Nanoscale Imaging and Mechanical Analysis of Fc Receptor-Mediated

Tao Yue, Ki Ho Park, Benjamin E. Reese, Hua Zhu, Seth Lyon, Jianjie Ma, Peter J. Mohler, and Mingjun Zhang . Quantifying Drug-Induced Nanomechanics an...
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Nanoscale Imaging and Mechanical Analysis of Fc ReceptorMediated Macrophage Phagocytosis against Cancer Cells Mi Li,†,‡ Lianqing Liu,*,† Ning Xi,*,†,§ Yuechao Wang,† Xiubin Xiao,∥ and Weijing Zhang∥ †

State Key Laboratory of Robotics, Shenyang Institute of Automation, Chinese Academy of Sciences, Shenyang 110016, China University of Chinese Academy of Sciences, Beijing 100049, China § Department of Mechanical and Biomedical Engineering, City University of Hong Kong, Hong Kong, China ∥ Department of Lymphoma, Affiliated Hospital of Military Medical Academy of Sciences, Beijing 100071, China ‡

ABSTRACT: Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and singlemolecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

1. INTRODUCTION In 1997, the U.S. Food and Drug Administration (FDA) approved the world’s first monoclonal antibody (mAb) drug, rituximab, for treating B-cell lymphomas. Since then, mAbs have rapidly developed into a clinically important drug class: more than 25 mAbs are approved, and more than 240 mAbs are currently in clinical studies for a wide range of diseases,1 including cancer, immunological disorders, infection, and neurological diseases.2 The use of mAbs has become a mainstream strategy for the treatment of cancer.3 Many mAbs are specific for antigens expressed by the tumor itself, and some mAbs target the tumor microenvironment.4 Antibodies kill the tumor cells through several mechanisms after their binding to the tumor cells, including the direct action of the antibody, immune-mediated cell killing, and specific effects of the antibody on tumor vasculature and stroma.5 In the immune-mediated killing, the Fc portion of antibodies can recruit immune effector cells (e.g., NK cells, macrophages, and dendritic cells) through Fc receptors, allowing for the activation of effector functions that can then cause the lysis of tumor cells.6 Biological responses induced via the Fc portions of antibodies are powerful and unusual, which can be exploited via the engineering of therapeutic monoclonal antibodies to improve their activity in vivo.7 In fact, the modification of Fc receptor-mediated activities is emerging as one of the most promising ways to increase the clinical potential of antibodies.1 © 2014 American Chemical Society

Consequently, investigating the Fc receptor-mediated cellular cytotoxicity activity is of great significance for developing new antibodies with enhanced efficacies, which can then be used to improve the quality of life of patients. In Fc receptor-mediated cellular cytotoxicity activities, macrophage phagocytosis plays an important role. Phagocytosis is a principal component of the body’s innate immunity in which macrophages and other antigen-presenting cells internalize particulate targets whose size exceeds about 1 μm.8,9 Current research about Fc receptor-mediated macrophage phagocytosis was commonly based on optical microscopy,10−12 which cannot reveal detailed information on cell ultramicrostructures because of the 200 nm resolution limit. In recent years, atomic force microscopy (AFM)13 has emerged as an invaluable multifunctional instrument in the study of biological systems.14 AFM cannot only visualize the submicroscopic structures of biological samples in their native states but also can acquire various biophysical and chemical properties (e.g., mechanical properties, adhesion forces, deformation, and energy dissipation),15 which act as important determinants in the process of life activities, opening up new possibilities for us to uncover the mysteries of life. Though we have learned much Received: November 4, 2013 Revised: January 8, 2014 Published: January 24, 2014 1609

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Figure 1. Integrated AFM fluorescence microscopy experimental setup in this study. (A) Schematic diagram of the hybrid system. Cells are grown on a substrate (e.g., Petri dish, glass slide). AFM probes the cells in liquids. A beam of top-down light is released and transmitted through the cells to generate the optical image. A beam of bottom-up exciting light is released to obtain the fluorescence image. (B) The actual photograph. The inset shows the Petri dish. Cells are grown in Petri dishes. The AFM probe is immersed in the medium in the Petri dish to probe the cells under the guidance of optical (fluorescence) microscopy. (Beyotime Institute of Biotechnology, Haimen, China) was added to the centrifuge tube and incubated for 10 min at 37 °C. After incubation, Raji cells were centrifuged to remove the free CFSE molecules. After the supernatant was removed, fresh PBS was added to stir the cell suspension. Next, rituximab solution was added to the centrifuge tube and incubated for 10 min at 37 °C. Then, Raji cells were incubated with RAW 264.7 cells for 30 min at 37 °C. After incubation, serial optical images and fluorescent images (the time interval was 10 min) of RAW 264.7 cells and Raji cells were recorded at room temperature in 2 h. 2.4. Atomic Force Microscopy. AFM experiments were performed using a Bioscope Catalyst AFM (Bruker, Santa Barbara, CA, USA) that was set on an inverted fluorescence microscope (Ti, Nikon, Tokyo, Japan), as shown in Figure 1. The nominal spring constant of the probe was 0.01 N/m, and its exact spring constant was calibrated by the thermal noise method.16 To image the cellular ultramicrostructures during macrophage phagocytosis, the cells were chemically fixed with 4% paraformaldehyde. The imaging experiments were performed in PBS at room temperature. We can discern Raji cells according to the fluorescence, and then the AFM probe was moved to the macrophages in contact with Raji cells. The imaging mode was the contact mode, and the scan rate was 0.5 Hz. The scan line and the sampling point were 256. Both the height image and deflection image were recorded. For AFM living macrophage imaging, Petri dishes containing RAW 264.7 macrophages were placed directly on the AFM stage. Fifteen living macrophages were selected to obtain cellular morphologies with which to compute the cell height and surface roughness. For each cell, 1 × 1 μm2 and 5 × 5 μm2 local areas were imaged to obtain the cell surface roughness on different scales. Force curves were obtained on the surface of living macrophages to measure the cellular mechanical properties. For each condition (with Raji cells and without Raji cells), 5 macrophages were selected and 50 force curves were obtained in the central region of each cell. For contrast experiments, only rituximab was added to macrophages and force curves were obtained on macrophages before and after the addition of rituximab. To examine the mechanical properties of Raji cells, Raji cells were attached to poly-L-lysine-coated glass slides and then force curves were obtained in the central region of five Raji cells. All of the force curves were obtained at the same loading rate, and the sampling point

