Introduction:

Macrophages perform a wide range of tasks, including bacterial suppression, tumor suppression, immune-stimulation, tissue repair, angiogenesis, and cellular debris phagocytosis Behavior is controlled by a large number of chemo-stimulants, and broadly are classified into two polarization groups, M1 (classically activated, pro-inflammatory) and M2 (alternatively activated, anti-inflammatory). Understanding the macrophage phenotype is essential in the development of pharmacological agents designed to influence macrophage behavior. Due to the plethora of macrophage stimulants, it is desirable to do analysis under label-free conditions.

Materials and Methods:

The murine macrophage cell line, J774A.1 was analyzed untreated (M0), stimulated with LPS to drive M1, and stimulated with IL-4 to drive M2. Efficacy of the stimulations was monitored by PCR analysis of the expression levels of iNOS and TNF-Alpha (up-regulated in the M1 state) and Arginase-1 and IL-10 (up-regulated in the M2 state). Imaging data was obtained on a holographic imaging cytometer Holomonitor® M4 (Phase Holographic Imaging, Lund, Sweden). The instrument permits acquisition of time-lapse holographic image sets of unstained cells in standard vessels placed in the incubator. Images are segmented, and quantitative features are recorded for individual cells, including optical volume, thickness, area amongst others. Positional information includes X and Y positions, as well as X and Y center of mass. Data can be presented as positional track maps, position vs. feature graphs, and feature vs. feature graphs. We also obtained time-lapse videos and generated 4-dimensional (X-pos., Y-pos., cell height and time) plots in ImageJ software.

Results:

In untreated cells, we were able to observe the morphological and behavioral changes in cells involved in the phagocytic process. These include elongation and development of bilateral symmetry of the cells, and formation of podosomes involved in the phagocytosis of cellular remnants - all hallmarks of the M1 polarization state. We detected the same effects in cells treated with LPS under conditions known to induce M1, but without the influence of target cells. Cells present an oscillating phenotype, which we believe was caused by the redistribution of the center of mass within the cell as the polarity of the cell reverses. In Cartesian time course plots and 4D images, M1 polarized cells present linear tracking profiles. Cells treated with IL4 under conditions known to induce M2, maintained radial symmetry observable in cell images and videos. In Cartesian time course plots and 4D images, M2 polarized cells presented initial “starburst” patterns, suggesting oscillations with rotations occurring around the center of mass during cycles.

Conclusions:

Recently developed label-free imaging techniques provide a new tool for classifying macrophages based on cellular behavior.

Citation Format: Ed Luther, Meghna Talekar, Qijun Ouyang, Mansoor Amiji. Characterization of macrophage behavior by 4-dimensional label free, quantitative holographic imaging. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2341. doi:10.1158/1538-7445.AM2015-2341

©2015 American Association for Cancer Research.
2015