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  • br In this study Y was used to


    In this study, Y27632 was used to enhance phagocytosis, which was previously known as the Rho kinase inhibitor that blocks RhoA/ROCK signaling (Nam et al., 2018). In agreement with the previous report, the result of the phagocytosis assay using CellTracker dye showed that the percentage (%) of the double positive signal (deep red and green) per the total number of macrophages, which indicates that the extent of phagocytosis increased in the Y27632-treated group compared to the control group(Fig. 2c).
    Next, we performed an alternative image-based phagocytosis assay using a pHrodo-SE dye that enabled the measurement of the fluores-cence of engulfed cancer Leupeptin (Fig. 2d). The pH sensitive, pHrodo-SE dye emits weak fluorescence at natural pH but changes to bright fluorescence at acidic pH. The pHrodo-SE-stained non-engulfed cancer cells emit light at a low fluorescence, while cancer cells engulfed by BMDMs emit bright florescence at a lower phagosome pH, which can easily be detected through fluorescence microscopy.
    To analyze the extent of phagocytosis, targeted cells (CMFDA-stained BMDMs and cancer cells labeled with pHrodo-SE dye) were co-incubated for 2 h, followed by washing with basic PBS (pH 10) for re-moving unengulfed tumor cells. The percentage of phagocytosis was calculated by the number of BMDMs that phagocytized cancer cells (green containing red) per total number of BMDMs (green). Similar to the results from the flow cytometry–based phagocytosis assay, treat-ment of BMDMs with Y27632 led to a significant increase in the pha-gocytosis of cancer cells (Fig. 2d).
    Note that it is difficult to exclude the possibility of cell-to-cell binding in the double positive signal of flow cytometric results utilizing CellTracker dyes. As shown in Fig. 2, the measured phagocytosis of 4 T1-Luc cells determined by the flow cytometry–based method was slightly higher than that determined by the fluorescence micro-scopy–method (Fig. 2c-d). This result indicates that the CellTracker-method might overestimate the baseline of cancer phagocytosis. Therefore, the appropriate methods should be selected to measure the degree of phagocytosis of cancer cells with strong aggregation such as 4 T1-Luc cells.
    3.2. Evaluation varied states of phagocytosis
    Current methods that utilize CellTracker dyes to verify phagocytosis are largely limited to flow cytometry– and manual image–based assays, providing limited information in spite of varying phagocytosis status. However, more detailed information beyond the measured phagocy-tosis percentage can be successfully assessed by a fluorescent micro-scopic method employing pHrodo-SE dye; the phagocytic index (PI) is calculated as the ratio of engulfed tumor cells (red) per total BMDMs (green), and the phagocytic capacity (PC) indicates engulfed cancer cells among double-positive BMDMs.
    To confirm whether pHrodo-based microscopy images comprise an advantageous method with which phagocytosis can be accurately analyzed, we used anti-CD47 antibodies as a phagocytosis agonist. In agreement with a previous study (Lee et al., 2018), phagocytosis was significantly enhanced by anti-CD47 antibodies that block the interac-tion between CD47-SIRPα (Fig. 3a, b-left). Notably, the PI and PC were
    Fig. 3. Several methods of analyzing phagocytosis of cancer cells stained with pHrodo-SE through fluorescent images. (a) Representative microscopic images of pHrodo-SE phagocytosis assays performed using CMFDA-stained BMDMs (green) against engulfed HT29 cells (red), the enlarged image of which represents BMDMs' phagocytosis of multiple cancer cells (insets). Scale bars: 100 μm. (b) Left: Phagocytosis calculated as the percentage of BMDMs containing cancer cells per total BMDMs. Middle: PI calculated as the ratio of engulfed cancer cells (pHrodo-SE, red) per total BMDMs (green). Right: PC calculated as the ratio of engulfed cancer cells per BMDMs containing cancer cells. Data are presented as means ± S.D. (n = 3–7). **p < .01 by Student's t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    considerably escalated in the anti-CD47-treated group compared with the untreated control (Fig. 3a, b). Remarkably, macrophages exposed to anti-CD47 exhibit enhanced phagocytic ability for simultaneous en-gulfment of multiple cells (Fig. 3a, inset). These results indicate that the proposed fluorescence microscopy-based method using pHrodo-SE is capable of representing the degree of phagocytosis, e.g., the number of engulfed cancer cells by a single macrophage.
    4. Discussion
    Macrophages, which are specialized phagocytes, are particularly active in the phagocytosis process (Chimini and Chavrier, 2000; Poon et al., 2014). Of late, the phagocytic function of macrophages has be-come increasingly recognized as an attractive target in cancer im-munotherapy (Brown et al., 2017). Given that an understanding the molecular interactions between phagocytes and tumor cells is critical for a better comprehension of their tumor-modulating action, there is an increasing demand for in vitro methods to study the role of macro-phages in tumor cell phagocytosis.