Statistical analysis of immuno-functionalized tumor-cell behaviors on nanopatterned substrates
© Kim et al.; licensee Springer. 2012
Received: 3 September 2012
Accepted: 11 November 2012
Published: 22 November 2012
Laser scanning cytometry has been proven as a powerful technology for high-content, high-throughput quantitative analysis of cellular functions in a fully automated manner. It utilizes a large-area fluorescence imaging scheme and rigorous image quantitation algorithms to enable informative analysis of cell samples attached to solid substrates. While this technology represents a powerful approach for high-content screening using cell lines, it has not been applied to the study of tumor-cell behaviors on these solid nanopatterned substrates after several hours of incubation. Herein, we statistically demonstrated functional cellular morphology information, including size, shape, and distribution of the captured cells after 0.5 to 45 h of incubation on nanopatterned substrates, such as silicon nanowires and quartz nanopillars, along with planar glass substrates. With increasing incubation time up to 45 h, we observed that the nanopatterned substrates could have not only increased adhesion and traction forces between cells and nanopatterned substrates, but also limited cell spreading on the substrates compared to the planar glass substrates. On the basis of our results, we suggest that the most important factors to influence the cell behaviors on the three solid substrates are the degree of dimension on cell behaviors and cell traction force.
KeywordsNanowire arrays Cell adhesion Circulating tumor cells Filopodia Cell migration Cell capture efficiency
Nanostructures have been increasingly used for studies on cell interaction with solid nanostructures because unique properties of nanostructured surfaces enable a variety of novel functions of immobilized cells on the nanostructured surfaces[1–3]. For example, Qi et al. recently reported that nanometer-scale topography influences diverse cell behaviors, including cell adhesion, motility, proliferation, and differentiation. In addition, owing to high surface area and increased nanowire-cell surface interaction, nanowire arrays functionalized with capture agents were also demonstrated for high-yield capture of surface-bound cells including immune cell subsets and circulating tumor cells (CTCs)[5–7]. We have also demonstrated a novel platform for separating CD4+ T lymphocytes from mouse splenocytes using streptavidin (STR)-functionalized and vapor–liquid-solid-grown silicon nanowire (SiNW) as well as transparent quartz nanopillar (QNP) arrays having a higher separation efficiency of 93% to 95.3%. More recently, we reported on the development of nanowire substrate-enabled laser scanning cytometry (LSC) for cell analysis in order to achieve quantitative, automated, and functional evaluation of the circulating tumor cells where the captured rare cells were at the very early stage of incubation (<0.5 h). In a previous study, we clearly demonstrated that the LSC method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and protein levels) at the single-cell level. In addition, more detailed studies are still required on how the various size and shape-matched nanostructures and nanomaterials interact in the targeted cells at different times of incubation (>1 h) using pre-proven LSC technique.
Herein, we demonstrate microarray scanner-based imaging cytometry representing a high-content, high-throughput approach to statically characterize tumor cell captured behaviors on nanostructured microchips after 1 to 48 h of incubation on two sets of nanostructures: QNP and SiNW substrates.
Next, SiNW arrays (2.5 × 2.5 cm2) were prepared by Ag-assisted chemical wet-etching process of p-type Si wafer immersed into 10 wt% hydrofluoric (HF) acid for 5 min to remove the native oxide layer and sequentially treated in a boiled RCA solution (H2O2/NH4OH/H2O = 1:1:5) for 1 h to create a hydrophilic surface. An Ag film with a thickness of 30 nm was coated onto (100) Si substrates with the resistivity of 1 to 10 Ω·cm, which were cleaned by electroless deposition in an aqueous solution. The cleaned Si samples were placed in 10% HF and 5 × 10−3 M AgNO3 solution at room temperature for 5 min. The Ag-coated Si samples were then immersed in an aqueous solution containing 10% HF and 0.3% H2O2 at room temperature for 30 min. The Ag metals remaining on the Si substrates were removed in boiling aqua regia (HCl/HNO3 = 3:1) for 1 h and by additional amorphous Si etching for 30 s in buffered oxide etchant (NH4F/HF = 6:1). Figure1b shows the SEM images of wet chemically etched SiNW arrays with tilted view.
