Abstract B31: Autofluorescence imaging of macrophage metabolism during tumor-mediated 3D migration

Abstract

Abstract Macrophages are key regulators of tumor progression, and can be targeted for cancer treatment. However, reprogramming macrophages for anti-tumor activity is challenging due to their plasticity. Standard assessment of macrophage function (e.g., ELISA) lacks the sensitivity to capture cell-level heterogeneity in living macrophages. Therefore, single cell imaging and analysis tools are needed to study heterogeneous macrophage behavior within the tumor microenvironment. Macrophages adapt their metabolism to alter both phenotype and function. Thus, we propose the combination of optical metabolic imaging (OMI) and microdevice co-culture to observe cellular level macrophage heterogeneity in response to tumor stimuli. OMI enables noninvasive, dynamic imaging of intact, living samples by measuring autofluorescence from the metabolic co-enzymes NAD(P)H and FAD. Cellular level redox balance is evaluated with the optical redox ratio, defined as the ratio of NAD(P)H to FAD intensity. Additionally, NAD(P)H and FAD fluorescence lifetimes report on metabolic enzyme binding activities. Three-layer microdevices consisting of murine PyVMT breast carcinoma cells, collagen (200 micron thickness), and RAW 264.7 macrophages, respectively, were designed to monitor tumor-mediated macrophage polarization and migration. Metabolic changes with macrophage migration were measured with OMI at 24, 48, and 72 hours after culture. Redox ratio and NAD(P)H mean lifetime increased over time in co-cultured macrophages (p <0.05). Macrophage mono-cultures exhibited lower redox ratio and NAD(P)H lifetime (p<0.05) than in co-culture, consistent with the metabolism of naïve and cytokine-stimulated M2-like macrophages, respectively. Phenotype and function of co-cultured macrophages were confirmed with polymerase chain reaction (PCR) gene expression analysis and immunostaining. Furthermore, volumetric imaging visualized macrophage migration distance-dependent changes in NAD(P)H autofluorescence intensity. These results demonstrate that OMI and microdevice co-culture can capture dynamic changes in macrophage metabolism throughout 3D tumor-stimulated polarization and migration. Overall, this approach could enhance our understanding of macrophage metabolism and function, thus revealing novel therapeutic targets within the tumor microenvironment. Citation Format: Tiffany M. Heaster, Jiaquan Yu, David J. Beebe, Melissa C. Skala. Autofluorescence imaging of macrophage metabolism during tumor-mediated 3D migration [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B31.