Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and... Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often con- sidered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detri- mental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better compre- hension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage func- tions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor micro- environment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different typesofphagocytosisagonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer. Keywords: tumor biology; RAW264.7 macrophages (ATCC Cat# TIB-71, RRID:CVCL_0493); THP-1 monocytes (ATCC Cat#TIB-202, RRID:CVCL_0006); CL-1 and PC3 (ATCC Cat# CRL-7934, RRID:CVCL_0035) prostate cancer cells; phagocytosis; cell imaging Introduction situations have been widely described, accumulating evidence yet suggests that macrophages also affect cancer initiation, develop- Macrophages are innate immune cells that play critical roles in the clearance of pathogens and the maintenance of tissue homeostasis ment, and progression [2]. Tumor-associated macrophages (TAMs) are the main population of inflammatory cells present in many [1]. While macrophages modes of action in nonpathological Received: 29 September 2016; Revised: 14 January 2017; Editorial decision: 18 January 2017; Accepted: 1 February 2017 V The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2| Martinez-Marin et al. human solid tumors. TAMs are derived from circulating monocytes (Manassas, VA, USA). Both cell lines were cultured as recom- and their mobilization toward tumor tissues is regulated through mended, between passages 1 and 12, to block gene loss and im- multiple microenvironmental signals such as cytokines, chemo- pair macrophage immune function [17]. Human CRPC CL-1 and kines, extracellular matrix (ECM) components, and hypoxia [2, 3–5]. PC3 DsRed Express cells were established as we described in In response to environmental stimuli, TAMs can appear with a range [18]. Briefly, PC3 DsRed Express were derived from PC3 cells of different phenotypes and exhibit both beneficial and detrimental (ATCC Cat# CRL-7934, RRID:CVCL_0035) and cultured according roles in various functions such as regulation of tumor growth, angio- to the ATCC recommended conditions. The CL-1 DsRed Express genesis, metastasis, matrix remodeling, and immune evasion cells were derived from the CL-1 CRPC cell line (kind gift from Dr through the release of different cytokines [2, 6, 7]. In this context, A. Belldegrun; University of California, LA, CA, USA) [19]. CL-1 two main subsets designated as M1 and M2 have been identified [8, cells were derived from the parental androgen-sensitive LNCaP 9]. While the M2 (alternatively activated) macrophages, closely re- cells grown under androgen deprivation to develop androgen- sembling TAMs, induce an anti-inflammatory response, wound independent variant. As observed in CRPC patients, CL-1 cells healing, and pro-tumorigenic properties, M1 (classically activated) express androgen receptors but lost transcriptional regulation macrophages are involved in the inflammatory response, pathogen of prostate-specific genes in response to the androgen depriva- clearance, and anti-tumor immunity. Recent studies have high- tion, therefore representing an excellent experimental model lighted the role of TAMs in resistance to chemotherapy and protec- to study CRPC. CL-1 DsRed Express cells were grown, between tion against radiotherapy emphasizing the therapeutic importance passages 4 and 12, in RPMI1640 supplemented with 5% heat- of a better understanding of the role of TAMs in the tumor microen- inactivated charcoal-dextran-treated serum (androgen depri- vironment [8–11]. Still, the functional role of macrophages in tumor vation to keep the expression of the androgen receptor up), progression and their prognosis value remain conflicting [12]. 2mM L-Gln, 1 NEAA acids and 1 mM NaP. Prostate cancer Further investigation regarding macrophages functional activities in cells with similar expression level of the DsRed Express fluo- the context of tumor progression are therefore urgently needed. rescent protein (whole, uncloned population) were selected Phagocytosis, from Ancient Greek /aci (phagein), meaning “to by fluorescence-activated cell sorting to reach a purity >96%. devour,” jts  o1 (kytos), meaning “cell,” and -osis, meaning “process,” The expression and homogeneity level of the DsRed Express is a receptor-mediated process by which phagocytes, such as mac- fluorescent protein were then verified by fluorescence mi- rophages, neutrophils, and monocytes, engulf and kill invading croscopy as we described in [18]. Importantly, we demon- pathogens, remove foreign particles, and clear cell debris (>0.5mm strated that both CL-1 and PC3 DsRed expressing cells in diameter). Phagocytosis is important in fighting infections and maintain the characteristics of the parental cell lines CL-1 maintaining tissues homeostasis [13, 14]. On the other hand, resis- and PC3, respectively, therefore validating DsRed expressing tance to phagocytosis has also been recognized for its role in the cells suitability [18]. growth and metastasis of human solid tumors [9]. Surprisingly, the detailed mechanisms of tumor cell phagocytosis by macrophages Co-culture of CRPC cells and macrophages remain incompletely understood urging for more studies. While ob- serving cell behavior in their native environment may represent the 1. Five days before imaging, gently scrape the monocytes/mac- best option to attain increased level of knowledge, in vivo data may rophages from the maintaining culture flask. After centrifu- be also extremely challenging to obtain because of the complexity gation, resuspend the cells in fresh complete medium and of the model and of the native environment [15]. In contrast, a two- seed the 2.5  10 cells into a 10-cm cell culture dish in dimensional (2D) co-culture system is a cell culture method in 10 ml of