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Efficient Capture of Cancer Cells by Their Replicated Surfaces Reveals Multiscale Topographic Interactions Coupled with Molecular Recognition Wenshuo Wang, Haijun Cui, Pengchao Zhang, Jingxin Meng, Feilong Zhang, and Shutao Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b01147 • Publication Date (Web): 06 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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ACS Applied Materials & Interfaces

Efficient Capture of Cancer Cells by Their Replicated Surfaces Reveals Multiscale Topographic Interactions Coupled with Molecular Recognition Wenshuo Wang,†,§ Haijun Cui,†,§ Pengchao Zhang,‡,§Jingxin Meng,† Feilong Zhang,‡,§and Shutao Wang*,†,§ †

CAS Key Laboratory of Bio-inspired Materials and Interfacial Science, CAS Center for

Excellence in Nanoscience, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China. ‡

Beijing National Laboratory for Molecular Sciences, Key Laboratory of Organic Solids,

Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, P. R. China. §

University of Chinese Academy of Sciences, Beijing 100049, P. R. China.

* E-mail Address of Corresponding Author: [email protected].

ACS Paragon Plus Environment

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ABSTRACT: Cell-surface topographic interactions can direct the design of biointerfaces, which have been widely used in isolation of circulating tumor cells or fundamental cell biological researches. By using of three kinds of cancer cell-replicated surfaces with differentiated structures, we uncover that multiscale-cooperative topographic interactions (at both nanoscale and microscale) coupled with molecular recognition enable efficient and specific isolation of cancer cells. The cell replicas precisely inherit the structural features from the original cancer cells, providing not only preferable structures for matching with cancer cells, but also a unique platform to interrogate whether certain cancer cells can optimally match with their own replicated surfaces. The results reveal that cancer cells do not show preferential recognitions to their respective replicas, while the capture agent-modified surfaces with hierarchical structures exhibit improved cancer cell capture efficiencies. Two levels of topographic interactions between cancer cells and cell replica surfaces exist. Nanoscale filopodia on cancer cells can topographically interact with different nanostructures on replica surfaces. In addition, microscale concave/convex on surfaces provide suitable sites for trapping cancer cells. This study may promote smart design of multiscale biofunctional materials that can specifically recognize cancer cells.

KEYWORDS: biointerface, cancer cell recognition, topographic interaction, cell mineralization, hierarchical structure


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INTRODUCTION Topographic interactions between cells and surfaces can direct the design of biointerfaces,1-6 which have been wildly applied to isolation of cancer cells,7-13 regulation of stem cells,14-17 antibiofouling,18,19 etc. For example, several types of nanostructures, such as nanowires,7 nanodots,8 nanofibers,9 and hierarchical structures,10 have been introduced into construction of biointerfaces that can specifically recognize cancer cells. To recognize target cells and regulate cell-material interactions, a variety of molecules such as peptide,20,21 antibody22,23 and aptamer,24-32 have been employed. Rather than solely depending on molecular recognition (e.g., for capturing circulating tumor cells, molecular recognition between epithelial cell adhesion molecule on cell membrane, EpCAM, and its antibody anti-EpCAM is usually employed22,33), those biointerfaces can achieve highly capture efficiency of rare cancer cells by exploiting topographic interactions, which result from structural matching between nanoscale filopodia on cellular surface and nanostructures on biointerfaces. Recently, fractal nanostructured biointerfaces have been engineered to further match surface fractal nanostructures of cancer cells, apparently improving their capability of specific recognition than previous nanowire-arrayed substrates.34,35 Despite that the reported nanostructures for cancer cell isolation are inspired by nanoprotrusions or fractal dimension of cancer cells, the artificial biointerfaces are far from optimal surfaces for matching with cancer cells, which possess multiscale and complex surface structures. Therefore, the effect of structural matching remains elusive, a question that arises is whether a certain type of cancer cells can optimally match with the biointerface owing their featured surface structures? It is challenging to obtain biointerfaces that can precisely replicate the multiscale and complex surface structures of cancer cells. Although cell imprinting as a typical method has been


