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Regulating MDA-MB-231 breast cancer cell adhesion on laser-patterned surfaces with micro- and nanotopography

Breast cancer is the most common type of cancer observed in women. Communication with the tumor microenvironment allows invading breast cancer cells, such as triple negative breast cancer cells, to adapt to specific substrates. The substrate topography modulates the cellular behavior, among other factors. A number of different materials and micro/nanofabrication techniques have been employed to develop substrates for cell culturing. Silicon-based substrates present a lot of advantages as they are amenable to a wide range of processing techniques and they permit rigorous control over the surface structure. We investigate and compare the response of triple negative breast cancer cells (MDA-MB-231) on laser-patterned silicon substrates with two different topographical scales, i.e., the micro- and the nanoscale, in the absence of any other biochemical modification. We develop silicon surfaces with distinct morphological characteristics by employing two laser systems with different pulse durations (nanosecond and femtosecond) and different processing environments (vacuum, SF6 gas, and water). Our findings demonstrate that surfaces with micro-topography are repellent, while surfaces with nano-topography are attractive for MDA-MB-231 cell adherence.


I. INTRODUCTION
Breast cancer is the most common type of cancer observed in women, being the second leading cause of death [0,2]. Among all subtypes of breast cancer, the triple negative one (Triple Negative Breast Cancer-TNBC) shows increased heterogeneity and lacks the expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor Receptor-2 (HER2) overexpression, therefore becomes very aggressive and resistant to several anti-cancer agents [3,4]. Proliferation and migration constitute two major hallmarks of cancer, being the main factors for cancerrelated deaths worldwide. The mechanisms underlying the dissemination of cancer cells to distant sites still remain poorly understood, despite the increasing evidence that have shed light to the factors affecting this procedure. Communication with the tumor microenvironment allows TNBC invading cancer cells, such as MDA-MB-231, to adapt to specific substrates in order to grow, proliferate, and disseminate to distant sites. 3 To elucidate the mechanisms underlying cancer cell survival, proliferation, and metastatic potential in vitro, a number of parameters should be taken into consideration such as the surface chemistry, surface stiffness, and topographic characteristics [5][6][7][8].
The ideal substrate for tumor cell culturing should be able to combine effectively mechanical properties and macro/nano scale topography in order to achieve the optimal conditions for cell survival and, most importantly, to mimic the microenvironment cells naturally reside in. This way, it can promote interactions between cells and the external environment (such as exchange of biochemical signals, oxygen supply, etc.), which are equivalent to the ones cells encounter in real tissues [9][10][11][12]. The substrate topography modulates the cellular behavior, as it has been shown that topographical features of different length-scales, ranging from nano-to micrometer ones, have a strong influence on cell adhesion, morphology, alignment, and contact guidance [13]. Even though it is understood that micro-and nanoscale topographic patterns resemble the extracellular environment, the topography of which constitutes a major biophysical regulator of cell behavior, to date cell reaction to topography remains not fully understood. It is anticipated that the optimum topography varies depending on the cell type, substrate material, and structure. Especially for cancer cells, the tumor microenvironment can greatly influence cell behavior and dissemination [14].
Recently, it has been shown that micro-and nanofabrication techniques have provided invaluable tools for studying various processes, such as cell proliferation [15], differentiation [16,17], adhesion [18][19][20][21][22], and migration [23][24][25][26][27][28][29][30] through the introduction of topographical features on cell-culturing platforms [31]. At the same time, a number of different materials have been employed as substrates for cell culturing [32,33]. Amongst them, silicon-based substrates present a lot of advantages as they are amenable to a wide range of processing techniques common to silicon 4 microfabrication, thus providing access to a big variety of tailored geometries and surface patterns, while at the same time permitting rigorous control over the structure and composition of the surface [34]. Furthermore, silicon can be surface-modified and replicated in polymeric scaffolds, which, coupled with the fact that it is not toxic to cells, makes it an ideal substrate to study several cellular properties such as adhesion, morphology, proliferation, and migration [35,36] on patterned surfaces and devices [37].