Prior to the surface functionalization, three as-prepared nanopatterned substrates (QNP, SiNW, and planar glass substrates for control samples) were carefully cleaned with H2O2/H2SO4 (1:1) for 10 min to remove all of the organic materials and impurities on the surface. Then, the substrates were washed using a conventional three-step cleaning process (acetone, isopropyl alcohol, and DI water) and dried with air. The surface was treated with O2 plasma for 20 s to confer the hydroxyl groups on the substrate surfaces. The surface of the two-nanopatterned substrates with planar glass substrates was functionalized by STR-immobilization method we developed previously (Figure1c). During this procedure, we first applied (3-aminopropyl)-triethoxysilane to aminate the nanowire surface, which can be further functionalized with STR via a two-step aldehyde/amine reaction using glutaraldehyde as the linker. Finally, biotinylated anti-human monoclonal anti-human epithelial cell adhesion molecule (EpCAM), where the targeted cells were pre-mixed, was introduced to the STR-functionalized nanowires through the high-affinity biotin-STR binding. Cell-capture chambers with nine circular wells (5 mm in diameter, Figure1c) were made by molding a polydimethylsiloxane (PDMS) elastomer. The solidified PDMS mold was cut to the size of 2.5 × 2.5 cm2, which is the same size of the surface-functionalized nanopatterned substrates. A solution of the A549 cells (human lung carcinoma cell line, CCL-185) purchased from American Type Culture Collection (ATCC, VA, USA), which are conjugated with biotin-EpCAM-Abs in F12K:DMEM (500 mL, Invitrogen Corporation, NY, USA), with a final volume of approximately 50 μL was then pipetted into each of the nine cell-capture chambers with cell populations of approximately 103 cells/chamber. Three samples with nine cell-capture chambers were prepared in each group (0.5, 21, and 45 h), and the average spreading areas and sizes of each fixed cell on three different substrates were consequently calculated with standard deviation (n = 9).
For the image of surface-bound cells on the nanopatterned substrates, an Axon Genepix microarray scanner 4000B (Molecular Devices, LLC, CA, USA) was used. Green and red YAG lasers (532 and 635 nm) were used to visualize the captured cells (actin, green 532 nm) and nuclei (red 635 nm) on the three different STR-functionalized substrates with approximately 5-μm resolution. The cell-capture platform was automatically scanned, and the scanned images of PDMS cell-capture chambers that contained the captured cells were transported into CellProfilerTM (http://www.cellprofiler.org) cell image software for rapidly quantitation of the captured cells on STR-functionalized dual-nanopatterned substrates with planar glass substrates.
Results and discussion
To further quantify the shape of the cells immobilized on the nanostructures and to support the migration behaviors of the nanostructures, the eccentricity of the cultured cells on the substrates was calculated using CellProfiler software. Figure4d,e,f show the summary of eccentricities of the cells for the three different substrates after 0.5 to 45 h of incubation. As we mentioned previously, the value in eccentricity is between 0 and 1. (0 and 1 are degenerate cases; an ellipse whose eccentricity is 0 is actually a circle, while an ellipse whose eccentricity is 1 is a line segment). Therefore, the average cell shapes on both SiNWs (approximately 0.44 ± 0.25) and QNP (approximately 0.58 ± 0.24) substrates were more circular compared to the planar glass substrates (approximately 0.72 ± 0.15), indicating the cells on planar glass were well spread and freely cultured on the substrates.
In summary, we statistically demonstrated the functional cellular morphology parameters including size, shape, and distribution of the captured cells after 0.5 to 45 h of incubation on nanopatterned substrates of SiNW and QNP, along with planar glass substrates, using a powerful high-content LSC method. With increasing incubation time up to 45 h, we observed that the nanopatterned substrates could not only increase the adhesion and traction force between the cells and nanopatterned substrates, but also limit the cell spreading on the substrates compared to the planar glass substrates. On the basis of our results, we found that the most important factors influencing the cell behaviors on the three sets of solid substrates are the degree of dimension (2-D or 3-D migration) in cell behaviors and the cell traction force. All together, this work demonstrates the utility of microarray scanner-based high-content imaging for post-capture characterization of tumor cells captured on nanostructured microwells and the versatility of this approach for functional characterization of cell behaviors including filopodia or lamellipodia evolution with nanostructured surfaces.
This study was supported by the Priority Research Centers Program and by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010–0029706 and 2010–0019694). This work was partially supported by the Human Resources Development Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) (20104010100660) and by a grant from the Global Excellent Technology Innovation R & D Program funded by the Ministry of Knowledge Economy, Republic of Korea (10038702-2010-01). SKL thanks Prof. Rong Fan at Yale University for the fruitful discussion.
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