complete medium. After 24 h, remove the medium which only two or more different populations of cells are grown on from the dish and treat for 48 h with 10 ml of fresh com- planar surfaces allowing some levels of cell contact [16]. plete medium that contains 50 nM phorbol 12-myristate Investigating these cellular interactions could provide insight into 13-acetate (PMA, Sigma-Aldrich). As we previously demon- cell functions and mechanisms that are only apparent when an- strated, PMA treatment activates RAW264.7 cells (reduced other population of cells is present. Therefore, the aim of the pre- expression of PKCa), and induces the differentiation into sent study was to develop a 2D in vitro live co-culture model to macrophages and the adherence of THP-1 cells [20]. As a re- functionally assess and quantify the phagocytosis of tumor cells by sult of PMA treatment, both cell lines express IL10 , High macrophages using a combination of Confocal and Nomarski imag- IL12 , TNFa , iNOS , and Arginase-1 suggesting a Low Low Low High ing microscopy. While the proposed co-culture model was specifi- M2/alternatively activated pathway Note 1: before seeding cally applied to prostate tumor cells in the present study, we the RAW264.7 macrophages, make sure no differentiation emphasized that this sensitive, measurable, and highly reproduc- has occurred (dendrite-like processes and/or increased body ible model can also be used with multiple types of cancer. size). If such cell morphology signs are visible, disregard the maintaining flask and thaw a new cell vial from an earlier passage. Note 2: the number of cells should be decided ac- Materials and methods cording to the cells used. Over-confluent cells may not be sensitive to PMA action leading to flaws in macrophage acti- In the present method, we developed an uncomplicated assay vation/differentiation. to measure and characterize phagocytosis in live human 2. Split the tumor cells (purified DsRed Express fluorescent castration-refractory prostate cancer (CRPC) cells (PC3 and CL-1) cells) 24 h after monocytes/macrophages were treated with and macrophages co-cultures (RAW264.7 and THP-1) using PMA. Before seeding the tumor cells, place 25 mm micro- Confocal and Nomarski Microscopy. scope sterile glass coverslips into wells of 6-well plate. The tumor cells should be 80% confluent before splitting. Seed Cells and cell culture 4 2.5  10 cells into each well of a 6-well plate, containing a Murine macrophage cell line RAW264.7 (ATCC Cat# TIB-71, coverslip, in 2 ml of complete medium and place the 6-well RRID:CVCL_0493) and human monocytic cell line THP-1 (ATCC dish into a CO (5%) incubator at 37 C for 24 h. Note 3: alter- Cat#TIB-202, RRID:CVCL_0006) were purchased from the ATCC natively, non-fluorescent tumor cells may be dyed using the Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Phagocytic activity in live macrophages-tumor cells | 3 Cell Tracker Red CMTPX dye (Life Technologies) as recom- Results mended by the manufacturer. In the present assay, we analyzed the phagocytosis of DsRed 3. One day after the prostate cancer cells have been seeded Express fluorescent tumor cells (PC3 and CL-1) by RAW264.7 into the 6-well plate, aspirate the medium from the 10-cm (Fig. 1A) or THP-1 (Fig. 2) macrophages through confocal micros- cell culture dishes containing the differentiated/activated copy using the Nikon T1-E microscope (60) in combination macrophages. Rinse the macrophages with sterile PBS, then with the appropriate filters. After imaging, the Z-stacks were gently scrape the cells from the dishes and transfer the cell merged using the NIS-Elements AR 4.00.03. The number of suspension into a single conical tube. Count the cells, trans- DsRed Express-positive macrophages, as well as the total num- fer the desired amount of cells (putting into account an extra ber of macrophages was assessed (150 macrophages per con- well) into a fresh conical tube. Centrifuge the cell suspension dition). Percent phagocytosis was then calculated and the data for 8 min at 100 g at 4 C. Carefully aspirate the superna- were represented on a boxplot graph using the IBM SPSS tant. Resuspend the cell pellet in RPMI-1640 (serum-free) Statistics 23 software (Figs 1B, 2). No agonist (-PEDF) was used as containing 1% (v/v) penicillin/streptomycin, 50 nM PMA, a control. As expected, no significant phagocytosis occurred in with or without the agonist of phagocytosis designated as the absence of the agonist (Figs 1A–B, 2). In contrast, we showed the Pigment Epithelium-Derived Factor (PEDF; 10 nM), for a 5 that under stimulatory treatment (in the presence of a phagocy- final concentration of 3.5–7.5  10 cells/2 ml medium [21, tosis agonist/PEDF), the engulfment of cell material was mark- 22]. Finally, aspirate the complete medium from the 6-well edly induced in both macrophages cell lines tested (Fig. 1A-2, plate containing the cancer cells and add 2 ml of the macro- white arrows). Stimulation of phagocytosis was demonstrated phages suspension into each 6 well. Place the 6-well plate by an increased number of fluorescent macrophages and fluo- into a CO (5%) incubator at 37 C, and image live cells after rescent vesicles within the cytoplasm of macrophages, but also 48 h. Note 4: our experience demonstrated that when the by an elevated level of fluorescence in each phagocytosis vesicle cells in the co-cultures are too confluent (>70%), phagocyto- when compared with the control (Fig. 2). The experiment was sis is drastically reduced. Thus, the ratio macrophages: tu- repeated 20 to verify reproducibility. Specificity of the endo- mor cells needs to be adjusted depending on the some/phagosome/phagolysosome/lysosome degradation path- aggressiveness of the tumor cells and the origin (cell lines or way was controlled using the Rab5a and LAMP-1 markers primary cells) of the macrophages. Note 5: the incubation (Invitrogen). Figure 3 shows that both markers overlaid with the time before imaging may vary depending