LAUSR

Glucose metabolism is fundamental to the function and survival of both cancer cells and immune cells within the tumor microenvironment. Cancer cells often exhibit altered metabolic pathways to meet the elevated energy and anabolic demands of rapid proliferation, including increased glucose uptake for enhanced glycolysis and glycogen storage. This can deplete glucose availability for infiltrating immune cells, restricting their ability to execute an effective anti-tumor response and promoting an immunosuppressive environment. Understanding glucose metabolism in cancer cells and T cells is important for elucidating the role of nutrient competition in immunotherapy effectiveness. To investigate these dynamics, we developed sensitive bioluminescence assays capable of measuring multiple aspects of glucose metabolism in both cancer and immune cells, including glycogen synthesis and utilization. Glycogen serves as a glucose reservoir that can rapidly respond to glucose supply and demand to maintain cellular functions under fluctuating nutrient conditions. Changes in glycogen levels (both increases and decreases) were observed in cancer cell lines in response to medium composition (e.g., glucose concentration) and drug treatments. Glycogen was also measured in T cells upon activation, providing a useful method for studying requirements for glycogen synthesis in these cells and its relevance for T cell function. The increased sensitivity of the glycogen assay required low cell numbers and was amenable to reduced volumes, in 384-well plates, further facilitating these studies. In addition to glycogen synthesis and degradation, other glucose metabolic pathways, including glycolysis, oxidative phosphorylation, and the pentose phosphate pathway, were analyzed. We used assays to measure intermediates and products in these pathways, including lactate, pyruvate and malate, as well as assays for measuring dehydrogenase enzyme activity. Dehydrogenases representing all three pathways exhibited increased activity upon T cell activation, indicating enhanced engagement of these pathways. These assays should provide a valuable method for studying the impact of limited glucose on T cell activation and suggesting approaches to address it. To better understand the role of glucose availability and implications of nutrient competition in the tumor microenvironment for anti-tumor immune responses, we used sensitive bioluminescent assays to investigate metabolic profiles of cancer and immune cells. The assays can facilitate identification of potential approaches and targets to eliminate limited access to glucose and other nutrients as a barrier to efficient immunotherapies. Donna Marie Leippe, Natasha Karassina, Mike Valley, Kayla Sylvester, Anthony Lauer, Jolanta Vidugiriene. Investigating glucose utilization in cancer and immune cells using bioluminescent metabolite and enzyme assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4178.