3D Biomimetic Chips for Cancer Cell Migration in Nanometer-Sized

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3D Biomimetic Chips for Cancer Cell Migration in Nanometer-Sized Spaces Using “Ship-in-a-Bottle” Femtosecond Laser Processing Felix Sima, Hiroyuki Kawano, Atsushi Miyawaki, Lorand Kelemen, Pal Ormos, Dong Wu, Jian Xu, Katsumi Midorikawa, and Koji Sugioka ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00487 • Publication Date (Web): 09 Oct 2018 Downloaded from http://pubs.acs.org on October 11, 2018

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ACS Applied Bio Materials

Title: 3D Biomimetic Chips for Cancer Cell Migration in Nanometer-Sized Spaces Using “Ship-in-a-Bottle” Femtosecond Laser Processing Felix Sima*, Hiroyuki Kawano, Atsushi Miyawaki, Lorand Kelemen, Pal Ormos, Dong Wu, Jian Xu, Katsumi Midorikawa, Koji Sugioka* Dr. Felix Sima, Dr. Dong Wu, Dr. Jian Xu, Prof. Katsumi Midorikawa, Prof. Koji Sugioka RIKEN Center for Advanced Photonics, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan E-mail: [email protected], [email protected] *Tel : +81-48-467-9495 Fax : +81-48-462-4682 Dr. Hiroyuki Kawano, Prof. Atsushi Miyawaki BSI, RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan Dr. Lorand Kelemen, Prof. Pal Ormos Biological Research Centre, Institute of Biophysics, Hungarian Academy of Sciences, Temesvári krt. 62, 6726 Szeged, Hungary Dr. Felix Sima CETAL, National Institute for Lasers, Plasma and Radiation Physics, Magurele, Ilfov, 00175, Romania E-mail: [email protected] Keywords: femtosecond laser processing, photosensitive glass, lab-on-a-chip, two-photon polymerization, nanofabrication, biomimetics, cancer cell migration

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Abstract Cancer cells undergo dramatic morphology changes when migrating in confined spaces narrower than their diameter during metastasis, and thus it is necessary to understand the deformation mechanism and associated molecular events in order to study tumor progression. To this end, we propose a new biochip with three-dimensional (3D) polymer nanostructures in a closed glass microfluidic chip.“Ship-in-a-bottle” femtosecond laser processing is an exclusive technique to flexibly create 3D small details in biochips. The wavefront correction by the spatial light modulator significantly improves a fabrication resolution of this technique. The fabricated device could then accomodate defect-free 3D biomimetic nanoconfigurations for the evaluation of prostate cancer cell migration in confined spaces. Specifically, polymeric channels with widths of ~900 nm, which is more than one order of magnitude smaller than the cell size, are integrated by femtosecond laser inside glass channels. The cells are responsive to an in-channel gradient of epidermal growth factor and can migrate a distance greater than 20 µm. After migration, the cells suffer partial cytokinesis, followed by fusion of the divided parts back into single cell bodies. Introduction Lab-on-a-chip devices have been intensively developed as an approach for realizing large-scale laboratory processes with high sensitivity and low reagent consumption. Such devices require advances in emerging fabrication technologies 1-5. Currently, the micro- and nano-scale processing of biocompatible materials offers multiple possibilities for the manufacture of reliable devices that allow the testing of biochemical reactions in nanoliter volumes 6-7. The challenge of validating and consolidating specific biochemical protocols on chips by user-friendly manipulation can be addressed by material choice and biochip architecture 8-9. The manipulation of cells inside such devices is a promising approach for developing platforms for biological investigations such as high-throughput drug and cell phenotype screening 10-13. In particular, innovative microfluidic platforms have been proposed for cancer cell detection and characterization 14, and to mimic the tumor niche in vitro in order to elucidate carcinogenesis mechanisms and explore novel therapeutics 15. For example, it is known that metastatic cancer cells can migrate in confined spaces (even much narrower than their nucleus), resulting in dramatic morphology changes 16. In order to mimic the hierarchical organization of tissues and employ alternatives to laboratory experiments, cancer cell migration and invasiveness tests are usually conducted in vitro using Boyden chambers 17-18. In a Boyden chamber assay (also known as a transwell assay), the cells are placed in the upper chamber on a filter with defined pore sizes of 3 to 8 µm in diameter and a solution containing a chemoattractant compound is used to fill the lower chamber in order to stimulate active cell transmigration by chemotaxis 19. Recently, a new configuration allowing the production of a linear concentration gradient was proposed for the direct visualization of cell migration 20. Dysregulated cell motility shows a specific characteristic of invasive tumors in which cells can migrate through neighboring tissues due to chemotaxis by extracellular signals from growth factors such as epidermal growth factor (EGF), leading to cell invasiveness 21-23. The development of microfluidic lab-on-a-chip devices allows the testing of cancer cell chemotaxis through confined spaces 24-26. It was previously demonstrated that cancer cell migration through 2 µm-wide spaces affected nuclear envelope integrity and DNA content, further inducing DNA damage 27. Such biochips based on polydimethylsiloxane (PDMS) are the most frequently used to configure complex microfluidic architectures 24, 28 as they exhibit several advantages, such as biocompatibility, good optical transparency, and ease of use. However, these biochips also have disadvantages, such as non-