Patterned and/or bio-modified silicon has been employed to investigate the effect of topography on fibroblasts [38], neuronal [39,40], and epithelial cells [41,42] as well as on various cancer cell lines [43][44][45][46][47][48], including mammary tumor cells, such as MCF-7 and MDA-MB-231. MCF-7 and MDA-MB-231 cell lines are derived from the pleural effusions of a breast cancer patient with metastasis but exhibit diverse phenotypic features. MCF7 cells are ER+,PR+,HER2-, they are estrogen-dependent and express markers for the luminal epithelial phenotype. MCF-7 breast tumor cells form multicellular nodules (foci) over a confluent monolayer in the presence of estradiol (E2), they are poorly aggressive, and exhibit low metastatic ability. On the contrary, MDA-MB-231 do not express epithelial markers, they are ER-,PR-,HER2-(triple negative) and have acquired a highly aggressive mesenchymal phenotype [49,50]. The rate of adhesion and growth of breast cancer MCF-7 cells was studied on mesoporous silicon [44], while MDA-MB-231 cells have been co-cultured with MCF10A non-tumorigenic human breast cells on microstructured silicon substrates with arrays of etched microcavities to study their adhesion pattern [51,52], on micromachined arrays of silicon walls to study their plasticity [53], and a few other  [51,52]. In addition, another study has shown that various cancer cell lines applied on 3D silicon micromachined structures (SMS), with arrays of 3-µm-thick silicon walls separated by 50-µm-deep, 5-µm-wide gaps, exhibited different behavior according to their invasiveness. The more invasive cancer types gave higher fractions of cells in the gaps, indicating a direct correlation between the aggressiveness of tumor cells and their capacity to grow into the narrow gaps of the 3D-SMS [53].
In this work, we investigate and compare the response of MDA-MB-231 cells on laser-patterned silicon substrates with two different topographical scales, i.e., the micro-and the nanoscale, in the absence of any other biochemical modification. We were able to develop silicon surfaces with distinct morphological characteristics by employing two laser systems with different pulse durations (nanosecond and femtosecond) and different processing environments (vacuum, SF6 gas, and water).
Laser processing is a single-step, maskless, tabletop method to create uniformly micro/nanopatterned surfaces over large areas, which does not require a clean room.
Laser processing of silicon has been shown to generate a variety of structures at the 6 micro-and nanoscale by tuning the fabrication parameters, such as wavelength, pulse duration, fluence, gas or liquid environment, and the number of incident laser pulses [54][55][56]. Laser-patterned silicon substrates have been used for the culture of Schwann cells [57], fibroblasts [47], and rat pheochromocytoma (PC12) cells [58], but have not been applied to TNBC (MDA-MB-231) cell culturing before. Furthermore, laser patterning of surfaces allows for localized surface modification and high spatial resolution of the formed structures, thus paving the way for the design of co-culturing substrates and devices based on topographical cues. In this way, invaluable information on the proliferative and migrating behavior of breast cancer cells will be provided, which will enable the formulation of novel treatment approaches.  Laser-structured silicon substrates. Silicon wafers ((100), thickness 500 ± 25 μm) were cleaned in an ultrasonic bath of acetone and methanol for 15 min and dried in nitrogen (N2) gas flow before laser structuring. Microstructured silicon substrates were fabricated by nanosecond laser processing in a gas environment of sulfur hexafluoride (SF6) or in vacuum. A pulsed Q-switched Nd:YAG laser system was used with 532 nm wavelength, 5 ns pulse duration, and 10 Hz repetition rate. Silicon wafers were placed in a vacuum chamber, which was evacuated to 9*10 -3 mbar (vacuum conditions) or filled with 0.6 bar SF6. The laser beam was focused on the silicon surface through a quartz window using a lens of 200 mm focal length and the vacuum chamber was raster scanned with respect to the laser beam using a computer-controlled set of xy translation stages. Nanostructured silicon substrates were fabricated by femtosecond laser processing in water. An ultrashort fiber-based laser system was used with 1064 nm wavelength, 370 fs pulse duration, maximum average power 1W, maximum pulse energy 1μJ, and variable pulse repetition rate up to 1 MHz. The laser beam was focused by a 4× microscope objective lens with NA = 0.10 on the surface of the silicon wafers that were placed at the bottom of a container filled with 6 ml of distilled water, which covered the surface of the wafer. The container was mounted on a computer-controlled set of xy linear translation stages with resolution 1 nm and maximum velocity 20 mm/s.  