about macrophage phagocytosis from the ensemble experiments based on optical microscopy, a knowledge of detailed situations of macrophage phagocytosis at the single-cell level is still scarce, and here we applied integrated AFM fluorescence microscopy to investigate quantitatively the nanoscale cellular morphological and mechanical properties during the Fc receptor-mediated macrophage phagocytosis against cancer cells.

2. MATERIALS AND METHODS 2.1. Cell Lines and Antibody. The macrophage RAW 264.7 cell line and lymphoma Raji cell line were used in this study. RAW 264.7 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China), and Raji cells were obtained from the Affiliated Hospital of the Military Medical Academy of Sciences (Beijing, China). Rituximab (10 mg/mL) was also obtained from the Affiliated Hospital of the Military Medical Academy of Sciences (Beijing, China). RAW 264.7 cells were cultured in DMEM containing 10% fetal bovine serum at 37 °C (5% CO2). Raji cells were cultured in RPMI-1640 containing 10% fetal bovine serum at 37 °C (5% CO2). RAW 264.7 cells were grown in Petri dishes, and Raji cells were grown in flasks. Cells were cultured 24 h before experiments. 2.2. Optical Microscopy. Optical bright field imaging was used to observe the antibody-dependent Fc receptor-mediated macrophage phagocytosis qualitatively against cancer cells. The RPMI-1640 medium containing Raji cells was transferred from flasks to a centrifuge tube and then centrifuged at 1000 rpm for 5 min. After removing the supernatant from the centrifuge tube, fresh phosphatebuffered saline (PBS) was added to stir the cell suspension. Next, rituximab solution was added to the centrifuge tube and incubated for 10 min at 37 °C, which allowed Raji cells to be coated with rituximabs. After incubation, Raji cells were incubated with RAW 264.7 cells for 3 h at 37 °C, and optical bright field images of RAW 264.7 cells and Raji cells were recorded. 2.3. Fluorescence Microscopy. Fluorescence microscopy was used to observe the dynamic process of the antibody-dependent Fc receptor-mediated macrophage phagocytosis against cancer cells. Raji cells were harvested into a centrifuge tube. Then CFSE solution 1610

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Figure 2. Optical images of macrophages and cancer cells after coincubation. (A, B) 10 min after coincubation. (C, D) 3 h after coincubation. (B, D) Enlarged views denoted by the squares in (A, C). The black arrows indicate cancer cells, and the red arrows indicate macrophages. of the force curves was 512. Before acquiring force curves on cells, force curves were obtained on the bare area of the substrate to calibrate the deflection sensitivity of the probe. 2.5. Data Analysis. The cell height and surface roughness of the macrophages were computed from the AFM height images of macrophages by using offline software Nanoscope Analysis (Bruker, Santa Barbara, CA, USA). According to the manual of the AFM supplier, the computed roughness Rq is root-mean-square average of height deviations taken from the mean image data plane, expressed as

Rq =

∑ Zi 2 N

approach curve to the indentation curve according to the contact point visually determined in the approach curve.

3. RESULTS AND DISCUSSION Figure 1 shows the integrated AFM fluorescence microscopy experimental system used in this study. Figure 1A is the schematic diagram, and Figure 1B is the actual photograph of the hybrid experimental system. With the AFM fluorescence microscopy platform, we can use AFM to probe the nanoscale activities of interesting cells under the guidance of fluorescence microscopy.18,19 We first used optical microscopy to observe the coincubation results of macrophages and cancer cells, and the results are shown in Figure 2. Lymphoma cells were first coated with rituximab and then were incubated with macrophages. The Fab domains of rituximab can bind to the CD20 antigen on the surface of lymphoma cell, and then the Fc domains of rituximab can bind to the Fc receptor on the surface of macrophages, which activates macrophage phagocytosis.20 Figure 2A,B shows the optical bright field images of the cells 10 min after coincubation. Figure 2B is an enlarged view denoted by the square in Figure 2A. We can discriminate cancer cells (denoted by the black arrows) and macrophages (denoted by the red arrows) in the optical images according to the cell morphology. A macrophage is a type of adherent cell, and the lamellipodium was clearly discernible. A lymphoma cell is a type of mammalian suspended cell and exhibits a round shape.