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reusability, the requirement for multiple-stacking processing, and the adsorption of organic compounds which might induce artefacts 29. In this paper, we propose the use of a glass-based biochip integrated with biomimetic polymeric features for the study of cancer cell migration. This biochip is fabricated using our innovative “ship-in-a-bottle” laser technology, which combines unique features of subtractive femtosecond laser assisted chemical etching (FLAE) of glass and additive two-photon polymerization (TPP). The fabrication resolution of this technique can be significantly improved by wavefront correction using a spatial light modulator (SLM).This hybrid approach is now proposed for the first time to create three-dimensional (3D) environments at the sub-micrometric level inside a closed glass microfluidic chip. Specifically, FLAE can be used to flexibly fabricate 3D microfluidic structures embedded in glass microchips without requiring the complicated procedure of stacking and bonding glass substrates. Successive TPP can directly integrate complex shapes of polymeric structures with a sub-micrometer feature to create biomimetic structures inside the glass microfluidics. Historically, FLAE using Foturan glass was utilized to create nano-aquariums for the analysis of microorganisms, such as 3D observation of the rapid flagellar motion of Euglena gracilis 30 and elucidation of the gliding mechanism of phormidium in soil for accelerating vegetable growth 31. Furthermore, the integration of micro-electronic components in 3D microfluidic glass channels by laser selective metallization was proposed for the electrical stimulation of biological samples in nano-aquariums 32-33. The “ship-in-a-bottle” method was further developed to add new functionalities to 3D glass microfluidic biochips, allowing fabrication of a multi-functional ﬁlter-mixer integrated biochip for fast fluid interspersion within a short channel distance 34 and of an optofluidic unit integrated with a 3D microlens array and center pass units for counting Euglena cells 35-36. FLAE can provide a robust, easy-to-handle device with full transparency to VIS-NIR for optical interrogation. Simultaneously, “ship-in-a-bottle” integration will enable the creation of unique 3D tissue-like environments with sub-micrometer precision for cell culture onto glass platforms. Thus, these novel biochips will be applicable for high-throughput screening of small amounts of biomolecules in order to characterize human cells in nanoliter spaces. In the present study, prostate cancer (PC3) cells were cultivated inside 3D biochips and monitored over short (hours) and long (days) periods to evaluate specific behaviors of cancer cells, such as migration and invasiveness. This is the first report of experiments with mammalian cells adhering to a glass microfluidic platform with embedded channels, confirming the huge potential of such biochips for advancing in vitro testing. To our knowledge, the polymeric channels of the present biochips fabricated by the “ship-in-abottle” femtosecond laser processing, with a width of ~900 nm integrated onto the glass substrate, are the narrowest demonstrated to date for cancer cell migration studies. Moreover, this is the first experiment of this kind conducted in a true 3D microfluidic configuration which is not possible to create by alternative conventional methods. Specifically, 3D sub-micrometer-scale polymeric structures integrated in a closed glass platform allow the construction of more biomimetic structures and the generation of a chemoattractant diffusion-based gradient stable over several hours. Use of a glass platform for biochip fabrication provides further advantages, such as multiple reusability, since the glass does not require any special coating treatment. The closed environment of the glass microfluidic structure is also important when handling biohazardous materials such as tumor-derived cells.



