. We used this assay to study cancer cell cytotoxicity induced by immunotherapeutic treatments, including antibody-drug conjugates (ADC) and antibody-dependent cell-mediated cytotoxicity (ADCC). The high sensitivity of the assay permitted the use of fewer cells, in 2D and 3D cultures, and the earlier detection of small changes (10 lysed cells in medium lacking serum). These sensitive bioluminescent assays require small amounts of sample and minimal sample processing to facilitate (1) identification of unique cancer cell metabolic signatures and potential therapeutic targets, (2) studies of connections between metabolism and effective immune cell function, and (3) the evaluation of cytotoxic immunotherapies.
Citation Format: Donna M. Leippe, Natasha Karassina, Michael P. Valley, James J. Cali, Jolanta Vidugiriene. Bioluminescent non-lytic metabolite and cytotoxicity assays for cancer and immunotherapy research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 373.