br The most widely used
The most widely used approaches for flow cytometry-based
phagocytosis studies involve the use of CellTracker and CFSE dyes due to their staining eﬃciency and ease of use. However, the significant deviations in data caused by cell-cell aggregation may limit the use of these conventional methods. To overcome this limitation, we present optimized methods of analyzing cancer phagocytosis by utilizing CellTracker or pHrodo-SE fluorescence dyes. The former suggests a more precise evaluation protocol than the conventional method, which exhibited no significant diﬀerence than the latter suggested method. Note that to eradicate the feasibility of detection of cancer 41994-02-9 bound to the surface of the phagocytes require pHrodo-SE staining. As well, the fluorescence microscopy–based method that utilizes pHrodo-SE guarantees more detailed reporting of phagocytosis; this enables the assessment of the definite number of cancer cells engulfed by each phagocyte.
Furthermore, these in vitro phagocytosis assays can be used to analyze the role of DCs that play important roles in antitumor im-munotherapy by evaluating engulfment, which is also the primary function of DCs. In order to initiate cancer immunity, it is important to uptake cancer cells and present antigens, which leads to the generation of tumor-specific T cells. Unlike phagocytes such as macrophages, DCs
can preserve antigenic peptides for a longer duration after phagocytosis (Lennon-Dumenil et al., 2002), and this plays a key role in the forma-tion of adaptive immunity through eﬀective antigen processing and presentation (Gordon, 2016).
The ability to reinforce innate immune responses is an important factor that aﬀects the therapeutic eﬃcacy of cancer immunotherapy. To eﬀectively elicit tumor antigen-specific immunity, immunotherapeutic candidates must potentiate the function of antigen-presenting cells at the initial stages of the antitumor immunity cycle. Therefore, we highlighted that these methods for detecting phagocytic function are expected to be utilized for further investigations into regulating the activity of DCs in antitumor therapy. Furthermore, these optimized methods that determine the engulfment of cancer cells by phagocytes can be applied to multiple types of tumors and used as a screening assay for a variety of phagocytosis agonists.
Declaration of interest
The authors declare no competing financial interest.
This work was supported by grants from the National Research Foundation of Korea (NRF) funded by the Korean government (2019R1A2B5B03004360 and 2017R1A3B1023418), the KU-KIST Graduate School of Converging Science and Technology Program, and the KIST Institutional Program.
Chimini, G., Chavrier, P., 2000. Function of Rho family proteins in actin dynamics during phagocytosis and engulfment. Nat. Cell Biol. 2, E191–E196. Garg, A.D., Romano, E., Rufo, N., Agostinis, P., 2016. Immunogenic versus tolerogenic phagocytosis during anticancer therapy: mechanisms and clinical translation. Cell Death Diﬀer. 23, 938–951.
Jeong, C., Yang, Y., Kim, I.S., 2018. Nanocage-therapeutics prevailing phagocytosis and immunogenic cell death awakens immunity against cancer. Adv. Mater. 30.
Palucka, K., Banchereau, J., 2012. Cancer immunotherapy via dendritic cells. Nat. Rev.
32 ORIGINAL RESEARCH
An Organoid-Based Preclinical Model of Human Gastric Cancer
Nina G. Steele,1 Jayati Chakrabarti,2 Jiang Wang,3 Jacek Biesiada,6 Loryn Holokai,4 Julie Chang,5 Lauren M. Nowacki,7 Jennifer Hawkins,8 Maxime Mahe,8 Nambirajan Sundaram,8 Noah Shroyer,7 Mario Medvedovic,6 Michael Helmrath,8 Syed Ahmad,9 and Yana Zavros3
1Department of Cell and Developmental Biology, University of Michigan, Ann Arbor, Michigan; 2Department of Pharmacology and Systems Physiology, University of Cincinnati, Cincinnati, Ohio; 3Department of Pathology and Lab Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio; 4Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, Ohio; 5Department of Biomedical Engineering, University of Cincinnati, Cincinnati, Ohio;
6Department of Environmental Health, Division of Biostatistics and Bioinformatics, University of Cincinnati College of Medicine, Cincinnati, Ohio; 7Department of Medicine, Section of Gastroenterology and Hepatology, Baylor College of Medicine, Houston, Texas; 8Department of Pediatric Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; and 9Department of Surgery, University of Cincinnati Cancer Institute, Cincinnati, Ohio