Mechanical and Morphological Analysis of Cancer ... - ACS Publications

Subscriber access provided by University of Otago Library

Article

Mechanical and morphological analysis of cancer cells on nanostructured substrates Dandan Ning, Binh Duong, Gawain Thomas, Yong Qiao, Liyuan Ma, Qi Wen, and Ming Su Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04469 • Publication Date (Web): 26 Feb 2016 Downloaded from http://pubs.acs.org on February 27, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Submitted to Langmuir Mechanical and morphological analysis of cancer cells on nanostructured substrates Dandan Ning1,4, Binh Duong3, Gawain Thomas2, Yong Qiao3, Liyuan Ma1,3, Qi Wen2*, Ming Su1,3,4* 1

2

Department of Chemical Engineering, Northeastern University, Boston, Massachusetts 02115

Department of Physics, and 3Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts 01609

4

Wenzhou Institute of Biomaterials and Engineering, Chinese Academy of Sciences, Zhejiang 325001

Cancer metastasis is a major cause of cancer induced deaths in patients. Mimicking nanostructures of extracellular matrix surrounding cancer cells can provide useful clue for metastasis. This article compares the morphology, proliferation, spreading, and stiffness of highly aggressive glioblastoma multiforme cancer cells and normal fibroblast cells seeded on a variety of ordered polymeric nanostructures (nanopillars and nanochannels). Both cells lines survive and proliferate on nanostructured surface, and show more similarity on nanostructured surfaces than on flat ones. Although both show similar stiffness on nanochannel surface, glioblastomas are softer, spread to a larger area and elongates less than fibroblasts. The nanostructured surfaces are useful in vitro model of extra-cellular matrix to study phenotype of cancer cell migratory. Keywords: nanoimprinting, nanochannels, nanopillars, cancer cell migration Electronic mail: [email protected]

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Introduction Cells in native environments are surrounded by extracellular matrix (ECM) that can affect cell behaviors through chemical, biological and mechanical signals.1 Among these types of signals, mechanical stress can cause re-distribution of integrins and cytoskeleton, which activates integrin receptors through inter-cellular pathway to control cell development and behavior.2 ECM consists of many nanoscale features including nanopores, nanofibers, nanochannels and nanoparticles,3 but cells in culture conditions are placed onto a two dimensional smooth surface, which does not reflect the true nanoscale features of ECM. A variety of nanostructures has been made to mimic the ECM structures, and their impacts on the morphology, proliferation, differentiation, migration, cytoskeletal organization, as well as gene expression has been studied.4 It has been found nanostructures or nanostructured materials can influence epithelial cell morphology and cytokine production. For instance, osteoblasts seeded on carbon nanofibers proliferate better and have higher alkaline phosphatease activity than those seeded on flat substrates.5 Fibroblast and kidney epithelial cells seeded on nanofibers have fewer stress fibers and smaller focal adhesion than those on flat surfaces.6 Nanostructures can also alter gene expression,7 and affect cell attachment, proliferation and spreading.8 Metastasis involves the movement of cells from one site to another.9 Cancer cell migration is crucial in tumor invasion, which starts once cancer cells detach from original sites and invade surrounding parenchymal cells and ECM.10 Compared to normal cells, cancer cells have different mechanical properties such as reduced stiffness and intercellular adhesiveness, both are related to tumor motility.11 Tumor invasion is also regulated by topographical feature of ECM.12 Mimicking the nanostructures of ECM surrounding cancer cells can provide useful clue


