A novel approach to analyze cellular metabolism outside the body, in complex living tissues.
- Cancer immunology
- Bioanalytical assay development
Metabolic activity and immune function are tightly linked, and in fact “immunometabolism” is emerging as an exciting new area of study, with the potential to lead to new treatment strategies for both autoimmune diseases and cancer. For example, tumor cells and immune cells both respond to stress by changing how they produce energy, with large changes in consumption of fuels like glucose and downstream metabolic processes such as glycolysis and oxidative phosphorylation. Additionally, studies have found that much of the immune suppression within the tumor microenvironment is driven by changes in metabolic function in innate and adaptive immune subsets.
Few technologies are available to measure metabolism in living tissues over time. Most measurements of metabolic activity in immune cell types are performed using cell cultures, which lack the complex microenvironment, spatial separation and stromal influence of the intact organ. Importantly, living tissues are highly organized, with many cell types functionally dependent upon close communication with extracellular matrix structures. It is therefore not likely that cells in a plastic dish residing in a two-dimensional space can represent the true behavior of an intact tissue. Confounding metabolic studies even further are the extreme plasticity of subsets of innate immune cells, such as macrophages and natural killer (NK) cells, that rapidly change function after sorting from tissue and tumor microenvironments. These characteristics make rigorous metabolic studies of immune cell types challenging.
Here, we will combine the expertise of three UVA faculty members, Rebecca Pompano, Melanie Rutkowski, and Michelle Bland, to detect metabolic activity and assess immune function in complex intact live tissues. The Pompano lab, in the Department of Chemistry in the College of Arts and Sciences, has shown that by applying a fluorescently-labelled derivative of glucose to intact tissue samples, individual regions of high glucose uptake can be visualized. This method was first tested in live lymph node tissue, revealing that glucose uptake is localized to specific regions within lymph nodes in response to cell-specific stimulation - for example, localizing to expected T cell regions after stimulation with CD3 ligation. In this proposal, we will adapt this assay for use with two new types of tissue for the first time: tumors from mice and the larval fruit fly fat body, which coordinates both immune and metabolic functions. The Rutkowski lab, in the Microbiology, Immunology, and Cancer Biology department in the School of Medicine, is an expert in tumor immunology, and has been developing methods to culture tumor slices over several days to assess differences in immune function and ex vivo response to immune therapies. The Bland lab, in the Pharmacology department in the School of Medicine, has expertise in studying metabolism driven by insulin signaling and activation of the innate immune system using powerful genetic approaches in the model organism Drosophila melanogaster. Together, we will work to develop the methods to reproducibly visualize glucose uptake in live organ cultures of these samples according to the following two aims:
Aim 1: Test the feasibility of longitudinal evaluation of glucose metabolism in ex vivo organ cultures. We will evaluate the duration of metabolic activity without stimulation after removal from animals. Additionally we will work to optimize the culture conditions to accommodate the unique cell types that are present within each tissue.
Aim 2: Combine visualization of glycolysis with cell specific immunostains, to determine what regions of tissue or organ are most active at baseline and in response to specific stimuli. For the tumor, we will test the hypothesis that T cells residing on the edges of the tumor will have more metabolic activity compared to T cells residing within the hypoxic core. We will use methodologies optimized in the Pompano lab to evaluate CD3+ CD4 and CD8 T cells, CD11b myeloid cells and RFP tumors. In the fly fat body, we will use genetic approaches to drive expression of constitutively-active insulin or Toll receptors in individual cells in an otherwise wild type organ to test the hypothesis that these two signaling pathways alter glucose metabolism cell autonomously and in different directions.
This work will show the feasibility of a novel assay to assess glucose metabolism in live organs and complex tissues and will form the basis of a new three-way faculty collaboration at UVA. The project will lead to one or more collaborative papers describing the methods, presentations at national conferences, and will support submission of grants for further funding from the National Institutes of Health, the American Diabetes Association, and the American Cancer Society. Once optimized, we expect to combine the three model systems (ex vivo lymph node slices and imaging, tumor metabolism, and in vivo Drosophila genetics to manipulate metabolism) to develop new tools (Pompano lab) to investigate novel metabolic pathways perturbed in response to challenges such as commensal dysbiosis in tumor-bearing individuals (Rutkowski lab) and bacterial infection in different genetic backgrounds (Bland lab). In the future, these assays will be used in our laboratories and shared with others to provide a new ways to look at metabolism in contexts of immunity, cancer, and autoimmunity, and to rapidly test the effects of proposed therapies on metabolic changes and immune function.
Undergraduate and graduate students from each of our three laboratories will be involved in every step of the research, giving them a unique opportunity to collaborate across fields and learn to communicate to an interdisciplinary audience.