(1)

where N is the number of the points within the selected region of the image, Zi is the current height values. The Young’s modulus of macrophages were calculated according to the conical Hertz model17

F=

2Eδ 2 tan θ π(1 − υ2)

(2)

where υ is the Poisson ratio of the cell (0.5), δ is the indentation depth, θ is the half-opening angle of the AFM tip, E is the cell Young’s modulus, and F is the applied loading force. The Young’s modulus of macrophages was obtained by running the software package (programmed by ourselves using Matlab) on the approach curves. From the original force curves, we can obtain the deflection of the cantilever that can be used to compute the loading force according to Hooke’s law. The indentation can be obtained by converting the 1611

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Figure 3. CFSE staining of lymphoma Raji cells. (A, C) Optical bright field images. (B, D) Corresponding fluorescent images.

There were five cancer cells in Figure 2B, but the phagocytosis phenomenon was not clear. Then we extended the coincubation time, and Figure 2C,D shows the optical field images of the cells 3 h after coincubation. Figure 2D is the enlarged view denoted by the square in Figure 2C. In this case, we can distinctly observe macrophage phagocytosis from the optical images. In Figure 2D, there are three cancer cells. We can see that one cancer cell (denoted by the II black arrow) was engulfed by the macrophage, whereas one cancer cell (denoted by the I black arrow) was in contact with the macrophage and the third cancer cell (denoted by the III black arrow) was moving toward the macrophage. Research on macrophage phagocytosis against polystyrene particles has showed that macrophage phagocytosis is closely related to the target geometry, such as local shape and the initial contact points.9 Here we can see similar experimental phenomena. When cancer cells (coated by antibodies) and macrophages are coincubating, several cancer cells might adhere to a macrophage via antibodies, but only the cells that have correct local shapes and initial contact points with the macrophage can be successfully engulfed. The results of Figure 2 qualitatively show macrophage phagocytosis against cancer cells. To investigate macrophage phagocytosis against cancer cells better, we need to recognize the two interacting cells accurately. Carboxyfluorescein succinimidyl ester (CFSE) is a commonly used living cell fluorescent dye,21,22 and cells still have biological activity after

CFSE staining. Hence we used CFSE for cell recognition. The working concentration of CFSE followed the supplier’s recommendations. First lymphoma Raji cells were tested by CFSE staining, and the results are shown in Figure 3. CFSEstained Raji cells were dropped onto glass slides (coated with a layer of poly-L-lysine to adhere the cells to the glass slide), and fluorescent images were recorded. Figure 3A,C shows the optical bright field images of Raji cells, and Figure 3B,D shows the corresponding fluorescent images. We can see that all of the Raji cells exhibit uniform green fluorescence. CFSE molecules can infiltrate the cell membrane and then bind the lysine of proteins inside the cell, which makes the cell exhibit fluorescence after the stimulation of excitation light. Then we used the established staining procedure to recognize cancer cells specifically during macrophage phagocytosis. After the CFSE-stained Raji cells were coated with rituximabs, Raji cells were incubated with macrophages. The cells were then serially observed with optical and fluorescence microscopy, and the results are shown in Figure 4. Figure 4A,B shows the optical bright field image and the corresponding fluorescent image, respectively. In the optical image, there were macrophages and cancer cells, and in the fluorescent image, several cancer cells exhibited fluorescence. Figure 4C is the overlay image, and Figure 4D is the enlarged view denoted by the black square. We can clearly see that a cancer cell (denoted by a black arrow in Figure 4D) is in contact with a macrophage (denoted by a red arrow in Figure 4D), and the identity of the 1612

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Figure 4. Dynamic process of macrophage phagocytosis against cancer cells. (A) Optical image and (B) corresponding fluorescent image. (C) Overlay image. (D) Enlarged view of the optical image denoted by the square in C. The black arrow indicates the cancer cell, and the red arrow indicates the macrophage. The white arrows indicate the lamellipodia. The blue arrow indicate the filopodia. (D−O) Serial optical images. The insets are the corresponding fluorescent images of the cancer cell.