Page 2 of 20

Page 3 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

to understand tumor metastasis and migration.13 Despite numerous studies on non-malignant cells, there are limited researches of cancer cell behaviors on nanostructured surfaces. It is also desired to compare the effects of micro and nanoscale features on cancer cell mechanical properties and migrations. This article describes the morphology, proliferation, spreading and stiffness of metastatic cancer cells sitting on a variety of ordered polymeric nanostructures (nanopillars and nanochannels) which are generated with spin-on nanoimprinting (SNAP) technique (Fig. 1). Cancer cells used in this work is highly aggressive glioblastoma multiforme (GBM) which is a major cause of death of cancer patients.14 Normal health fibroblast cells (NIH 3T3) are used as a comparison. The stiffness and morphology of both cell lines on micro and nanostructured surfaces, and flat substrates are determined and compared to each other. It is expected that the polymeric nanostructures can be used to provide useful insights into biology of cancer cell migratory phenotype. 2. Materials and Methods 2.1 Spin-on nanoimprinting of polymeric nanostructures Polymeric substrates with ordered micro- and nano-structures were generated with spinon nanoimprinting technique.15 Polyacrylonitrile (PAN) dissolved in dimethylformamide (DMF) was heated at 150°C to partially cyclize (stabilize) PAN. A silicon master mold with nanofeatures was spin-coated with a thin layer of PAN solution. The film was then cured at 150°C for 1 min before transferred onto substrate. Fibronectin solution (with a concentration of 0.1 mg/ml) in phosphate buffer saline (PBS) was added to cover the polymer surface, incubated at 37 ºC for 2 h, and washed with PBS for 3 times. The modified surfaces were sterilized by exposing to ultraviolet (UV) light at 365 nm for 0.5 h for sterilization.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2.2 Calcein AM/EthD-1 assay A172 (glioblastoma) and NIH 3T3 fibroblasts were cultured in standard conditions (5% CO2 in air at 37°C) that contained RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin. After reaching about 70-80% confluence, cells were trypsinized with 0.25% trypsin/0.53 mM EDTA solution at 37°C for 3 min, followed by adding fresh medium to neutralize trypsin. After centrifugation and re-suspension into fresh medium, cell viability was examined with Trypan blue stain, and cell number was counted with hemocytometer (Horsham, PA). After seeding the cells onto surfaces, calcein AM/EthD-1 assay was performed as follows: 100 µl of PBS was added in each well to wash cells and to dilute serum containing esterase. 100 µl of calcein AM/EthD-1 assay reagent was added into each well and incubated for 30 min at room temperature before fluorescence measurement. A cell-free control was used to determine the background fluorescence and these signals were subtracted before calculations. A fluorescence microscope from Olympus (BX51M) was used to take fluorescent images. The percentages of live cells and dead cells were derived using the equation provide by Invitrogen. 2.3 Immunostaining assay of γ-H2XA and F-actin After seeding on surfaces, cells were fixed with 4% paraformaldehyde for 20 min, washed three times with PBS, blocked in 3% bovine serum albumin solution for 1 h at room temperature, and made permeable with 0.1% Triton X-100 in PBS solution for 15 min. In one sample, cells were incubated for 4 h with primary antibodies against γ-H2XA. After rinsed with PBS for three times, cells were incubated for an additional 1 h with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (anti-rabbit IgG-FITC antibody produced in goat). After rinsed with PBS for three times, cells were stained with 0.2 µg/ml 4,6-diamidino-2-phenylindole


Page 4 of 20

Page 5 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(DAPI) for 15 min in the dark. The samples were washed with PBS prior to acquiring fluorescence images. In another sample, cells were incubated for 30 min with F-actin staining solution. After washing with PBS for three times on shaker, cells were stained with 0.2 µg/ml DAPI for 15 min in the dark. The samples were washed with PBS prior to taking fluorescence images. 2.4 Scanning electron microscopy (SEM) Cells seeded on surfaces were incubated overnight at 37ºC and rinsed by pre-heated Hank’s balanced salt solution (HBSS) for three times. Cells were incubated in primary fixative containing 3% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4 with 0.1 M sucrose for 45 min at room temperature. After removing the primary fixative, cells were washed with and then incubated in cacodylate-sucrose buffer (0.1 M sodium cacodylate, 0.1 M sucrose, pH 7.4) for 5 min, and then washed for 5 min. After removing cacodylate buffer, cells were fixed with the secondary fixative (1% osmium tetroxide in cacodylate-sucrose buffer pH 7.4) for 1 h at 4°C and rinsed twice (5 min each) with cacodylate-sucrose buffer. After removing washing buffer, ethanol was added gently in a graded series of concentrations (35%, 50%, 70%, 95%, and 100%) for 10 min each. After replacing the last ethanol solution with hexa-methyldisilazane (HMDS), cells were incubated for 10 min, air-dried for 20-30 min and kept in a desiccator prior to SEM (JEOL) observation. 2.5 Atomic force microscope (AFM) The mechanical properties of cells seeded on surfaces were measured using an atomic force microscope (AFM, MFP-3D) from Asylum Research (Santa Barbara, CA). AFM microcantilevers with nominal spring constants of 0.08 N/m were used for indentation tests. The real spring constant of each microcantilever was calibrated with a thermal method prior to each