on the phagocyto- DsRed Express-positive cell debris suggesting that the degrada- sis agonist used in this assay. A preliminary kinetics study is tion of ingested cellular material involves phagosome matura- therefore strongly recommended. Note 6: to confirm that the tion through fusion of early endosomes, late endosomes, and fluorescent endocytosis vesicles are targeted to the endo- lysosomes. In this model, we also used Nomarski/DIC some/phagosomes/phagolysosomes/lysosomes degradation (Differential Interference Contrast) to investigate the macro- pathway, the CellLight Endosome/Early Phagosome-GFP phage cell morphology associated with phagocytosis activity. (targeting Rab5a – Ras-related protein 5a, a small GTPase as- Pronounced differentiation of the macrophages (increased body sociated with early phagosomes) and Late Phagosome/ size level and dendritic processes length) was observed in the Lysosomes-GFP (targeting LAMP-1 – lysosomal-associated presence of the agonist suggesting the present method as possi- membrane protein 1, a protein that is utilized to show phag- ble selection tool for phagocytosis agonists. osomes after lysosomal fusion; all from Invitrogen) were In conclusion, our data indicate that the presented in vitro used as recommended by the manufacturer [23]. model in correlation with Confocal and Nomarski imaging may Alternatively, other markers such as Mac1, transferrin re- be used to characterize functionally and quantify the phagocy- ceptor can be used [23–25]. tosis of tumor cells by macrophages in live co-cultures. Nomarski and Confocal microscopy imaging Discussion Before imaging, prepare the cell suspension buffer (CSB): 0.35 mM Na HPO , 110 mM NaCl, 0.44 mM KH PO , 5.4 mM KCl, Macrophages have been acknowledged as the major inflamma- 2 4 2 4 1 mM MgSO , 1.3 mM CaCl , 25 mM HEPES, pH: 7.4 [26]. Carefully tory cell type present in the microenvironment of most human 4 3 remove the 25 mm microscope cover glass using forceps and solid tumors including prostate tumors. Furthermore, interac- V R place it into the bottom of an Attofluor Cell Chamber (Life tions between cancer cells and macrophages are important in Technologies, A-7816). Adjust the cover glass in the center of allowing cancer cells to avoid immunological surveillance and the chamber and gently screw on the top chamber. Finally, add creating a favorable environment for tumor growth and dissemi- 1 ml CSB to the top chamber. nation. While TAMs are usually considered beneficial for tumor Nomarski/DIC images and confocal images were obtained us- cell migration, invasion, and metastasis, depending on the tumor ing the Nikon T1-E microscope with A1 confocal and STORM microenvironment it is now recognized that macrophages can super-resolution with a 60 objective (N.A. 1.4; oil; Z-stack). After also be detrimental to the tumor through cytotoxic and/or phago- imaging, Z-stacks were merged. Percent phagocytosis was deter- cytic activities [6]. Since macrophages are particularly important mined by counting the number of DsRed Express positive macro- for tumor development, analyses of the molecular interactions phages over the total number of macrophages. More than 150 between macrophages and tumor cells, and quantification of the macrophages per condition treatment were investigated to con- phagocytic activity of macrophages are essential for the determi- trol statistical significance within a single experiment. The exper- nation of their functional activities. Still, the characterization of iment was repeated at least five times to verify reproducibility. these molecular mechanisms in vivo remains complicated and re- Note 7. To validate that the red fluorescent present within quires expensive high-technology equipment. There is therefore the cytoplasm of macrophage does come from the DsRed an increasing demand for in vitro methodologies to study the role Express-positive tumor cells, spectral imaging microscopy can of cell–cell interactions in the tumor microenvironment. Using be used. cell co-cultures has been recognized as a suitable model to study Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4| Martinez-Marin et al. Figure 1: The phagocytosis agonist PEDF increases the engulfment of tumor cells by RAW264.7 macrophages. DsRed Express fluorescent prostate cancer cells (PC3 or CL-1) and PMA-activated macrophages were co-cultured as described in the “Materials and methods” section. After 24 h, the co-cultures were treated with 50 nM PMA in the presence or absence of PEDF (10 nM). Phagocytosis was analyzed by Confocal and Nomarski/DIC microscopy 48 h after the initiation of the treatment. (A) Representative pictures of prostate tumor cells (Red) – macrophages (unstained) co-cultures showing an increase in the number of DsRed Express-positive macro- phages, as well as in the number of DsRed Express-positive vesicles within the macrophages treated with PEDF. Top panel: Red fluorescence; middle panel: Nomarski; bottom panel: overlay Red fluorescence/Nomarski. White arrows: intracellular vesicles located within the cytoplasm of macrophage which contains DsRed Express fluorescent tumor cell debris. (B) Quantification of % Phagocytosis. Number of macrophages positive for the DsRed Express protein and the total number of macro- phages (>150 per treatment condition) were counted. Percent phagocytosis was then calculated. Data were represented using a boxplot graph showing the median, in- ter-quartile range, upper and lower quartiles, and whiskers. Statistical analysis was performed using one-way ANOVA followed by Games–Howell post-hoc test. *P < 0.05. Graphs and statistical analysis were performed using the IBM SPSS Statistics 23 software. the tumor microenvironment and as such, the impact of this prostate tumor cells by macrophages using Confocal and study using a combination of Nomarski and Confocal microscopy Nomarski microscopy. The present protocol presents multiple is highly significant [16]. advantages compared to other pre-existing