μm, the average half-height width 11 ± 2 μm, and the average distance between neighboring spikes 16 ± 2 μm center-to-center, which results in a spike density of 8.6*10 5 spikes/cm 2 . This morphology was obtained when irradiating silicon in SF6 ambient with a fluence of 1 J/cm 2 and translating the silicon surface with respect to the laser beam with a speed such that each spot on the silicon surface was irradiated by 1000 laser pulses [59,60]. At this fluence, this is the result of melting, ablation, and interference effects that occur upon nanosecond laser irradiation of silicon in SF6 environment [55,56]. The results of MDA-MB-231 cell attachment on the microstructured silicon surface, relative to a flat silicon surface (Fig. 1c) and a control glass coverslip (Fig. 1d), 10 are shown in Fig. 2. Figure 2a shows a fluorescent microscope image of cell attachment on the coverslip. We observe that cells were uniformly spread throughout the surface, which was identical to the behavior of the cells on the flat silicon surface (Fig. 2b). On the contrary, surface microstructuring, through the introduction of anisotropic microcones, seemed to act as a repellent for TNBC cells (Fig. 2c). For a direct comparison, Fig. 2d shows the results of cell adherence on the border between a flat (right) and a microstructured (left) silicon surface. We clearly observe the flat silicon side is cell-adhesive and the microstructured silicon side is cell-repellent. These Similar structures have been employed for the growth of neuronal cells, where they have been shown to promote and direct neuronal outgrowth [61], however, in the case of TNBC MDA-MB-231 cells, they hinder cell attachment. This is probably due to the inherent hydrophobicity of these surfaces [62], which act as a barrier for migratory cells, a finding which is further confirmed by other studies where microstructures and their effect on cell behavior were investigated [26]. Laserstructured silicon microspikes in SF6 have been shown to be covered by a surface layer with a sulfur concentration of 0.5 -0.7% [63,64]. Additionally, because the samples used in this work were exposed to air for several days before being used as cell culture substrates, a native oxide layer was also formed on the surface, thus the MDA-MB-231 cells interact with the oxidized surface. Therefore, the presence of sulfur underneath Increasing the nanosecond laser fluence, we created crater structures on the surface of silicon. Figure 3 shows an area on the silicon surface, which has been irradiated by 1000 pulses with a laser fluence of 2 J/cm 2 in SF6 ambient. The employment of a high laser fluence led to the formation of a crater at the center of the irradiated area, with the appearance of various other surface topographies as we move towards the periphery of the crater, due to the Gaussian profile of the laser beam fluence. The major diameter of the elliptical crater is 870 μm and its depth 530 ± 20 μm. As the laser fluence decreases radially, the central crater is surrounded by a zone of silicon microspikes, similar to the ones generated with a lower laser fluence (Fig. 1), which is succeeded by a zone of microripples, followed by a zone of re-solidified silicon after melting [65]. Examination of cell attachment in and around these craters revealed the cells tend to concentrate more in the circumference of the crater (Fig. 4). Indeed, the cell population density appears slightly lower inside the crater rather than on the circumference and on the flat silicon area surrounding the crater. 14 The crater effect on cell behavior becomes more pronounced for another crater morphology, which can be obtained by changing the environment in which laser irradiation takes place. Figure 5 shows an area on the silicon surface, which has been irradiated in vacuum, while the rest of the experimental conditions were kept identical to the conditions employed for the crater shown in Fig. 3 (1000 pulses, fluence 2 J/cm 2 ).

A. MDA-MB-231 cell culturing on microstructured substrates
For this crater, too, we observe the same effect as in Fig. 3, i.e., an elliptical crater with major diameter 860 μm and depth 130 ± 20 μm at the center of the irradiated area, followed sequentially by microspikes, microripples, and finally molten and resolidified silicon towards the edges. However, the fact that the crater shown in Fig. 5 was fabricated upon laser ablation in vacuum, results in a strikingly different morphology at the center, relatively to the crater fabricated in SF6 ambient, as the former is shallower with vertical walls surrounding a central area which is decorated with microripples.  We studied the behavior of the cells around the crater shown in Fig. 5 in more detail by creating a series of four such craters side-by-side, as shown in Fig. 7. The four craters present a similar morphology to the single crater shown in Fig. 5, with a central flat cavity, surrounded by a zone with microspikes, followed by a zone of microripples.