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

test.16 The indentations were done at three different locations in the perinuclear region of each cell. The AFM curves were analyzed using at MATLAB program, which fits the forceindentation data to the Hertz model.17 Only the initial 300 nm of indentation of each force curve were fitted to minimize the effects of nonlinear elasticity and ensure the availability of the Hertz model.18 For each surface condition more than 60 cells were measured. 2.6 Statistical analysis ANOVA tests were performed to determine the statistical significance of the differences between experimental results. Quantitative data are presented as mean ± standard deviation (SD) of the mean. A value of p < 0.05 was considered to be statistically significant. 3. Results and Discussion 3.1 Nanoimprinted polymeric structures Micro and nanostructures were produced with spin-on nanoimprinting technique as shown in Fig. 1A. 8% (wt) of PAN solution in DMF was spun onto a silicon master mold to form a thin film. The film was cured at 150°C for 1 min before transferred on substrate. SEM operated at 3 kV voltage was used to image the structures after depositing a thin film of gold-palladium. Fig. 1 (B-I) show images of crack-free nanostructures imprinted on PAN films. The cross sections show 3D features of structures. The dimension such as diameter, height and spacing of each sample was derived from SEM image. The diameter and height of nanopillars in Fig. 1F were 200 and 300 nm, respectively, with a center-to-center spacing of 700 nm. The depth and width of nano-channels in Fig. 1H were 140 and 150 nm, respectively. The dimensions of micro/nanosturctures were selected to be comparable to those of cell diameters (~10 µm) or distances between receptors (~100 nm) expressed on cell membranes. Table 1 shows the detailed dimension of each sample.


Page 6 of 20

Page 7 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table1. The height, wide and spacing of micro- and nano-structure Micropillars (MP)

Microchannels (MC)

Nanopillars (NP)

Nanochannels (NC)

Height

1.5 µm

1.2 µm

300 nm

140 nm

Dia./Wid.

12 µm

2.4 µm

200 nm

150 nm

Spacing

25 µm

2 µm

700 nm

555 nm

3.2 Viability and proliferation Calcein AM/EthD-1 was used to derive the viabilities of fibroblast and glioblastoma seeded on PAN surface. Ethidium homodimer-1(EthD-1) stain can detect integrities of cell membranes; while calcein acetoxymethyl ester (CalceinAM) stain can detect activities of intracellular esterase. Fluorescent microscopy results indicated both cell lines were green colored, meaning cells are in healthy condition (Fig. 2A). γ-H2XA protein repairs DNA double strand break, and the expression level of γ-H2XA protein can indicate the level of DNA double strand break.19 Immuno-staining assay (not shown) did not show green colored γ-H2XA, suggesting that PAN film does not cause DNA double strand break. Surface topography can affect cell proliferation through cytoskeletal organization.20 Fig. 2 showed cell proliferation rates determined by counting cells grown on each surface. More cells were found on flat surface than on nanostructured one over 3 days. The numbers of cells on nano-structured surfaces increased after the second day, meaning nanostructured surfaces can maintain cell viability and sustain cell growth. Further, more cells were found on nanochannel surfaces than on nanopillar surfaces, indicating that cells like to be orientated in a preferred direction. The results were in accordance with previous cell growth on micropatterns, where smooth muscle cells (SMCs) proliferation rate decreased.21