in vitro assays. First, There are currently a number of assays available to assess this assay is easy to perform with no required specialization in phagocytic activity in vitro. Most of these assays include using cell culture. While we recognize the equipment price and avail- fluorescence labeled latex beads, fluorescent polystyrene micro- ability may be limiting (similarly to other technologies such as spheres [27], zymosan, IgG-opsonized or IgM/complement com- intravital multiphoton imaging), this protocol remains inexpen- ponent 3-opsonized erythrocytes [28]or Escherichia coli particles sive without requiring the purchase of additional kits, specific [29], and microscopes or flow cytometers [17] to measure the supplies, reagents, or mice. This assay does not involve any pro- phagocytic activity of particular phagocytes. The present proto- cessing of the samples (single-step protocol) allowing the re- col describes the materials and methods, and notes for the searcher to study functionally live cells. A second advantage of quantification and characterization of the phagocytosis of working with live cells may be a possible combination with time Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Phagocytic activity in live macrophages-tumor cells | 5 Figure 2: Stimulation of the phagocytosis of CL-1 prostate tumor cells by THP-1 macrophages. PMA-differentiated THP-1 were cultured alone (left panel) or with DsRed Express-fluorescent CL-1 cells (center and right panels) þ/ a phagocytosis agonist (10 nM PEDF) as described in the “Materials and methods” section. Cells were im- aged using Nomarski (left and center panels) or Confocal (right panel) microscopy. Number of macrophages positive for the DsRed Express protein and the total num- ber of macrophages (>150 per treatment condition) were counted. Percent phagocytosis was showed using a boxplot graph. Data were represented using a boxplot graph showing the median, inter-quartile range, upper and lower quartiles, and whiskers. Statistical analysis of the data was performed using the Student’s t-test. *P < 0.05. Graphs and statistical analysis were performed using the IBM SPSS Statistics 23 software. Figure 3: DsRed Express-positive macrophagic vesicles co-localized with markers of the Phagosome/phagolysosome/lysosome degradation pathway. RAW264.7 – CL-1 co-cultures were incubatedþ/ a phagocytosis agonist in the presence of the CellLight Endosome/Early Phagosome-GFP (Rab5a, A) or Late Phagosome/Lysosomes-GFP (LAMP-1, B; both green). Co-cultures were then imaged by Nomarski (left panels) or Confocal (right panels) microscopy. Thin white arrows show Rab5a and LAMP-1 lo- cation within the cytoplasm of unstimulated macrophages. In contrast, thick white arrows show that DsRed Express-positive vesicles within the stimulated macro- phages (þ phagocytosis agonist) were also positive for Rab5a and LAMP-1, therefore identifying these vesicles as part of the endosome/early phagosome and late phagosome/lysosomes, respectively. lapse microscopy. To further characterize this assay, we have antibodies, cell conditioned media, or chemotherapeutic drugs applied this technology to different tumor cell experimental have been extensively tested by our laboratory (Martinez-Marin models including human and mouse melanoma cell lines et al., 2016, submitted for publication; Jarvis et al., 2016, manu- (Plebaneck et al., 2016, submitted for publication). Different script in revision). For all of these agonists, we were able to types of phagocytosis agonists such as recombinant protein, functionally detect and quantify the phagocytosis of tumor cells Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6| Martinez-Marin et al. in a highly reproducible manner. Furthermore, the sensitivity of to complement in vivo studies using primary macrophage line- the assay was high with the detection of phagocytosis for doses age cells. of chemotherapeutic drugs significantly inferior to their serum concentration. Finally, the use of the present assay in combina- Acknowledgements tion with other tests such as competition assays using phagocy- tosis agonist and antagonist, gain/loss of function studies using Data were generated in the Image Analysis Core Facility mammalian expression plasmid, siRNA, shRNA or either path- supported by TTUHSC. ways activators or inhibitors make it a good tool to select and characterize new TAMs targeting drugs for anti-neoplasic treatments. Funding For this study, we chose to use RAW264.7 (most commonly The present study was performed with the financial support used macrophage line) macrophages and THP-1 monocytic cell of the NIH/NCI grant R15CA161634, the School of Medicine lines. Guo and collaborators recently demonstrated in a murine at TTUHSC and The CH Foundation (Lubbock, TX). phagosome proteomics study that several phagosomal func- tions such as acidification and proteolysis are significantly Conflict of interest statement. 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Enomoto R, Imamori M, Seon A, Yoshida K, Furue A, Tsuruda 39. Wyckoff JB, Wang Y, Lin EY et al. Direct visualization of H, et Lee-Hiraiwa E. Proposal for a new evaluation of phago- macrophage-assisted tumor cell intravasation in mammary cytosis using different sizes of fluorescent polystyrene micro- tumors. Cancer Res 2007;67:2649–56. spheres. Advances in Biological Chemistry 2013;3(6):556–563. 40. Egeblad M, Ewald AJ, Askautrud HA et al. Visualizing stromal 28. Hamczyk MR, Villa-Bellosta R, et Andres V. In Vitro cell dynamics in different tumor microenvironments by spin- Macrophage Phagocytosis Assay. Methods in molecular biology ning disk confocal microscopy. Dis Model Mech 2008;1:155–67; 2015;1339:235–246. discussion 165. Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology Methods and Protocols Oxford University Press

Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

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Abstract

Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often con- sidered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detri- mental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better compre- hension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage func- tions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor micro- environment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different typesofphagocytosisagonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer. Keywords: tumor biology; RAW264.7 macrophages (ATCC Cat# TIB-71, RRID:CVCL_0493); THP-1 monocytes (ATCC Cat#TIB-202, RRID:CVCL_0006); CL-1 and PC3 (ATCC Cat# CRL-7934, RRID:CVCL_0035) prostate cancer cells; phagocytosis; cell imaging Introduction situations have been widely described, accumulating evidence yet suggests that macrophages also affect cancer initiation, develop- Macrophages are innate immune cells that play critical roles in the clearance of pathogens and the maintenance of tissue homeostasis ment, and progression [2]. Tumor-associated macrophages (TAMs) are the main population of inflammatory cells present in many [1]. While macrophages modes of action in nonpathological Received: 29 September 2016; Revised: 14 January 2017; Editorial decision: 18 January 2017; Accepted: 1 February 2017 V The Author 2017. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2| Martinez-Marin et al. human solid tumors. TAMs are derived from circulating monocytes (Manassas, VA, USA). Both cell lines were cultured as recom- and their mobilization toward tumor tissues is regulated through mended, between passages 1 and 12, to block gene loss and im- multiple microenvironmental signals such as cytokines, chemo- pair macrophage immune function [17]. Human CRPC CL-1 and kines, extracellular matrix (ECM) components, and hypoxia [2, 3–5]. PC3 DsRed Express cells were established as we described in In response to environmental stimuli, TAMs can appear with a range [18]. Briefly, PC3 DsRed Express were derived from PC3 cells of different phenotypes and exhibit both beneficial and detrimental (ATCC Cat# CRL-7934, RRID:CVCL_0035) and cultured according roles in various functions such as regulation of tumor growth, angio- to the ATCC recommended conditions. The CL-1 DsRed Express genesis, metastasis, matrix remodeling, and immune evasion cells were derived from the CL-1 CRPC cell line (kind gift from Dr through the release of different cytokines [2, 6, 7]. In this context, A. Belldegrun; University of California, LA, CA, USA) [19]. CL-1 two main subsets designated as M1 and M2 have been identified [8, cells were derived from the parental androgen-sensitive LNCaP 9]. While the M2 (alternatively activated) macrophages, closely re- cells grown under androgen deprivation to develop androgen- sembling TAMs, induce an anti-inflammatory response, wound independent variant. As observed in CRPC patients, CL-1 cells healing, and pro-tumorigenic properties, M1 (classically activated) express androgen receptors but lost transcriptional regulation macrophages are involved in the inflammatory response, pathogen of prostate-specific genes in response to the androgen depriva- clearance, and anti-tumor immunity. Recent studies have high- tion, therefore representing an excellent experimental model lighted the role of TAMs in resistance to chemotherapy and protec- to study CRPC. CL-1 DsRed Express cells were grown, between tion against radiotherapy emphasizing the therapeutic importance passages 4 and 12, in RPMI1640 supplemented with 5% heat- of a better understanding of the role of TAMs in the tumor microen- inactivated charcoal-dextran-treated serum (androgen depri- vironment [8–11]. Still, the functional role of macrophages in tumor vation to keep the expression of the androgen receptor up), progression and their prognosis value remain conflicting [12]. 2mM L-Gln, 1 NEAA acids and 1 mM NaP. Prostate cancer Further investigation regarding macrophages functional activities in cells with similar expression level of the DsRed Express fluo- the context of tumor progression are therefore urgently needed. rescent protein (whole, uncloned population) were selected Phagocytosis, from Ancient Greek /aci (phagein), meaning “to by fluorescence-activated cell sorting to reach a purity >96%. devour,” jts  o1 (kytos), meaning “cell,” and -osis, meaning “process,” The expression and homogeneity level of the DsRed Express is a receptor-mediated process by which phagocytes, such as mac- fluorescent protein were then verified by fluorescence mi- rophages, neutrophils, and monocytes, engulf and kill invading croscopy as we described in [18]. Importantly, we demon- pathogens, remove foreign particles, and clear cell debris (>0.5mm strated that both CL-1 and PC3 DsRed expressing cells in diameter). Phagocytosis is important in fighting infections and maintain the characteristics of the parental cell lines CL-1 maintaining tissues homeostasis [13, 14]. On the other hand, resis- and PC3, respectively, therefore validating DsRed expressing tance to phagocytosis has also been recognized for its role in the cells suitability [18]. growth and metastasis of human solid tumors [9]. Surprisingly, the detailed mechanisms of tumor cell phagocytosis by macrophages Co-culture of CRPC cells and macrophages remain incompletely understood urging for more studies. While ob- serving cell behavior in their native environment may represent the 1. Five days before imaging, gently scrape the monocytes/mac- best option to attain increased level of knowledge, in vivo data may rophages from the maintaining culture flask. After centrifu- be also extremely challenging to obtain because of the complexity gation, resuspend the cells in fresh complete medium and of the model and of the native environment [15]. In contrast, a two- seed the 2.5  10 cells into a 10-cm cell culture dish in dimensional (2D) co-culture system is a cell culture method in 10 ml of complete medium. After 24 h, remove the medium which only two or more different populations