Figure 5. AFM images reveal the changes in cellular ultramicrostructures during the different steps of macrophage phagocytosis against cancer cells. The cancer cells are identified by the green fluorescence. (A, G, M) Overlay images of optical images and fluorescent images. Cancer cells exhibit green fluorescence. (A−F) Initial contact stage between a cancer cell and macrophage. (G−L) Beginning of engulfing. (M−R) Some proportion of the cancer cell has been engulfed. AFM (B, H, N) height images and (C, I, O) deflection images on a large scale. AFM (D, J, P) height images and (E, K, Q) deflection images on a small scale denoted by the red squares. (D, J, P) Asterisks indicate the cancer cells. (F, L, R) Schematic diagrams of phagocytosis.

every 10 min. The insets are the corresponding fluorescent images of the cancer cell. At the beginning (0 min), the boundary between the cancer cell and macrophage was

cancer cell was verified by the fluorescent image. Figure 4D−O shows the dynamic changes in macrophage phagocytosis against the cancer cell in 2 h. The images were obtained 1613

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Figure 6. AFM images of macrophages engulfing several cancer cells simultaneously. (A−J) One macrophage engulfing three cancer cells. (K−R) One macrophage engulfing six cancer cells. (A) Overlay of optical image and fluorescence image. AFM (B) height image and (C) deflection image of the macrophage. (D, E) AFM local scanning images of cancer cell I (denoted by the red square in C). (F, G) AFM local scanning images of cancer cell II (denoted by the blue square in C). (H, I) AFM local scanning images of cancer cell III (denoted by the green square in C). (J) Schematic diagram. (K) Overlay of the optical image and fluorescence image. AFM (L) height image and (M) deflection image of the macrophage. (N, O) AFM local scanning images denoted by the red square in M. (P, Q) AFM local scanning images denoted by the blue square in M. (R) Schematic diagram. The asterisks indicate the cancer cells.

apparent, and the fluorescence intensity of the cancer cell was uniform. Then the macrophage began to deform and engulf the cancer cell. After 60 min, we can clearly see that a small proportion of the cancer cell had been swallowed into the macrophage (Figure 4J). We can see that the fluorescence intensity in Figure 4J is nonuniform. This is because the macrophage coated some part of the cancer cell, and this influenced the fluorescence intensity of the cancer cell. After 90 min, an interesting phenomenon occurred. The macrophage did not continue to swallow but released the cancer cell (Figure 4M−O). The fluorescence intensity in Figure 4O is again uniform because the cancer cell has been released from the macrophage. During phagocytosis, we can see the remarkable reorganization of cell lamellipodia (denoted by the white arrows in Figure 4D,O) and filopodia (denoted by the blue arrow in Figure 4J). This is because phagocytosis is a complex process driven by a finely controlled rearrangement of the actin cytoskeleton.23 The rearrangement of the actin cytoskeleton caused the deformation of and morphological changes in the macrophages. From Figure 4, we can see that the macrophage engulfed the cancer cell in the first 90 min but finally released the cancer cell. Research has shown that after 30−60 min of coincubation at 37 °C (5%CO2) antibody-dependent Fc receptor-mediated macrophage phagocytosis against cancer cells could be successfully observed.10,11 The experiments here were done at room temperature under ambient conditions, which may influence the physiological activity of the cells. In future studies, we can improve the experiments by using a temperature-controlled stage14 that will allow us to investigate cell behavior better during cellular biological functions. Besides, in antibody-dependent macrophage phagocytosis, researchers have found that trogocytosis (trogocytosis is the fast uptake of

membranes and associated molecules from one cell by another24) can occur simultaneously,25 and this may also influence phagocytosis. The results in Figure 4 showed the dynamics during the macrophage phagocytosis against cancer cells, but the detailed cellular morphological changes cannot be revealed by optical microscopy because of its 200 nm resolution limit. We then used AFM to image the nanoscale cellular morphological changes during macrophage phagocytosis, and the results are shown in Figure 5. After coincubating the cancer cells and macrophages for 3 h, the cells were chemically fixed and then imaged by AFM under the guidance of fluorescence microscopy. First, the optical bright field images and fluorescent images were obtained. Then AFM images were obtained. Figure 5A−F shows the cellular morphologies at the initial contact step between a cancer cell and macrophage. From the overlay of the optical image and fluorescent image (Figure 5A), we can clearly discern the cancer cell by the fluorescence. Figure 5B,C shows the AFM height image and deflection image of the two cells, respectively. Figure 5D,E shows the AFM height image and deflection image of the contact area (denoted by the red square in Figure 5C), respectively. From the optical image, we cannot see the detailed situation of the contact area between the two cells, although the AFM images clearly show that the cancer cell docked on the macrophage and the engulfing of the macrophage did not occur. Figure 5F is a schematic diagram of phagocytosis at the initial contact step. Figure 5G−L shows the cellular morphologies at the beginning of engulfing. Figure 5G is the overlay image. The cancer cell was identified by the fluorescence. Figure 5H,I shows the AFM height image and deflection image, respectively. Figure 5J,K shows the local-area AFM images (denoted by the red square in 1614

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Figure 7. AFM images of living macrophages. Optical image of a macrophage with a cancer cell (A) before and (B) after AFM scanning. The cancer cell was denoted by the black arrow. (C) Optical image of a living macrophage. AFM (D) height image and (E) deflection image of the macrophage. AFM (F) height image and (G) deflection image of the local area on the macrophage (denoted by the square in E). The white arrows indicate the lamellipodium and filopodium. (H) Profile lines taken in the AFM height images (D, F) along the dashed lines.