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

3.3 Cell morphology A172 glioblastoma and 3T3 fibroblast cells were seeded on micro/nanostructured surfaces and flat surfaces coated with fibronectin. Micro and nanochannel surfaces caused elongation and nuclear polarization in both cell lines, in which F-actin fibers were mostly stretched along the long axis of cells (Fig. 3C and E). Cell nuclei were elongated, and aligned to the long axis of each cell in most cases. More F-actin fibers were found in fibroblasts than in glioblastoma, suggesting high adhesion in fibroblasts, because tension generated by actin fibers was responsible to produce focal adhesion. Fluorescence images also confirmed that cells in channels oriented their actin fibers in alignment with the long axis of channels. Cell orientation along both micro and nanochannels were seen clearly in SEM images (Fig. 3H and J), where cell membrane conformed to microchannels but bridged nanochannels. In comparison, cells seeded on micro and nanopillar surfaces showed no elongation or orientation (Fig. 3G and I) where round cells showed less organized cytoskeletons. Cells were randomly spread on flat surfaces and micro/nanopillar surface (Fig. 3F, G and I). These results indicated that cell membranes in direct contact with micro/nanostructured channels suffered tensile and rearranged their subcellular components including integrin complexes and signaling molecules by mechanical forces. 3.4 Cell spread area Cell spread area was known as a major contributor to metastatic ability.22 Cell spread area was derived from Image J software. Fig. 4A showed fibroblasts spread mostly on nanochannels with an average area of 1500 µm2, which was larger than those on nanopillars (1000 µm2) and flat surface (1000 µm2) with a p value of 0.01. Fiberblasts spread less on micropillars or microchannels with an average area of 750 µm2. Glioblastoma showed a larger


Page 8 of 20

Page 9 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

area than fibroblast. Glioblastoma spread to 3300 µm2 on flat surface, 3000 µm2 on nanochannels, and 3000 µm2 on nanopillars. In addition, cells spread randomly on micro or nanopillar surfaces, and spread along grooves of micro and nanochannels. Cells spread to a larger area on microstructured surfaces than on nanostrctured ones. Glioblastoma spread the most on the flat surface, while fibroblasts spread to the largest area on nanochannels. This was likely due to contact guidance, which caused fibroblasts to create more traction force and focal adhesion sites on nanochannels. In comparison, glioblastoma was not able to create the same traction forces on nanochannels, causing them to spread less. 3.5 Cell stiffness An AFM was used to measure the stiffness of cytoskeleton with indentation method. Fig. 4B showed that both cell lines were stiffest on the flat surfaces and nanochannels: the stiffness of glioblastoma was 1.5 kPa, and that of fibroblasts was 2.25 kPa; the stiffness was significantly less (1.5 for fibroblasts and 0.75 kPa for glioblastoma) on nanopillars, where more actin fibers were observed for fibroblasts than for glioblastoma. Glioblastoma were stiffest on nanochannels (1.75 kPa), while fibroblasts were stiffest on flat surface (2.4 kPa). The changes in cell stiffness were reflected in cellular mechanical properties. Previous research showed the mechanical properties of cells were strongly associated with their lineage.23 In our study, the stiffness of glioblastoma on all surfaces was less than that of fibroblasts. Glioblastoma as cancer cell had less F-actin and lower stiffness, which allowed easier movement. 3.6 Cell elongation factor Cells seeded on micro and nano-channels aligned and elongated themselves in the direction of channels. Cell elongation reflected organization of actin cytoskeleton, cytoskeletal stiffness and contractility. Elongation factor was derived by calculating the ratio of each cell’s


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 20

major axis to its minor axis, and normalizing with respect to a circle representing the value of one.24 Fig. 5 showed the elongation factor of each cell line on micro and nanochannels, where cells on nanochannels had the largest elongation factor (4.0 for both cell lines). In comparison, cells on nanopillars or flat substrates were broader rather than aligning in any particular directions. Cells on nanopillars had smaller elongation factors: fibroblasts of 3.5 and glioblastoma of 3.0; cells on flat surfaces had the smallest elongation factors: fibroblasts of 2.0 and glioblastoma of 1.5. The result that glioblastoma had less alignment than fibroblasts was in agreement with those on aligned actin fibers. The guided growth of cells supported the concept that micro or nanochannels could guide the growth of both cell lines into one direction. Conclusions Nanostructured surfaces made with spin-on nanoimprinting technique had been used as an in vitro model of extra-cellular matrix. Nanostructured surfaces had shown significant effects on the orientation, morphology and cytoskeleton arrangement of cancer cells. Cancer cells seeded on nanostructured surfaces can survive and proliferate. Compared to fibroblasts, glioblastoma were softer, spread to larger area and elongated less. Micro and nanochannel surface can also guide cell growth into certain direction due to contact guidance. Acknowledgments This work was supported by a Director’s New Innovator Award (1DP2EB016572) from National Institute of Health (NIH), and a Faculty Early Career Development Award (1055599) from National Science Foundation (NSF).


