Abstract 2785: Metabolic autofluorescence microscopy of 3D microscale macrophage-tumor co-cultures
Authors: Heaster Tiffany M. , Yu Jiaquan , Edman Margaret , Beebe David J. , Skala Melissa C. 
Published: August 2019

Abstract
Macrophages are ideal treatment targets due to their tumor-regulatory functions and high infiltration in the tumor microenvironment (TME). However, macrophage heterogeneity prevents effective therapeutic reprogramming. Macrophages have high plasticity and adopt phenotypes with diverse behavior and metabolism. The complex TME can drive this plasticity but is not well understood. Standard functional assays (e.g. flow cytometry, ELISA) lack sensitivity to heterogeneous cell populations. Also, their destructive sample processing limits spatial and temporal assessment. Thus, nondestructive, single cell imaging and analysis tools are needed to study macrophage heterogeneity within the TME.
Optical metabolic imaging (OMI) measures two-photon excited fluorescence from the coenzymes NAD(P)H and FAD to resolve cellular metabolism within intact, 3D samples. The optical redox ratio (NAD(P)H intensity/FAD intensity) and fluorescence lifetimes reflect cell redox state and intracellular protein binding, respectively. Previous studies have shown that OMI detects spatial and temporal changes in stromal cells across in vivo and 3D in vitro models. Microscale models closely mimic the TME, are high throughput, and enable precise environmental control. Thus, microenvironmental stimulation of macrophage polarization and migration was mimicked in 3D microscale cultures of mouse macrophages (RAW264.7) and mammary carcinoma cells (PyVMT) in a collagen matrix. OMI captured redox ratio, NAD(P)H and FAD mean lifetime (τm) changes in mono-cultured and co-cultured macrophages 24-72 hours post-seeding. Intensity and lifetime volumes of macrophage layers further assessed metabolic changes in macrophages during migration.
Co-cultured macrophages exhibited significantly increased (p&lt;0.05) redox ratio and NAD(P)H τm compared to mono-cultured macrophages by 24 hours. Decreasing redox ratio in co-cultures over time suggested an oxidative metabolic shift, characteristic of M2-like phenotype. Distribution curves of redox ratio revealed macrophage subpopulations after 24 hours co-culture that disappear by 72 hours. Immunofluorescence markers of M1- or M2-like macrophages indicated heterogeneous polarization at 24 hours then transition to a homogeneous M2-like phenotype by 72 hours, consistent with metabolic imaging. Migration increased in co-cultured macrophages over time. Decreased redox ratio and increased NAD(P)H and FAD τm in migratory macrophages suggested proximity to the tumor affects macrophage metabolism. These results establish OMI for noninvasive monitoring of cell-level macrophage dynamics in 3D, in vitro TME models. Microscale co-culture and OMI were also demonstrated with primary mouse macrophage/Panc02 co-culture and human THP-1/MDA-MB-231 co-culture. Overall, these tools could better characterize heterogeneous macrophage behavior to improve regulation of anti-tumor activity.
Citation Format: Tiffany M. Heaster, Jiaquan Yu, Margaret Edman, David J. Beebe, Melissa C. Skala. Metabolic autofluorescence microscopy of 3D microscale macrophage-tumor co-cultures [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2785.