image of cancer cell II (denoted by the blue square in Figure 6C), respectively. The boundary between cancer cell II and the macrophage is apparent, and the cancer cell has not been engulfed. Figure 6H,I shows the AFM local scanning height image and deflection image of cancer cell III (denoted by the green square in Figure 6C), respectively. The AFM images shows that the macrophage is beginning to wrap around cancer cell III. Figure 6J is the schematic diagram. Figure 6K−R shows phagocytosis where one macrophage engulfs six cancer cells. From the overlay image (Figure 6K), we can see that the macrophage also exhibits fluorescence, and we can see that five cancer cells are around the macrophage, although from the AFM deflection image (Figure 6M) we can clearly see that besides the five cancer cells around the macrophage there was also one cancer cell (denoted by the white arrow in Figure 6M) that had been swallowed by the macrophage. It was this cancer cell that make the macrophage exhibit fluorescence. Figure 6N−Q shows the AFM local scanning images of two cancer cells (denoted by the red and blue squares in Figure 6M), and the macrophage has begun to engulf these two cancer cells. Figure 6R is the schematic diagram. From Figure 6, we can see that when several cancer cells bind to the different regions of one macrophage, the macrophage can simultaneously engulf these cancer cells. Optical imaging sometimes cannot reveal how many cancer cells are engulfed by the macrophage, whereas AFM imaging can accurately reveal the cancer cells around the macrophage. Rituximab is a powerful mAb against B-cell lymphomas and has now become a mainstay in clinical therapy. Rituximab has been shown to induce apoptosis, complement-mediated lysis, and antibody-dependent cellular cytotoxicity in vitro.26 Traditional research on the rituximab’s killing mechanism was based on ensemble results from many cells.10−12 Detailed information about the rituximab-induced biological activity at single-cell levels is still scarce. Here, AFM-fluorescence microscopy was

Figure 5I). In this case, from the AFM images (Figure 5J,K), we can distinctly see that the macrophage opens its mouth to engulf the cancer cell (denoted by the white arrow in Figure 5J), although this detailed morphological feature could not be revealed by optical microscopy (Figure 5G). Figure 5L is a schematic diagram of the beginning of engulfing. Figure 5M−R shows the cellular morphologies at the step in which the macrophage has engulfed some proportion of the cancer cell. Figure 5M is the overlay image, and the cancer cell is identified by the fluorescence. Figure 5N,O shows the AFM height image and deflection image of the cells, respectively, and Figure 5P,Q shows the local-area AFM images. Figure 5R is a schematic diagram of this step. From the optical image (Figure 5M), we did not know whether the cancer cell had been engulfed by the macrophage, although from the AFM images we can clearly see that some proportion of the cancer cell had been engulfed by the macrophage. The results of Figure 5 indicate that AFM can reveal detailed situations on the nanoscale that were not be accessible by optical microscopy, thus providing novel information that can help us to understand cell biology better. In the experiments, we found that one macrophage could engulf several cancer cells simultaneously, as shown in Figure 6. Figure 6A−J shows phagocytosis where one macrophage engulfs three cancer cells. Figure 6A is the overlay of the optical image and fluorescent image. From the overlay image, we can discern that three cancer cells contacted the macrophage according to the fluorescence. Figure 6B,C shows the AFM height image and deflection image, respectively. Figure 6D,E shows the AFM local scanning height image and deflection image of cancer cell I (denoted by the red square in Figure 6C), respectively. From the local scanning AFM images, we can see that the boundary between cancer cell I and the macrophage was not clear and some proportions of cancer cell I had been coated by the macrophage. Figure 6F,G shows the AFM local scanning height image and deflection 1615

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of living macrophages, we can see the apparent morphological features of macrophages (e.g., the long lamellipodium, the corrugated surface morphology). We know that macrophages move in the tissues to kill the malignant cells, and the special morphology may be beneficial and suited to the phagocytosis function of macrophages. With AFM images, we can further extract quantitative data about the cellular topography. From the line profiles of cellular ultrastructures (Figure 7H), we can see that the height of the lamellipodium (red curve in Figure 7H) was in the range of 0.6−1 μm, and that the height of ruffles (blue curve and black curve in Figure 7H) was in the range of 0.2−1 μm. Then we imaged 15 macrophages to obtain quantitative topographical data about macrophages, as shown in Table 1. For each cell, the