Page 11 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure Captions Figure 1. Polymeric nanostructures made with spin-on nanoimprinting (A). Scanning electron micrographs of nanoimprinted polymer structures: micropillars (B and C), microchannels (D and E), nanopillars (F and G), and nanochannels (H and I). Figure 2. Proliferation rates of A172 glioblastoma and 3T3 fibroblast cells on micro- and nanostructures, where F stands for flat; MC stands for microchannels; MP stands for micropillars; NC stands for nanochannels; and NP stands for nanopillars. Figure 3. A172 glioblastoma and 3T3 fibroblast cells on micro- and nanostructures. Fluorescence images of DAPI (blue) and F-actin (green) stained cells on flat surface (A), micropillar (B), microchannel (C), nanopillar (D), and nanochannel (E). SEM images of cells on flat surface (F), micropillar (G), microchannel (H), nanopillar (I), and nanochannel (J). Figure 4. Cell spread areas of A172 glioblastoma and 3T3 fibroblast cells seeded on micro- and nano-structures (A). The stiffness of A172 glioblastoma and 3T3 fibroblast cells seeded on micro- and nano-structures (B). Figure 5. Cell elongation profiles of A172 glioblastoma and 3T3 fibroblast cells cultured on micro- and nano-structures.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 20

References 1.

Ozbek, S.; Balasubramanian, P. G.; Chiquet-Ehrismann, R.; Tucker, R. P.; Adams, J. C.,

The evolution of extracellular matrix. Mol. Biol. Cell 2010, 21, 4300. 2.

Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E., Geometric control

of cell life and death. Science (New York, N.Y.) 1997, 276, 1425. 3.

Gelain, F.; Bottai, D.; Vescovi, A.; Zhang, S., Designer self-assembling peptide nanofiber

scaffolds for adult mouse neural stem cell 3-dimensional cultures. PLoS One 2006, 1, e119. 4.

(a) Andersson, A. S.; Backhed, F.; von Euler, A.; Richter-Dahlfors, A.; Sutherland, D.; B.,

K., Nanoscale features influence epithelial cell morphology and cytokine production. Biomater. 2003, 24, 3427–3436; (b) Flemming, R. G.; Murphy, C. J.; Abrams, G. A.; Goodman, S. L.; Nealey, P. F., Effects of synthetic micro- and nano-structured surfaces on cell behavior. Biomater. 1999, 20, 573; (c) Hasirci, V.; Kenar, H., Novel surface patterning approaches for tissue engineering and their effect on cell behavior. Nanomedicine 2006, 1, 73; (d) Kim, D.-H.; Kim, P.; Song, I.; Cha, J. M.; Lee, S. H.; Kim, B.; Suh, K. Y., Guided three-dimensional growth of functional cardiomyocytes on polyethylene glycol nanostructures. Langmuir 2006, 22, 5419. 5.

Elias, K. L.; Price, R. L.; Webster, T. J., Enhanced functions of osteoblasts on nanometer

diameter carbon fibers. Biomater. 2002, 23, 3279. 6.

Schindler, M.; Ahmed, I.; Kamal, J.; Nur, E. K. A.; Grafe, T. H.; Young Chung, H.;

Meiners, S., A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture. Biomater. 2005, 26, 5624. 7.

Huang, C.-L.; Hsiao, I.-L.; Lin, H.-C.; Wang, C.-F.; Huang, Y.-J.; Chuang, C.-Y., Silver

nanoparticles affect on gene expression of inflammatory and neurodegenerative responses in mouse brain neural cells. Environ. Res. 2015, 136, 253.


Page 13 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

8.

Yin, M.; Yin, Y.; Han, Y.; Dai, H.; Li, S., Effects of uptake of hydroxyapatite

nanoparticles into hepatoma cells on cell adhesion and proliferation. J. Nanomater. 2014, 2014. 9.