of cells are grown on from the dish and treat for 48 h with 10 ml of fresh com- planar surfaces allowing some levels of cell contact [16]. plete medium that contains 50 nM phorbol 12-myristate Investigating these cellular interactions could provide insight into 13-acetate (PMA, Sigma-Aldrich). As we previously demon- cell functions and mechanisms that are only apparent when an- strated, PMA treatment activates RAW264.7 cells (reduced other population of cells is present. Therefore, the aim of the pre- expression of PKCa), and induces the differentiation into sent study was to develop a 2D in vitro live co-culture model to macrophages and the adherence of THP-1 cells [20]. As a re- functionally assess and quantify the phagocytosis of tumor cells by sult of PMA treatment, both cell lines express IL10 , High macrophages using a combination of Confocal and Nomarski imag- IL12 , TNFa , iNOS , and Arginase-1 suggesting a Low Low Low High ing microscopy. While the proposed co-culture model was specifi- M2/alternatively activated pathway Note 1: before seeding cally applied to prostate tumor cells in the present study, we the RAW264.7 macrophages, make sure no differentiation emphasized that this sensitive, measurable, and highly reproduc- has occurred (dendrite-like processes and/or increased body ible model can also be used with multiple types of cancer. size). If such cell morphology signs are visible, disregard the maintaining flask and thaw a new cell vial from an earlier passage. Note 2: the number of cells should be decided ac- Materials and methods cording to the cells used. Over-confluent cells may not be sensitive to PMA action leading to flaws in macrophage acti- In the present method, we developed an uncomplicated assay vation/differentiation. to measure and characterize phagocytosis in live human 2. Split the tumor cells (purified DsRed Express fluorescent castration-refractory prostate cancer (CRPC) cells (PC3 and CL-1) cells) 24 h after monocytes/macrophages were treated with and macrophages co-cultures (RAW264.7 and THP-1) using PMA. Before seeding the tumor cells, place 25 mm micro- Confocal and Nomarski Microscopy. scope sterile glass coverslips into wells of 6-well plate. The tumor cells should be 80% confluent before splitting. Seed Cells and cell culture 4 2.5  10 cells into each well of a 6-well plate, containing a Murine macrophage cell line RAW264.7 (ATCC Cat# TIB-71, coverslip, in 2 ml of complete medium and place the 6-well RRID:CVCL_0493) and human monocytic cell line THP-1 (ATCC dish into a CO (5%) incubator at 37 C for 24 h. Note 3: alter- Cat#TIB-202, RRID:CVCL_0006) were purchased from the ATCC natively, non-fluorescent tumor cells may be dyed using the Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Phagocytic activity in live macrophages-tumor cells | 3 Cell Tracker Red CMTPX dye (Life Technologies) as recom- Results mended by the manufacturer. In the present assay, we analyzed the phagocytosis of DsRed 3. One day after the prostate cancer cells have been seeded Express fluorescent tumor cells (PC3 and CL-1) by RAW264.7 into the 6-well plate, aspirate the medium from the 10-cm (Fig. 1A) or THP-1 (Fig. 2) macrophages through confocal micros- cell culture dishes containing the differentiated/activated copy using the Nikon T1-E microscope (60) in combination macrophages. Rinse the macrophages with sterile PBS, then with the appropriate filters. After imaging, the Z-stacks were gently scrape the cells from the dishes and transfer the cell merged using the NIS-Elements AR 4.00.03. The number of suspension into a single conical tube. Count the cells, trans- DsRed Express-positive macrophages, as well as the total num- fer the desired amount of cells (putting into account an extra ber of macrophages was assessed (150 macrophages per con- well) into a fresh conical tube. Centrifuge the cell suspension dition). Percent phagocytosis was then calculated and the data for 8 min at 100 g at 4 C. Carefully aspirate the superna- were represented on a boxplot graph using the IBM SPSS tant. Resuspend the cell pellet in RPMI-1640 (serum-free) Statistics 23 software (Figs 1B, 2). No agonist (-PEDF) was used as containing 1% (v/v) penicillin/streptomycin, 50 nM PMA, a control. As expected, no significant phagocytosis occurred in with or without the agonist of phagocytosis designated as the absence of the agonist (Figs 1A–B, 2). In contrast, we showed the Pigment Epithelium-Derived Factor (PEDF; 10 nM), for a 5 that under stimulatory treatment (in the presence of a phagocy- final concentration of 3.5–7.5  10 cells/2 ml medium [21, tosis agonist/PEDF), the engulfment of cell material was mark- 22]. Finally, aspirate the complete medium from the 6-well edly induced in both macrophages cell lines tested (Fig. 1A-2, plate containing the cancer cells and add 2 ml of the macro- white arrows). Stimulation of phagocytosis was demonstrated phages suspension into each 6 well. Place the 6-well plate by an increased number of fluorescent macrophages and fluo- into a CO (5%) incubator at 37 C, and image live cells after rescent vesicles within the cytoplasm of macrophages, but also 48 h. Note 4: our experience demonstrated that when the by an elevated level of fluorescence in each phagocytosis vesicle cells in the co-cultures are too confluent (>70%), phagocyto- when compared with the control (Fig. 2). The experiment was sis is drastically reduced. Thus, the ratio macrophages: tu- repeated 20 to verify reproducibility. Specificity of the endo- mor cells needs to be adjusted depending on the some/phagosome/phagolysosome/lysosome degradation path- aggressiveness of the tumor cells and the origin (cell lines or way was controlled using the Rab5a and LAMP-1 markers primary cells) of the macrophages. Note 5: the incubation (Invitrogen). Figure 3 shows that both markers overlaid with the time before imaging may vary depending on the phagocyto- DsRed Express-positive cell debris suggesting that the degrada- sis agonist