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polydimethylsiloxane (PDMS),36 polyurethane,37,38 and so on,39-41 it can hardly preserve surface nanostructures of mammalian cells.42-44 Thus, this approach limits the exposing of topographic interactions between cancer cells and their replicas during the recognition process. Recently, silica bioreplication has been utilized to faithfully replicate the structural features of mammalian cells.45 Therefore, it provides us not only with more suitable structures to isolate cancer cells, but also with an opportunity to interrogate whether a certain type of cancer cells can be most specifically and efficiently captured by biointerfaces composing of their own replicated structures. Herein, by using of three kinds of cancer cell replica surfaces with differentiated structures prepared via silica bioreplication, we demonstrate that highly efficient and specific recognition of the biointerfaces to cancer cells originates from molecule-guided multiscale topographic interactions. These cancer cell-replicated surfaces exhibit featured structures ranging from rich to sparse, which are beneficial to clarifying topographic interactions between cancer cells and surfaces at nanoscale and microscale. The results reveal that cancer cells do not show preference for their respective replicas. Structural matching from two scales – nanoscale and microscale – gives rise to topographic interactions between cancer cells and their replicated surfaces. Nanofilopodia on cancer cells can topographically interact with and adhere to functionalized surfaces with different nanostructures. Also, the microscale concave/convex on surfaces provide suitable sites for trapping the whole cells, leading to better cell-surface interactions. Therefore, cell-replicated surfaces that are equipped with hierarchical and complex structures exhibit more efficient cancer cell capture performance under molecular guidance. These findings are helpful


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for guiding the design of smart cell-material biointerfaces with specific recognition to cancer cells. EXPERIMENTAL SECTION Materials: Hydrochloric acid (HCl, 36%-38%, AR) and anhydrous ethanol (≥ 99.8%, GR) were purchased from Beijing Chemical Works. Doubly distilled water (> 18.2 MΩ cm, MilliQ system) was used. Tetramethyloxysilane (98%, TMOS), 3-mercaptopropyl trimethoxysilane (95%, MPTMS), 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS) and CalceinAM were purchased from Sigma-Aldrich. Streptavidin (SA) was purchased from Prospec Tany TechnoGeno Ltd. Biotinylated anti-human EpCAM antibody (anti-EpCAM) was purchased from R&D. Glutaraldehyde was purchased from J&K Scientific Ltd. Cell culture media, Dulbecco's phosphate buffered saline (PBS) and penicillin-streptomycin were purchased from Thermo Scientific. Fetal Bovine Serum (FBS) was purchased from Fisher Scientific. Trypsin-EDTA (0.25%) was purchased from Invitrogen. Six well cell culture plate was purchased from Corning Incorporated (Costar). Cell culture: The human breast cancer cell line (MCF7), human prostate cancer cell line (PC3), human pancreatic cancer cell line (T24), cervical cancer cell line (HeLa), human T lymphocyte cell line (Jurkat T) and Burkitt’s lymphoma cell line (Daudi) were purchased from Beijing Xiehe Hospital. Cells were cultured in media (DMEM for MCF7; F-12K for PC3; MaCcoy’5A for T24; R1640 for HeLa, Jurkat T and Daudi) supplemented with 10% FBS and 1% penicillin-streptomycin at 37℃ and 5% CO2, and were passaged at approximately 90% confluency. Preparation of cell replica surfaces: The fabrication procedure of the cell replica surfaces was described in detail as below. First, the cells (1.2 × 106 cells per well) were added into six