Results Hierarchical biochip design and downsizing concept. The glass biochip has a relatively simple design. It consists of 4 open micro-reservoirs that are connected by embedded T-shape channels (Fig. 1a). The lower side micro-reservoirs (µR1 and µR2) are used as cell inlets, while the upper side micro-reservoirs (µR3 and µR4) are used for injecting the chemoattractant. The connecting channels are labeled in Fig. 1b, which shows a topview of Fig. 1a. Channel 1 (GCh1) connects µR1 and µR2 and is used as a platform for cell attachment. Channel 3 (GCh3) is the link between µR3 and µR4 and is necessary for delaying chemoattractant diffusion towards channel 2 (GCh2). The novelty of the biochip consists of the unique combination of glass with polymer that allows downsizing the dimension from millimeters to nanometers by integrating biomimetic polymeric scaffolds in GCh2. Micro-reservoir µR3 is intended to both delay chemoattractant diffusion to the scaffold and as an additional chamber for collecting migrating cells for downstream analysis, such as real-time quantitative PCR or single cell genomics/proteomics studies. The sizes of the micro-reservoirs and channels are designed based on the scale-up, scale-down approach . Specifically, a micro-reservoir diameter of 1 mm was selected to permit easy pipetting, while the channel space was narrowed to extend the diffusion length, in particular for observation of the region of interest (zoom in Fig. 1c). In GCh2, we grew polymeric scaffolds using TPP with the aim of reproducing biomimetic environments suitable as model structures for cancer cell migration in confined spaces. More precisely, we developed submicrometer-wide channels with a panpipe architecture, which has channels arranged in a step-wise manner with increasing length. The polymeric scaffold is composed of six channels with lengths of 6, 8, 11, 14, 18, and 21 µm (Fig. 1c). The channels are placed at the bottom of GCh2, and the scaffold is then surrounded by a polymeric wall that covers the entire cross-section of GCh2 (50 × 90 µm2) to seal and separate the cell platform (µR1-GCh1-µw2) from the chemoattractant reservoirs (µR4-GCh3-µR3). For better stability, the wall thickness was designed to be ~5 µm (top-view in Fig. 1c and cross-sectional view in Fig. 1d). Thus, the cell platform and the chemoattractant reservoir connect only through the polymer microchannels in the scaffold. This configuration ensures the efficient generation of a diffusion-based gradient of the chemoattractant along the sub-micrometric channels during hours of experimental observation. Additionally, the panpipe architecture allows the generation of different chemical gradients in each channel, thereby allowing more insightful investigations into specific pathways controlling cancer cell migration and/or invasion.


