br Appendix A Supplementary data br References br D
Appendix A. Supplementary data
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Contents lists available at ScienceDirect
Journal of Immunological Methods
journal homepage: www.elsevier.com/locate/jim
An optimized protocol to determine the engulfment of cancer U-73122 by T
phagocytes using flow cytometry and fluorescence microscopy
Gi-Hoon Nama,b,1, Yeonsun Honga,b,1, Yoonjeong Choia,b, Gi Beom Kima,b, Yoon Kyoung Kima,b, Yoosoo Yangb,c, , In-San Kima,b,
a KU-KIST Graduate School of Converging Science and Technology, Korea University, 145, Anam-ro, Seongbuk-gu, Seoul 02841, Republic of Korea
b Biomedical Research Institute, Korea Institute of Science and Technology (KIST), Seoul 02792, Republic of Korea
c Division of Bio-Medical Science &Technology, KIST School, Korea University of Science and Technology, Seoul 02792, Republic of Korea
The engulfment of cancer cells by macrophages is an important cellular process in innate cancer immunity. Antitumor immunotherapy that utilizes the enhanced engulfment of cancer cells by phagocytic cells has at-tracted much attention. Therefore, there is a growing demand for methods of measuring cancer cell phagocy-tosis. Quantifying the various stages of phagocytosis is invaluable for elucidating cancer-immune responses during this process. Here, we describe two phagocytosis assays, a flow cytometric assay and a fluorescent mi-croscopic assay; the flow cytometric method utilizing CellTracker dye provides a simple, measurable, and highly reproducible functional assay to measure the phagocytosis eﬃciency of cancer cells by bone marrow-derived macrophages. As an alternative method of evaluating various states of cancer cell phagocytosis, a fluorescent microscopic method that employs a pH-sensitive dye (pHrodo-SE dye) is also described in this paper. Image-based analysis using this labeling approach enables researchers to measure phagocytic indices that indicate the number of cancer cells engulfed by each macrophage. We have highlighted that these assays can be applied to multiple tumor types and used as selection tools for a variety of phagocytosis agonist types. The results of this study may facilitate a better understanding of the interactions between tumor cells and phagocytes, which could lead to the identification of new therapeutic targets against cancer.
Cancer immunotherapies have led to unprecedented rates of posi-tive clinical responses in patients with various cancer types (Palucka and Banchereau, 2012). To induce antitumor immunity, innately im-mune cells such as macrophages or dendritic cells (DCs) must phago-cytose tumor cells and process tumor specific antigens (Palucka and Banchereau, 2012). Professional antigen presenting cells (APCs) among phagocytes specialize in presenting antigens to T cells in antitumor immune responses. In particular, macrophages are highly eﬃcient at internalizing cancer cell antigens, which represents a critical compo-nent of innate immunity against cancer.
The determination of the phagocytic activity of innately immune cells can be a reliable predictor for evaluating response rates to im-munotherapy (Garg et al., 2016). However, the evaluation methods that
have been used thus far have not led to a well-established under-standing of the process of phagocytosis of cancer cells. In addition, phagocytic activity cannot be measured clearly due to intercellular adhesion between cancer cells and macrophages, resulting in poor re-producibility and sensitivity of these methods (Miksa et al., 2009). Thus, there is an increasing need for the development of a precise and highly reproducible functional assay to evaluate the degree of phago-cytosis of cancer cells by macrophages.