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well cell culture plate, in which clean and sterilized glass substrates (1 cm × 1 cm, sterilized by 75% ethanol) were preloaded. After culture of cancer cells on glass substrates overnight, the glass substrates were occupied completely by cancer cells. Then, the substrates were rinsed by phosphate buffered saline (PBS) 3 times followed by fixing in 2.5% glutaraldehyde at least 20 minutes. After fixation, cells were rinsed 3 times with doubly distilled water and silicified in a solution of 0.1 M tetramethoxysilane (TMOS) at pH 3 and 40 °C for a certain time. Silicified cells were then rinsed with doubly distilled water and dehydrated by gradient ethanol of 50% and 100% (2 ×). Following dehydration and drying in the air, silicified cells were calcined at 550 °C for 2 hours. The morphologies of as-obtained cell replicas were characterized by a field-emission scanning electron microscopy (JSM-7500F, JEOL). Anti-EpCAM modification of silicified cell replica surfaces: The detailed modification procedure was described in our previous work.46 In brief, the cell-replicated substrates were first treated with oxygen plasma and followed by immersing in MPTMS/ethanol overnight. Then, the thiol-functionalized substrates were linked with SA via coupling agent GMBS. Finally, for capturing cancer cells, biotinylated anti-EpCAM was modified on the substrates via biotinstreptavidin interactions. Cell capture experiments: Following immobilization of anti-EpCAM, the cell replica surfaces were washed 3 times with PBS and then placed into a chamber of six well cell culture plate, in which 3 mL of cell suspension (i.e., MCF7, PC3 or T24, 105 cells mL-1) was loaded. After incubation at 37 °C and 5% CO2 for a certain time in a cell incubator (Thermo Forma Series II, Thermo Scientific), the cell replica surfaces were gently rinsed with PBS 3 times. To perform the washing step, one can pick up a substrate by using a tweezer and then dip it into the PBS solution for three times. The captured cells were then stained by Calcein-AM for 5 minutes


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in PBS. Finally, the captured cells were imaged and counted by an inverted fluorescence microscope (Nikon Ti-E, Japan). The cell capture efficiency was calculated by the ratio of the captured cell density on the substrate to the theoretical cell density.35 The theoretical cell density in a well of a six well plate was obtained by dividing the total cell number by the bottom area of the well, and the experimental captured cell density on the substrate was obtained by dividing the average cell number in one fluorescence image by the image area. SEM observation of captured cells on different cell replica surfaces: For characterization of captured cells on different cell replica surfaces, the captured cells were fixed and dehydrated as follows. First, captured cells were fixed with 2.5% glutaraldehyde in PBS for at least 4 hours, followed by a PBS wash. Then, the cells were dehydrated through a series of ethanol concentrations (30%, 50%, 70%, 80%, 95%, 100%, and 100%, 15 min each) at room temperature. Samples were then treated with critical CO2 drying and gold sputtering for subsequent scanning electron microscopy observation (QUANTA FEG 250, 45° tilt in Figure 5). RESULTS AND DISCUSSION In our experiment, three typical EpCAM-positive cancer cell lines with distinctive surface structures – human breast cancer cells (MCF7), human prostate cancer cells (PC3) and human pancreatic cancer cells (T24) – were chosen to fabricate cell replica-based substrates (SubMCF7, SubPC3 and SubT24) (Figure 1 and Figure S2 in the Supporting Information) via silica bioreplication.12,45 After confluent culture on clean glass substrates, cells were fixed in glutaraldehyde, silicified in tetramethoxysilane (TMOS), dehydrated in gradient ethanol and finally calcined to obtain cell replicas. We found that the silicified time is a key factor for precisely preserving the nanoscale structures of the fixed cells (Figure S1 in the Supporting Information). After long-time silicification (e.g., 20 h), the surface nanostructures of cells were


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covered by over deposited silicon oxide. On the other hand, the surface structures faded during calcination under short-time silicification (e.g., 5 h). The silicification time of 17 h was chosen for the following experiments. The silicified cell replica surfaces inherited the structural features from original cells (Figure S3) and showed distinctive structures: SubMCF7 exhibited long dendritic and irregularly sheet structures; SubPC3 revealed short but densely virgulate structures; SubT24 showed little nanostructures except that sparse apophysis were occasionally observed. Thus we obtained three kinds of surfaces with different structures that could be used to interact with specific cancer cells after further modification with capture agent (anti-EpCAM). We then evaluated the performance of structural matching between certain cancer cells and the as-prepared surfaces after modification with anti-EpCAM. We chose 45 min as the optimal cell incubation time since cell capture efficiencies on these substrates almost reached a plateau at this time (Figure 2a and Figure S4 in the Supporting Information). The captured cells were stained by Calcium-AM and counted with fluorescence microscope to calculate cell capture efficiencies. Figure S5 in the Supporting Information showed typical fluorescent images of captured cells on different surfaces at the optimal incubation time. For MCF7 cells, as shown in Figure 2b, their capture efficiency was 61% ± 16%, 59% ± 12%, 48% ± 5% and 7% ± 3% on SubMCF7, SubPC3, SubT24 and SubFlat, respectively. These results indicated that the capture efficiency of nanostructure-rich MCF7 cells on cell-replicated surfaces was much higher than that on flat glass. We suggest that surface structures are significant for enhancing the topographic interactions between surfaces and cells, and hence for increasing the cell capture efficiency. SubFlat exhibited the lowest cell capture efficiency because of the absence of topographic interactions between cells and flat surface. A close experiment setup further demonstrated the importance of structures for cancer cell capture (Figure 2c). In addition, the cell-replicated surfaces derived from different