The circumference of the craters is shown in more detail in Fig. 7c, which shows that the microspikes protrude above the surface of flat silicon, while the microripples have 16 significantly smaller height than the microspikes and a smaller curvature. Fig. 7d shows a magnification of the microripples, where we observe the shape of the microripples and the fact that they are decorated with sub-micron roughness on the surface. Cell adhesion on the series of four craters is demonstrated in Fig. 8, which shows again that the cells developed increased adherence capacity towards the areas with the microripples. Furthermore, the difference between microripples and conical microspikes in terms of cell preference becomes evident, as cells congregated in the rippled topography (indicated by the red rectangles), with a sub-micron surface roughness and a different aspect ratio than the microspikes, rather than the spiked one (indicated by the red circles). Similar to Fig. 6, quantification analysis showed that 994 ± 67.87 (N=3) cells adhered around the crater, while 691 ± 22.59 (N=3) cells localized inside the crater. The fact that different types of microstructures can affect cancer cell adherence, and consequently proliferation and growth, in various ways is further confirmed by the results obtained by other groups [66]. Therefore, the next step is to study cellular attachment on substrates with nanomorphology, as the above observations indicate that a substrate morphology exhibiting roughness at the nanoscale is significantly more favorable for cell adherence.

B. MDA-MB-231 cell culturing on nanostructured substrates
We obtained nanostructured silicon substrates by employing a femtosecond laser system. Femtosecond laser irradiation of silicon in water, as described in the Experimental Section, resulted in the formation of columnar nanopillars on the surface, 18 as shown in Fig. 9 [67,68]. This is the result of ultrafast melting and interference effects, which occur only with femtosecond laser irradiation and not with nanosecond irradiation [69,70]. The nanopillars were monolithically formed on silicon with a mean pillar diameter of 168 ± 33 nm, a mean height of 476 ± 68 nm, and a mean distance between neighboring nanopillars 257 ± 56 nm center-to-center.  The MDA-MB-231 cells showed a remarkable preference for the nanostructured areas and accumulated on the nanopillars to a great extent. Specifically, 493 ± 3.844 (N=3) cells (Fig. 10a) and 465 ± 2.517 (N=3) cells (Fig. 10b) were attached to the nanostructured areas while 3 ± 1.155 (N=3) and 12 ± 2.082 (N=3) cells, respectively, were found on the flat areas aside. This is in stark contrast to the behavior of the cells on the microspikes shown in Fig. 1, which proved to be cell-repellent compared to the surrounding flat silicon area. Therefore, the nanoscale proves to be a naturally favorable topographical scale for MDA-MB-231 cell adherence. Nano-roughness is considered 19 to be the closest to natural tissue morphology with a positive effect on cell adhesion, growth, and maturation. In human venous endothelial cells, it has been shown that increasing the roughness of the biomaterial surface at the nanometer scale can enhance cell adhesion and growth [22]. Mesenchymal MDA-MB-231 cells show maximal response to the influence of substrate morphology for cue dimensions close to the focal adhesion size [14].

IV. SUMMARY AND CONCLUSIONS
Employing laser-patterning methods, we were able to modify the surface of silicon, inducing two different topographical scales, i.e., microtopography and nanotopography, by varying the experimental parameters, most notably the laser pulse duration and the processing medium. Laser processing is a simple method to create micro/nanopatterned surfaces over large areas, without the need of a clean room. We 20 used these substrates in order to compare the effect of topography on the adherence of MDA-MB-231 breast cancer cells in the absence of any other biochemical modification. The microtopographies we obtain by nanosecond laser irradiation, either conical microspikes or micro-craters, prove to be cell-repellent. On the other hand, the nanotopography we obtain by femtosecond laser irradiation, consisting of silicon nanopillars, is cell-attractive. The same observation holds for smooth microtopographies, such as microripples, decorated with sub-micron surface roughness, introduced with the use of nanosecond laser irradiation. The response of MDA-MB-231 cells appears maximum for substrate cue dimensions close to their focal adhesion size. Regulating cell attachment by inducing adhesion or repellence at specific surfaces will thus provide invaluable information about the proliferative and migrating behavior of breast cancer cells and will aid in designing novel treatment approaches.
Gaining a deeper understanding of the topographical cues that govern TNBC adherence and elucidating cell responsiveness to mechanical signals, will enable further insights to be gained into prognostic markers, mechanically mediated metastasis pathways for therapeutic targets, and model systems required to advance cancer mechanobiology [71].

Conflicts of Interest
The authors have no conflicts to disclose.