used to image the cell ultramicrostructures during rituximabinduced macrophage phagocytosis against cancer cells, visually revealing detailed cellular morphological changes during phagocytosis. The information obtained by AFM is inaccessible by optical microscopy. From AFM images, we can discern the detailed contact situations between the macrophage and cancer cell, such as the initial contact, the beginning of engulfing, and the progress of engulfing. Besides, we can exactly discern how many cancers were engulfed by the macrophages by AFM imaging. Though electron microscopy can also reveal ultramicrostructures,27,28 it requires the cells be dried, which restricts its application. AFM can provide direct visual information (e.g., cell geometry,29,30 cell morphology31) in liquids, which is meaningful for us to understand the biological functions better. Researchers have used AFM to investigate cell−cell interactions (e.g., fungus-macrophage,21 bacteriaphage32,33) and virus−cell interactions.34 In this study, the nanoscale cellular morphological features during macrophage phagocytosis against cancer cells were visualized, improving our understanding of the macrophage phagocytosis against cancer cells. The results in Figures 5 and 6 were obtained on chemically fixed cells. We then tried to image the macrophage phagocytosis on living cells by AFM. Under the guidance of optical microscopy, the AFM tip was moved to a macrophage that had adsorbed a cancer cell (Figure 7A). The cancer cell in Figure 7A was denoted by the black arrow. Then AFM scanning was performed on the two cells, but we found that it is hard to obtain AFM images of living cancer cells. The optical image after scanning is shown in Figure 7B. We can see that the position of the cancer cell changed from one side of the macrophage to the other after scanning. Though cancer cells bind to macrophages via antibodies, the conjugations were not strong enough to withstand the force exerted by the scanning tip, and hence the cancer cells were pushed by the scanning tip. A lymphoma cell is a mammalian suspended cell that cannot grow on the substrate. To image mammalian suspended cells with AFM, we first need to immobilize them via microfabricated pillars35 or wells.36 Here, cancer cells can bind to macrophages via biological conjugation, but this trapping is still weak for AFM imaging. In the case in which a macrophage has engulfed some parts of the cancer cell, as shown in Figure 5O, the conjugation between the cancer cell and macrophage may become stronger and then it may be prone to AFM imaging. To investigate the detailed topography of living macrophages, we then used AFM to image living macrophages, and the results are shown in Figure 7C−H. Figure 7C is the optical image. Figure 7D,E shows the AFM height image and deflection image of the macrophage, respectively. The scan size was 60 μm. Lamellipodium and filopodium were evident in the AFM images (denoted by the white arrows). Then local area scanning was performed on the central region of the cell (denoted by the red square), and the AFM images are shown in Figure 7F,G. We can clearly see the corrugated morphology of the macrophage surface. A macrophage is an adherent cell and can spread on the substrate, which makes it easy to acquire AFM images of living macrophages. In the living organisms, there are various types of cells. Each type of cell has a unique geometry (e.g., shape and size), and the cell geometry is closely related to cellular physiological functions.37 The shape of motile cells is determined by many dynamic processes that involve many interacting elements (e.g., cytoskeleton, cell membrane, cell−substrate adhesions).38 From the AFM images

Table 1. Topographical Information Extracted from AFM Height Images of Macrophages cell id

cell height (μm)

1 × 1 μm2

5 × 5 μm2 roughness (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

3.6 5.9 5.8 6.4 5.0 4.3 5.8 6.4 5.6 6.0 3.9 5.0 3.8 4.9 6.5

13.1 13.9 20.4 33.8 18.3 14.3 15.4 34.0 24.7 20.3 17.0 10.1 18.3 31.0 16.5

80.1 112 149 101 176 162 112 121 113 170 124 88.5 125 98.7 134

whole cellular topography was first obtained. Then the AFM tip was localized to the central region of the cell, and local area scanning was performed on two scales (1 × 1 and 5 × 5 μm2). The cell height was computed from the AFM height image of the whole cell. The surface roughness was computed from the AFM height images of the local areas. The cell membrane roughness has been shown to be a biomarker in the cellular physiological activity (e.g., the membrane roughness of red blood cells indicates the disease status of diabetes patients39 and cell membrane roughness can identify oxidative stressinduced cellular apoptosis40 and also the expression of tumor suppressor protein41). Hence, investigating cell membrane roughness is useful for understanding the cell biological activity. From Table 1, we can see that the cell height of macrophages was in the range of 3−7 μm, the roughness of 1 × 1 μm2 areas was in the range of 10−40 nm, and the roughness of 5 × 5 μm2 areas was in the range of 80−200 nm. We obtained local area images on the central region of macrophages. Because the sizes of the local areas (1 × 1 and 5 × 5 μm2) were much smaller than the size of macrophages, we can approximately consider that the local areas are flat. Alsteens et al.42 have investigated the roughness of the mycobacterial cell surface, and the results showed that the roughness of the cell surface was related to the size of the cell areas. Here we can see that the roughness of macrophages was also related to the size of the cell areas. The cell roughness of 5 × 5 μm2 areas was significantly larger than the roughness of 1 × 1 μm2 areas. However, it should be noted that cell curvature may influence the computed roughness, and 1616

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Figure 8. Measuring the cellular Young’s modulus of macrophages by obtaining force curves for macrophages with or without cancer cells. (A, C, E) Macrophage with a cancer cell . (B, D, F) Macrophage without a cancer cell. Typical force curves obtained on a macrophage (A) with cancer cell or (B) without a cancer cell. The insets in A and B are the optical bright field images. (C, D) Histogram of the Young’s modulus computed from the approach curves. (E, F) Contrast in experimental indentation curve and fitting indentation curve.