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cancer cells presented different capture performances for MCF7 cells. As can be seen from Figure 2b, MCF7 cells showed lower capture efficiency on SubT24 than that on SubMCF7 and SubPC3, probably due to the lack of nanostructures on SubT24. Moreover, MCF7 cells displayed similar capture efficiencies on SubMCF7 and SubPC3, indicating that nanostructure-rich MCF7 cells exhibited improved recognitions to cell-replicated surfaces with hierarchical structures while did not show preferable structural matching with their own replicas. Immunofluorescent staining images (Figure 2d) displayed that captured MCF7 cells extended much more protrusions on SubMCF7 and SubPC3 while extended less protrusions on SubT24 and barely stretched out protrusions on flat substrates, further indicating the promoted topographic interactions between MCF7 cells and cell replicas with hierarchical structures. Apart from structural factors, we further uncovered the role of molecular recognition between cell membrane protein EpCAM and its antibody in cancer cell capture. On one hand, the surfaces without anti-EpCAM modification showed much lower cell capture efficiencies; on the other hand, the specificity was also assessed by introducing EpCAM-negative cell lines (Hela, Jurkat T and Daudi) to these surfaces. Inefficient capture of EpCAM-negative cells was verified on antiEpCAM modified surfaces (Figure 3). These results demonstrated the efficient cancer cell recognition was dependent on molecule-guided topographic interaction. We further evaluated the cell capture performance of the other two cancer cell lines owning different surface structures. For PC3 cells with short nanostructures, the capture efficiency on SubPC3 was 62% ± 13%, which was very close to that on SubMCF7 (61% ± 6%) but much higher than that on SubT24 (53% ± 2%) (Figure 4b). These results indicated that PC3 cells also exhibited the enhanced topographic interactions with hierarchically structured cell replicas while did not show preference to their own replicas. In addition, as shown in Figure 4c, the capture efficiency


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of T24 cells on their own replicated surface, SubT24, was lowest (62% ± 3%), much lower than that on SubMCF7 (72% ± 8%) and SubPC3 (70% ± 1%), indicating that T24 cells did not exhibit the best structural matching and recognition with their own replicas. Meanwhile, the cell-replicated surfaces did not bring about the highest capture efficiencies to their original cancer cells. For example, SubMCF7 showed higher capture efficiency for T24 cells (72% ± 8%) than that for MCF7 cells (61% ± 16%), and SubPC3 also showed higher capture efficiency for T24 cells (70% ± 1%) than that for PC3 cells (62% ± 13%), despite that T24 cells express lowest EpCAM among these three cancer cell lines.47 These results suggest that topographic interactions between cancer cells and their respective replicas are not the one to one relationship of structural matching like lock-and-key. To further elucidate the mechanism of topographic interactions between cancer cells and asprepared surfaces, we characterized the morphologies of captured cells on surfaces by scanning electronic microscopy (SEM). Typical SEM images of cells adhered to anti-EpCAM coated cell replica surfaces were shown in Figure 5 and Figure S6 in the Supporting Information. MCF7 cells exhibited many long filopodia (about 100 nm width, 5 µm length)34 on SubMCF7 and SubPC3, while they displayed fewer filopodia on SubT24. PC3 cells also showed more filopodia on SubMCF7 and SubPC3 than on SubT24, despite that their filopodia were shorter. T24 cells extended lamellipodia on all substrates, and at the front of lamellipodia, nanoscale filopodia and wrinkles could be observed. Cells of the same type showed the similar morphologies on SubMCF7 and SubPC3, which means that the captured cells showed similar topographic interactions with the two surfaces. Therefore, the capture efficiencies of each type of cells on SubMCF7 and SubPC3 revealed no significant difference. Compared with those on SubT24, the captured cells on SubMCF7 and SubPC3 showed much more filopodia. Since topographic interactions occurred between nanoscale