used in this assay. A preliminary kinetics study is tion of ingested cellular material involves phagosome matura- therefore strongly recommended. Note 6: to confirm that the tion through fusion of early endosomes, late endosomes, and fluorescent endocytosis vesicles are targeted to the endo- lysosomes. In this model, we also used Nomarski/DIC some/phagosomes/phagolysosomes/lysosomes degradation (Differential Interference Contrast) to investigate the macro- pathway, the CellLight Endosome/Early Phagosome-GFP phage cell morphology associated with phagocytosis activity. (targeting Rab5a – Ras-related protein 5a, a small GTPase as- Pronounced differentiation of the macrophages (increased body sociated with early phagosomes) and Late Phagosome/ size level and dendritic processes length) was observed in the Lysosomes-GFP (targeting LAMP-1 – lysosomal-associated presence of the agonist suggesting the present method as possi- membrane protein 1, a protein that is utilized to show phag- ble selection tool for phagocytosis agonists. osomes after lysosomal fusion; all from Invitrogen) were In conclusion, our data indicate that the presented in vitro used as recommended by the manufacturer [23]. model in correlation with Confocal and Nomarski imaging may Alternatively, other markers such as Mac1, transferrin re- be used to characterize functionally and quantify the phagocy- ceptor can be used [23–25]. tosis of tumor cells by macrophages in live co-cultures. Nomarski and Confocal microscopy imaging Discussion Before imaging, prepare the cell suspension buffer (CSB): 0.35 mM Na HPO , 110 mM NaCl, 0.44 mM KH PO , 5.4 mM KCl, Macrophages have been acknowledged as the major inflamma- 2 4 2 4 1 mM MgSO , 1.3 mM CaCl , 25 mM HEPES, pH: 7.4 [26]. Carefully tory cell type present in the microenvironment of most human 4 3 remove the 25 mm microscope cover glass using forceps and solid tumors including prostate tumors. Furthermore, interac- V R place it into the bottom of an Attofluor Cell Chamber (Life tions between cancer cells and macrophages are important in Technologies, A-7816). Adjust the cover glass in the center of allowing cancer cells to avoid immunological surveillance and the chamber and gently screw on the top chamber. Finally, add creating a favorable environment for tumor growth and dissemi- 1 ml CSB to the top chamber. nation. While TAMs are usually considered beneficial for tumor Nomarski/DIC images and confocal images were obtained us- cell migration, invasion, and metastasis, depending on the tumor ing the Nikon T1-E microscope with A1 confocal and STORM microenvironment it is now recognized that macrophages can super-resolution with a 60 objective (N.A. 1.4; oil; Z-stack). After also be detrimental to the tumor through cytotoxic and/or phago- imaging, Z-stacks were merged. Percent phagocytosis was deter- cytic activities [6]. Since macrophages are particularly important mined by counting the number of DsRed Express positive macro- for tumor development, analyses of the molecular interactions phages over the total number of macrophages. More than 150 between macrophages and tumor cells, and quantification of the macrophages per condition treatment were investigated to con- phagocytic activity of macrophages are essential for the determi- trol statistical significance within a single experiment. The exper- nation of their functional activities. Still, the characterization of iment was repeated at least five times to verify reproducibility. these molecular mechanisms in vivo remains complicated and re- Note 7. To validate that the red fluorescent present within quires expensive high-technology equipment. There is therefore the cytoplasm of macrophage does come from the DsRed an increasing demand for in vitro methodologies to study the role Express-positive tumor cells, spectral imaging microscopy can of cell–cell interactions in the tumor microenvironment. Using be used. cell co-cultures has been recognized as a suitable model to study Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4| Martinez-Marin et al. Figure 1: The phagocytosis agonist PEDF increases the engulfment of tumor cells by RAW264.7 macrophages. DsRed Express fluorescent prostate cancer cells (PC3 or CL-1) and PMA-activated macrophages were co-cultured as described in the “Materials and methods” section. After 24 h, the co-cultures were treated with 50 nM PMA in the presence or absence of PEDF (10 nM). Phagocytosis was analyzed by Confocal and Nomarski/DIC microscopy 48 h after the initiation of the treatment. (A) Representative pictures of prostate tumor cells (Red) – macrophages (unstained) co-cultures showing an increase in the number of DsRed Express-positive macro- phages, as well as in the number of DsRed Express-positive vesicles within the macrophages treated with PEDF. Top panel: Red fluorescence; middle panel: Nomarski; bottom panel: overlay Red fluorescence/Nomarski. White arrows: intracellular vesicles located within the cytoplasm of macrophage which contains DsRed Express fluorescent tumor cell debris. (B) Quantification of % Phagocytosis. Number of macrophages positive for the DsRed Express protein and the total number of macro- phages (>150 per treatment condition) were counted. Percent phagocytosis was then calculated. Data were represented using a boxplot graph showing the median, in- ter-quartile range, upper and lower quartiles, and whiskers. Statistical analysis was performed using one-way ANOVA followed by Games–Howell post-hoc test. *P < 0.05. Graphs and statistical analysis were performed using the IBM SPSS Statistics 23 software. the tumor microenvironment and as such, the impact of this prostate tumor cells by macrophages using Confocal and study using a combination of Nomarski and Confocal microscopy Nomarski microscopy. The present protocol presents multiple is highly significant [16]. advantages compared to other pre-existing in vitro assays. First, There are currently a number of assays available to assess this