the surfaces of cells.46 We then used AFM to obtain force curves on macrophages with and without cancer cells to investigate whether phagocytosis influence cellular mechanical properties. The process of computing the cellular Young’s modulus from a force curve is shown in Figure 8. Figure 8A is a typical force curve obtained on macrophages that trap cancer cells. Figure 8B is a typical force curve obtained on macrophages that do not adsorb cancer cells. From the optical image in Figure 8A, we can clearly see that a cancer cell is in contact with the macrophage, and from the optical image in Figure 8B, the macrophage did not adsorb cancer cells. Each force curve consists of two components: the approach curve and the retract curve. In force curve mode, the tip first approaches and contacts the cell and then retracts from the cell surface. The vertical motion of the cantilever was driven by a piezoelectric tube, and the vertical displacement of the cantilever can be obtained from the tube. When the tip did not contact the cell, the deflection of the cantilever remained constant and the shape of force curve was flat. After the tip contacted the cell, because of the interaction between the tip and cell the deformation of the cantilever changed and then the shape of the force curve became bent. The bending of the cantilever was detected by a laser that was reflected off the back

how to eliminate the influence of cell curvature is the issue that needs to be solved in the future. For the same size, we can see that the roughness is also variable. The variations in cell topographical information (cell height, surface roughness) may be related to the heterogeneity between cells. We know that cells are essentially dynamic, performing various physiological functions at every moment, and this causes the properties between cells to be variable. In fact, heterogeneity has been classically observed and speculated to be a fundamental property of cellular systems,43 and cell heterogeneity can also be observed in populations of monoclonal cells that have been cultured under identical conditions.44 Hence, heterogeneity accompanies the life of each cells. This is particularly important in cancer research. If every cancer cell is unique, then this needs to be taken into consideration for the improvement of cancer treatment and the design of clinical trials to evaluate new therapies.45 Viewed from this aspect, investigating the properties and behaviors of single cells and combining the detected information with ensemble measurements to analyze the underlying regulatory mechanisms are of great significance for uncovering the secrets of life. Besides imaging the topography of cells, AFM can measure the mechanical properties of cells by obtaining force curves on 1617

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Figure 9. Measuring the mechanical properties of macrophages during phagocytosis. (A) Young’s modulus of macrophages (with and without cancer cells) and cancer cells. (B) Young’s modulus of macrophages before and after the stimulation of rituximab.

kPa) into the Hertz model formula, we can obtain the fitting indentation curve (the red curve in Figure 8E), and the contrast in the experimental indentation curve and the fitting indentation curve is shown in Figure 8E. We can see that the experimental indentation curve was consistent with the fitting indentation curve, indicating that the Hertz model is appropriate for depicting the indentation process between the tip and cell. For the indentation curve in Figure 8F, the histogram of computed Young’s modulus values is shown in Figure 8D. The Gaussian fitting of the histogram in Figure 8D showed that the mean value of the histogram was 1.95 kPa, and the contrast in the experimental indentation curve and fitting indentation curve is shown in Figure 8F, indicating that the two curves were also consistent with each other. In the AFM measurement of the cell mechanical properties, an important issue is the influence of the substrate. To reduce the influence of the substrate, we obtained force curves in the central region of the cell.49 Recently, researchers have presented a correction for the Hertz model,50 providing a significant improvement in the estimation of local mechanical properties of thin samples.

side of the cantilever. When the tip and cell were in contact, the tip indents the cell and the indentation reflects the cell’s mechanical properties. By analyzing an approach curve, the mechanical properties (e.g., stiffness, Young’s modulus) of the cell can be obtained, and retract curves are used to analyze the adhesion forces.47 According to the contact point (denoted by the arrows in Figure 8A,B), the approach curve was converted to an indentation curve. The indentation is equal to the difference between the vertical displacement of the cantilever and its bending. The experimental indentation curves are shown in Figure 8E,F (the black curves). By applying the Hertz model, we computed the Young’s modulus of the cell from the indentation curve.48 Each indentation curve contained many discrete data points, and each point could be used to compute a Young’s modulus. Hence, we can often obtain hundreds of Young’s moduli for each indentation curve. For the indentation curve in Figure 8E, the histogram of computed Young’s modulus values is shown in Figure 8C. The Gaussian fitting of the histogram in Figure 8C showed that the mean value of the histogram was 2.55 kPa. After putting the mean value (2.55 1618

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caused changes in the cellular mechanical properties. In this situation, we can use AFM to quantify the changes in the cellular mechanical properties, but when only the Fc domains of rituximab bind to the Fc receptors of the macrophage (the Fab domains of rituximab are free), there was no actin cytoskeleton reorganization and the cell mechanical properties remained unchanged after the single stimulation of rituximab. Cell mechanical properties have proven to be an effective biomarker that indicate the physiological status of cells. Research have shown that cancer cells are softer than normal cells.49 When cells transform from health to malignancy, stiffness profiles of malignant tissues are quite different from those of normal and benign tissues.54 Besides, research on different types of cancer cells showed that aggressive cancer cells were softer than indolent cancer cells.55,56 With the AFM indentation technique, the mechanical properties of a wide range of types of cells were measured, such as breast cells (0.4− 12 kPa), lung cells (10−35 kPa), liver cells (0.3−20 kPa), epithelial cells (0.4−2 kPa), and fibroblasts (0.4−1 kPa).57 For macrophages, research has showed that the Young’s modulus of macrophages was in the range of 0.5−2 kPa.58 Recent research showed that the cell mechanical properties determined the macrophage functions.59 Here the measured Young’s modulus of macrophages was in the range of 1−3 kPa and became 2−5 kPa after the activation of phagocytosis, comparable to the measured values of the cell Young’s modulus by other groups. In the era of personalized medicine, investigating the properties of patients at single-cell levels is increasingly important. The success of personalized medicine depends on the diagnostic tests that identify patients who can benefit from targeted therapies,60 which requires us to characterize the physiological properties of each patient accurately. Because of the fact that cell mechanical properties play an important role in cellular physiological functions and can be used to indicate the cellular physiological status, investigating the cell mechanical properties at the single-cell level will produce meaningful information for developing methods for disease diagnosis and drug efficacy prediction. Here the changes in cell mechanical properties were observed and quantified during macrophage phagocytosis against cancer cells, but the underlying mechanisms is unknown and further research is needed.