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components of cells and nanostructures on cell replicas, the surfaces with abundant nanostructures (i.e., SubMCF7 and SubPC3) were capable of efficiently capturing these three cancer cell lines. Besides, on surfaces with high density of nanostructures, the filopodia could grasp them by molecular recognition between EpCAM and anti-EpCAM. While on surfaces with less nanostructures (i.e., SubT24), most of the filopodia retracted, leading to weak topographic and molecular interactions.48 Thus, cancer cells showed high affinities to the nanostructure-rich surfaces under molecular guidance. These results indicated that nanostructures on surfaces play a key role in the capture of target cancer cells by improving topographic interaction and molecular recognition between cells and surfaces at nanoscale. In addition to nanostructures, microstructures enhanced the topographic interaction between cancer cells and surfaces. As shown in SEM images, for SubT24 with a few nanostructures, these three different cells were all inclined to adhere to the concave region of SubT24. The cell profile-derived microscale concave/convex provided suitable sites that could trap the cells with the assistance of molecular recognition. This trapping effect enhanced the topographic interaction at microscale between captured cells and surfaces. Thus, the cell capture performance was better on SubT24 than that on flat glass. Comparison of the cell capture efficiencies at the incubation time of 45 min between SubT24 (48% ± 5%, 53% ± 2%, 62% ± 3% for MCF7, PC3 and T24 cells, respectively) and SubFlat (7% ± 3%, 6% ± 2%, 15% ± 4% for MCF7, PC3 and T24 cells, respectively) (Figure 4 and Figure S4 in the Supporting Information) proved that microscale structures were important for cell capture by providing trapping effect at microscale. T24 cells showed different morphology from MCF7 and PC3 cells: they recruited more lamellipodia and nanoscale filopodia and wrinkles at the front of lamellipodia. The spreading lamellipodia might enhance topographic interactions between nanoprotrusions at the front of lamellipodia and nanostructures


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on cell replicas in a greater scope, which could improve the capture efficiency of T24 cells on surfaces. To sum up, the cells could topographically interact with nanostructures and could be trapped by microscale structures on surfaces. The cooperative effect of multiscale topographic interactions and molecular recognition highly improved the isolation efficiency of target cancer cells. CONCLUSIONS In conclusion, we investigated topographic interactions between three cancer cell lines with differentiated morphologies and their replica surfaces. The results revealed that the cancer cells did not show obvious preference to their respective replica surfaces. There existed two levels of topographic interactions between cancer cells and their replica surfaces. The nanostructures on surfaces led to structural matching between nanoscale components on cell surface and these nanoscale structures on substrates. Apart from nanostructures, the microscale topography also enhanced the topographic interaction between cells and their replica surfaces by the trapping effect. The biointerfaces that replicated multiscale structures exhibited improved affinities with cancer cells by synergistic effect of cooperative topographic interactions and molecular recognition. This study may advance smart design of multiscale biofunctional materials with highly specific cell recognition and provides an alternative to investigate interfacial properties of cells. ASSOCIATED CONTENTS Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. SEM images of cell-replicated surfaces at different silicified time before and after calcination; SEM images of substrates replicated by three kinds of cancer cells at low magnification; capture