assay is easy to perform with no required specialization in phagocytic activity in vitro. Most of these assays include using cell culture. While we recognize the equipment price and avail- fluorescence labeled latex beads, fluorescent polystyrene micro- ability may be limiting (similarly to other technologies such as spheres [27], zymosan, IgG-opsonized or IgM/complement com- intravital multiphoton imaging), this protocol remains inexpen- ponent 3-opsonized erythrocytes [28]or Escherichia coli particles sive without requiring the purchase of additional kits, specific [29], and microscopes or flow cytometers [17] to measure the supplies, reagents, or mice. This assay does not involve any pro- phagocytic activity of particular phagocytes. The present proto- cessing of the samples (single-step protocol) allowing the re- col describes the materials and methods, and notes for the searcher to study functionally live cells. A second advantage of quantification and characterization of the phagocytosis of working with live cells may be a possible combination with time Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Phagocytic activity in live macrophages-tumor cells | 5 Figure 2: Stimulation of the phagocytosis of CL-1 prostate tumor cells by THP-1 macrophages. PMA-differentiated THP-1 were cultured alone (left panel) or with DsRed Express-fluorescent CL-1 cells (center and right panels) þ/ a phagocytosis agonist (10 nM PEDF) as described in the “Materials and methods” section. Cells were im- aged using Nomarski (left and center panels) or Confocal (right panel) microscopy. Number of macrophages positive for the DsRed Express protein and the total num- ber of macrophages (>150 per treatment condition) were counted. Percent phagocytosis was showed using a boxplot graph. Data were represented using a boxplot graph showing the median, inter-quartile range, upper and lower quartiles, and whiskers. Statistical analysis of the data was performed using the Student’s t-test. *P < 0.05. Graphs and statistical analysis were performed using the IBM SPSS Statistics 23 software. Figure 3: DsRed Express-positive macrophagic vesicles co-localized with markers of the Phagosome/phagolysosome/lysosome degradation pathway. RAW264.7 – CL-1 co-cultures were incubatedþ/ a phagocytosis agonist in the presence of the CellLight Endosome/Early Phagosome-GFP (Rab5a, A) or Late Phagosome/Lysosomes-GFP (LAMP-1, B; both green). Co-cultures were then imaged by Nomarski (left panels) or Confocal (right panels) microscopy. Thin white arrows show Rab5a and LAMP-1 lo- cation within the cytoplasm of unstimulated macrophages. In contrast, thick white arrows show that DsRed Express-positive vesicles within the stimulated macro- phages (þ phagocytosis agonist) were also positive for Rab5a and LAMP-1, therefore identifying these vesicles as part of the endosome/early phagosome and late phagosome/lysosomes, respectively. lapse microscopy. To further characterize this assay, we have antibodies, cell conditioned media, or chemotherapeutic drugs applied this technology to different tumor cell experimental have been extensively tested by our laboratory (Martinez-Marin models including human and mouse melanoma cell lines et al., 2016, submitted for publication; Jarvis et al., 2016, manu- (Plebaneck et al., 2016, submitted for publication). Different script in revision). For all of these agonists, we were able to types of phagocytosis agonists such as recombinant protein, functionally detect and quantify the phagocytosis of tumor cells Downloaded from https://academic.oup.com/biomethods/article-abstract/2/1/bpx002/3064667 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6| Martinez-Marin et al. in a highly reproducible manner. Furthermore, the sensitivity of to complement in vivo studies using primary macrophage line- the assay was high with the detection of phagocytosis for doses age cells. of chemotherapeutic drugs significantly inferior to their serum concentration. Finally, the use of the present assay in combina- Acknowledgements tion with other tests such as competition assays using phagocy- tosis agonist and antagonist, gain/loss of function studies using Data were generated in the Image Analysis Core Facility mammalian expression plasmid, siRNA, shRNA or either path- supported by TTUHSC. ways activators or inhibitors make it a good tool to select and characterize new TAMs targeting drugs for anti-neoplasic treatments. Funding For this study, we chose to use RAW264.7 (most commonly The present study was performed with the financial support used macrophage line) macrophages and THP-1 monocytic cell of the NIH/NCI grant R15CA161634, the School of Medicine lines. Guo and collaborators recently demonstrated in a murine at TTUHSC and The CH Foundation (Lubbock, TX). phagosome proteomics study that several phagosomal func- tions such as acidification and proteolysis are significantly Conflict of interest statement. None declared. reduced in the RAW264.7 cell line compared to Bone Marrow- Derived Macrophages (BMDMs) from C57/BL6 mice [30]. In two References other studies, Berghaus et al. and Chamberlain et al. agreed that RAW264.7 cells most closely mimic BMDMs, when compared to 1. Gordon S. The macrophage: past, present and future. Eur J splenic macrophages and bone marrow dendritic cells, in terms Immunol 2007;37(Suppl. 1):S9–17. of cell surface receptors and response to microbial ligands that 2. Chanmee T, Ontong P, Konno K et al. Tumor-associated mac- initiate cellular activation via Toll-like receptors 3 and 4/TLR3-4 rophages as major players in the tumor microenvironment. [31, 32]. Interestingly, TLR3-4 has been recently identified as im- Cancers 2014;6:1670–90. portant player in tumor cell phagocytosis [22]. The totality of 3. Mizutani K, Sud S, McGregor NA et al. 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Biology Methods and ProtocolsOxford University Press

Published: Mar 9, 2017

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