Chaffer, C. L.; Weinberg, R. A., A perspective on cancer cell metastasis. Science (New

York, N.Y.) 2011, 331, 1559. 10.

Grimstad, I. A., Direct evidence that cancer cell locomotion contributes importantly to

invasion. Exp. Cell Res. 1987, 173, 515. 11.

Suresh, S., Biomechanics and biophysics of cancer cells. Acta Biomater. 2007, 3, 413.

12.

Valastyan, S.; Weinberg, R. A., Tumor metastasis: molecular insights and evolving

paradigms. Cell Biol. Int. 2011, 147, 275. 13.

Balachander, G. M.; Balaji, S. A.; Rangarajan, A.; Chatterjee, K., Enhanced metastatic

potential in a 3D tissue scaffold toward a comprehensive in vitro model for breast cancer metastasis. Appl. Mater. Interface 2015, 7, 27810. 14.

Holland, E. C., Glioblastoma multiforme: the terminator. Proc. Natl. Acad. Sci. USA

2000, 97, 6242. 15.

Duong, B.; Khurshid, H.; Gangopadhyay, P.; Devkota, J.; Stojak, K.; Srikanth, H.; Tetard,

L.; Norwood, R. A.; Peyghambarian, N.; Phan, M.-H.; Thomas, J., Enhanced magnetism in highly ordered magnetite nanoparticle-filled nanohole arrays. Small 2014, 10, 2840. 16.

Thomas, G.; Burnham, N. A.; Camesano, T. A.; Wen, Q., Measuring the mechanical

properties of living cells using atomic force microscopy. J. Visualized Experiments 2013, 76, 50497. 17.

Yim, E. K. F.; Darling, E. M.; Kulangara, K.; Guilak, F.; Leong, K. W.,

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells. Biomater. 2010, 31, 1299.


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

18.

Page 14 of 20

Solon, J.; Levental, I.; Sengupta, K.; Georges, P. C.; Janmey, P. A., Fibroblast adaptation

and stiffness matching to soft elastic substrates. Biophys. J. 2007, 93, 4453. 19.

Qiao, Y.; Zhang, P.; Wang, C.; Ma, L.; Su, M., Reducing X-ray induced oxidative

damages in fibroblasts with graphene oxide. Nanomater. 2014, 4, 522. 20.

Papenburg, B. J.; Rodrigues, E. D.; Wessling, M.; Stamatialis, D., Insights into the role of

material surface topography and wettability on cell-material interactions. Soft Matter 2010, 6, 4377. 21.

Thakar, R. G.; Ho, F.; Huang, N. F.; Liepmann, D.; Li, S., Regulation of vascular smooth

muscle cells by micropatterning. Biochem. Biophys. Res. Comm. 2003, 307, 883. 22.

Chen, C. S.; Alonso, J. L.; Ostuni, E.; Whitesides, G. M.; Ingber, D. E., Cell shape

provides global control of focal adhesion assembly. Biochem. Biophys. Res. Comm. 2003, 307, 255. 23.

Darling, E. M.; Zauscher, S.; Block, J. A.; Guilak, F., A thin-layer model for viscoelastic,

stress-relaxation testing of cells using atomic force microscopy: do cell properties reflect metastatic potential? Biophys. J. 2007, 92, 1784. 24.

Dalby, M. J.; Riehle, M. O.; Yarwood, S. J.; Wilkinson, C. D.; Curtis, A. S., Nucleus

alignment and cell signaling in fibroblasts: response to a micro-grooved topography. Exp. Cell Res. 2003, 284, 274.


Page 15 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Langmuir

A

B

D

F

H

C

E

G

I

Figure 1 ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 16 of 20

A

B


Page 17 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Langmuir

A

B

G

D

50 μm

50 μm

50 μm

F

C

H

E

50 μm

I

50 μm

J


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

A

Page 18 of 20

B


Page 19 of 20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Langmuir

F

MP

MC

NP

NC


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

50 μm

Page 20 of 20

50 μm

Table of Content Graphic ACS Paragon Plus Environment

Mechanical and Morphological Analysis of Cancer ... - ACS Publications

Recommend Documents