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efficiencies of cancer cells on cell-replicated substrates and flat glass at different incubation time; typical florescence and ESEM images of capture cells; ESEM images of capture cells on flat glass. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Notes The authors declare no competing ﬁnancial interest. ACKNOWLEDGMENTS This research is supported by the National Natural Science Foundation of China (21501184, 21425314, 21434009, 21421061 and 21504098), the Key Research Program of the Chinese Academy of Sciences (KJZD-EW-M01), MOST (2013YQ190467), the Top-Notch Young Talents Program of China, and Beijing Municipal Science & Technology Commission (Z161100000116037). REFERENCES (1) Curtis, A.; Wilkinson, C. Topographical Control of Cells. Biomaterials 1997, 18, 1573-1583. (2) Bettinger, C. J.; Langer, R.; Borenstein, J. T. Engineering Substrate Topography at the Micro- and Nanoscale to Control Cell Function. Angew. Chem., Int. Ed. 2009, 48, 5406-5415. (3) Choi, C. K.; Breckenridge, M. T.; Chen, C. S. Engineered Materials and the Cellular Microenvironment: A Strengthening Interface between Cell Biology and Bioengineering. Trends Cell Biol. 2010, 20, 705-714. (4) Mager, M. D.; LaPointe, V.; Stevens, M. M. Exploring and Exploiting Chemistry at the Cell Surface. Nat. Chem. 2011, 3, 582-589.


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Figure 1. Characterization and modification of three kinds of as-prepared cell replica surfaces. (a–c) Schematic and scanning electron microscopy (SEM) images of cell replicas-based substrates – a) SubMCF7, b) SubPC3 and c) SubT24 – replicated from MCF7, PC3 and T24 cells, respectively. SubMCF7 shows rich and outstretched nanoscale protrusions, SubPC3 dense but short ones, and SubT24 sparse ones. d) The modification process of the as-prepared surfaces with antiEpCAM for cancer cell capture.


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Figure 2. Evaluation of MCF7 cells capture performances on the as-prepared cell replica surfaces after anti-EpCAM modification. a) With the increase of incubation time, the capture efficiencies of MCF7 cells increase significantly and reach a maximum value around 45 min. b) In comparison with other surfaces, SubMCF7 and SubPC3 show higher capture efficiencies of MCF7 cells at incubation time of 45 min. Meanwhile, the capture efficiencies of MCF7 on cellreplicated surfaces are much higher than that on the anti-EpCAM modified flat glass. c) A fluorescent image of captured MCF7 cells on SubMCF7 and SubFlat in a close experiment setup. d) Immunofluorescence images (actin, red; nuclear, blue) of captured MCF7 cells on different surfaces. MCF7 cells own more protrusions on SubMCF7 and SubPC3, while exhibit less protrusions on the other two surfaces. Arrows indicate the protrusions of MCF7 cells.


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Figure 3. The influence of molecular recognition on cell capture performance of various surfaces. a) Comparison between MCF7 cell capture efficiencies on anti-EpCAM modified surfaces and those on only streptavidin modified surfaces. It clearly shows that molecular recognition strongly promoted the cell capture efficiencies on cancer cell-replicated surfaces. b) Capture efficiencies of EpCAM-negative cells on different surfaces modified with anti-EpCAM at optimum incubation time. The results demonstrate that nonspecific cells show much lower adhesion on surfaces due to the lack of molecular recognition.


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Figure 4. Comparison of capture efficiencies of a) MCF7, b) PC3 and c) T24 cells on various anti-EpCAM modified surfaces. These data indicate that i) all these cells exhibit higher affinities with SubMCF7 and SubPC3, both of which own abundant multiscale structures; ii) on SubMCF7 and SubPC3, the capture efficiencies of MCF7 cells, as well as the other two cell types, show no significant difference; iii) compared with MCF7 and PC3, T24 cells display highest capture efficiency on each of the surfaces.


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Figure 5. Typical SEM images of three different types of cancer cells captured on anti-EpCAM coated surfaces. MCF7 and PC3 cells show similar spherical shape and both exhibit numerous filopodia on SubMCF7 and SubPC3, while they display fewer nano-filopodia on SubT24. T24 cells recruit more lamellipodia and nanoscale protrusions at the front of lamellipodia. The nanoscale topographic interactions between nano-filopodia and nanostructures on surfaces enhance cancer cell capture efficiency. In addition, these three different types of cancer cells all tend to adhere to the concave region of SubT24, indicating the trapping effect exists at the microscale between cells and surfaces. Scale bar: 10 µm.


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