According to the process of calculating the cell Young’s modulus described in Figure 8, all of the force curves obtained on macrophages with and without cancer cells were calculated, and the results are shown in Figure 9A. For each situation (with a cancer cell and without a cancer cell), five macrophages were measured. When macrophages adsorbed cancer cells, the Young’s modulus of macrophages was in the range of 2−5 kPa. When macrophages did not adsorb cancer cells, the Young’s modulus of macrophages was in the range of 1−3 kPa. We can see that after the binding of cancer cells the macrophages became stiffer. When the rituximab-coated cancer cells and macrophages were coincubated, cancer cells could come into contact with adherent macrophages simply by landing on or near them under the influence of gravity.25 After cancer cells came into contact with macrophages, the Fc domains of rituximab on the cancer cells then bound to the Fc receptors on the surface of macrophages, and this activated macrophage phagocytosis. The ability of eukaryotic cells to establish their asymmetrical shapes, to transport intracellular constituents, and to drive their motility depends on the cytoskeleton,51 an interconnected network of filamentous polymers and regulatory proteins.52 Actin cytoskeleton determines the mechanical properties of a cell.53 When macrophage phagocytosis was activated, a rapid accumulation of F-actin and associated proteins in the periphagosomal region occurred.8 This phenomenon can be clearly seen in Figure 7A,B. In Figure 7A, we can distinctly see the internalization of the cancer cell in the macrophage. When the cancer cell is pushed by the scanning tip, we can see a distinct concave area on the macrophage (Figure 7B). Hence, the actin cytoskeleton reorganized during macrophage phagocytosis, and this reorganization may lead to changes in the cell’s mechanical properties. To examine whether the introduction of Raji cells causes the stiffening of macrophages, we measured the Young’s modulus of Raji cells. Because a Raji cell is a type of suspended cell, we attached them to the glass slide via poly-L-lysine. Five Raji cells were selected to obtain force curves. From Figure 9A, we can see that the Young’s modulus of Raji cells was 1−3 kPa, comparable to the Young’s modulus of macrophages (without Raji cells). Although the Young’s modulus of macrophages became 2−5 kPa after engulfing the cancer cells, it is significantly larger than the Young’s modulus of Raji cells. Hence, we can conclude that the stiffening of macrophages was caused by the activation of phagocytosis activities of macrophages, not the introduction of cancer cells. For control experiments, we investigated whether the single stimulation of rituximab can influence the cell mechanical properties, and the results are shown in Figure 9B. Force curves were first obtained on five living macrophages. Then rituximab solution was added and incubated for 3 h at 37 °C (5% CO2). After the incubation, force curves were obtained on the five macrophages. From Figure 9B, we can see that before the stimulation of rituximab the Young’s modulus of macrophages was in the range of 1−3 kPa, consistent with the measured Young’s modulus in Figure 9A. After the stimulation of rituximab, the Young’s modulus of macrophages was still in the range of 1−3 kPa. This indicated that the stimulation of rituximab did not influence the mechanical properties of macrophages. In antibody-dependent Fc receptor-mediated macrophage phagocytosis against cancer cells, the cancer cells were linked to the macrophages via antibodies and the actin cytoskeleton rapidly reorganized to assist in the engulfing the cancer cells. The reorganization of the actin cytoskeleton then

4. CONCLUSIONS This work has demonstrated the use of integrated AFM fluorescence microscopy to visualize and quantify the nanoscale cellular properties (ultramicrostructure, surface roughness, and mechanical properties) successfully during antibody-dependent Fc receptor-mediated macrophage phagocytosis against cancer cells. The dynamic changes during macrophage phagocytosis against cancer cells were recorded by time-lapse optical (fluorescence) microscopy. AFM imaging illustrated the distinct ultramorphological features of macrophages during the different stages of phagocytosis. AFM living cell local scanning showed that the macrophages have a heterogeneous surface. The AFM mechanical property measurements indicated that the Young’s modulus of macrophages increased after the binding of cancer cells via rituximabs whereas the single stimulation of rituximab did not influence the cell mechanical properties. In future studies, we plan to visualize the real-time cellular morphological changes during the process of phagocytosis on living cells with time-lapse AFM. These studies will be particularly useful for us to understand macrophage phagocytosis against cancer cells. 1619

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AUTHOR INFORMATION

Corresponding Authors

*Phone: +86-24-2397-0540. Fax: +86-24-2397-0021. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (61175103) and the CAS FEA International Partnership Program for Creative Research Teams. We thank Hui Zhang and Professor Enlong Ma from Shenyang Pharmaceutical University for culturing the cells.

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