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Figure 1: Biochip design and concept: a) T-shape microfluidic platform with 4 open microreservoirs (µRs1 and 2 as cell inlets and µRs 3 and 4 as chemoattractant inlets) connected by embedded channels (tilt view); b) Top-view of the biochip shows scale-up, scale-down aspects of the microfluidic platform and the connecting channels (GCh1 connects µR 1 and µR 2; GCh3 connects µR 3 and µR 4; GCh2 connects µR 3 and GCh1). The observation area is depicted by a dashed square, emphasizing the position of the polymeric scaffold; c) Zoom-in top view of the observation area in GCh2 shows the architecture of the panpipe-shape polymeric scaffold consisting of 6 channels of different lengths (6, 8, 11, 14, 18 and 21 µm) placed at the bottom of GCh2, and a polymeric separation wall covering the entire cross-section of GCh2 (50 × 90 µm2); d) Magnified cross-sectional view of the observation area. Next, in order to develop an appropriate protocol for in vitro testing of the migration potential of PC3 cells in confined spaces, we evaluated the total volume of the glass microfluidic platform. For facile representation, we provide a schematic of the biochip together with the calculated volumes of each micro-reservoir and channel in Supplementary information (Figure S1a). Based on these values, we could estimate the number of PC3 cells to be loaded and the range of volumes of chemoattractant to be introduced. After loading the PC3 cells, the cells were cultured in µRs 1 and 2 until they completely occupied channel GCh1. When the cells were approaching the submicrometer channels in the panpipe scaffold (schematically shown in Supplementary information, Figure S2b), the chemoattractant was introduced from µR4 to start the experiment. The observation area remained fixed for all experiments.



Biochip fabrication. We employed two successive laser approaches (subtractive and additive processing) for biochip fabrication. The T-shape glass microfluidic platform was developed by subtractive FLAE in a controllable 3D manner (Fig. 2a). It was necessary to optimize the experimental parameters (e.g., laser power, scanning mode and speed, scanning pitch, glass annealing temperatures) in order to obtain smooth glass surfaces appropriate for both polymer growth on the bottom of the channels and for cell investigations (see Roughness evaluation in Supplementary information, Figures S2 and 3). A smooth surface is essential to induce efficient cell migration and to clearly observe the cells in the microchannel using an optical microscope. Thus, the bottom surface was kept at the same smoothness (±20 nm) for all micro-reservoirs and channels. An optical microscopy image of the microfluidic platform after the FLAE process is presented in Fig. 2c, together with an optical image (Fig. 2d) of the details of the area of interest (crossing of channels GCh2-GCh1). A clear, transparent, defect-free structure was obtained by FLAE. We then applied “ship-in-a-bottle” integration of polymeric structures using TPP inside the glass GCh2 (Fig. 2b). In brief, after platform fabrication by FLAE, the formed microfluidic structure was filled with SU-8, a negative tone photoresist, which was subsequently exposed to femtosecond laser irradiation. In order to achieve higher resolution, oil immersion objective lenses with higher numerical apertures (NA = 1.25 or 1.4) were used. Sub-micrometer-diameter channels with a panpipe architecture consisting of six channels with lengths of 6, 8, 11, 14, 18 and 21 µm were formed (Fig. 2e) at the bottom of GCh2 and surrounded by the polymeric wall with anchoring branches to maintain stability. The more important role of the wall was to isolate the cell platform and the chemoattractant injection site. This process allowed reduction of the inner diameter of the glass microchannel, resulting in a 3D biomimetic environment in terms of resolution and hierarchy similar to that of tissue organization. The hybrid laser processing of both glass and polymer permitted the fabrication of a functional integrated biochip more suitable than previous microchip architectures for cell testing.


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Figure 2: Biochip fabrication: Schematics of a) FLAE of Foturan glass followed by b) TPP of SU8 inside GCh2; c) optical microscopy image of the glass microfluidic platform with d) details of the observation area; e) panpipe-shape scaffold consisting of six channels with lengths of 6, 8, 11, 14, 18 and 21 µm formed at the bottom of GCh2 integrated by TPP. Tailoring downsizing: improvement of TPP resolution inside the glass microfluidic channel The most significant challenge in this study was to create the narrowest true 3D channels as confined spaces for cancer cell migration. TPP is often applied on surfaces and processing parameters are in most of the cases known. Optimization of the TPP parameters was first carried out on coverslip surfaces or in open glass channels. We began by constructing linear ridges that could be built on a polymeric flat scaffold (one layer at the bottom) as can be seen in Fig. 3a. By covering the ridges with a polymeric rooftop, one can obtain stable 3D channels on a glass surface, without the need for any supporting scaffold (Fig. 3b). Detailed SEM observation of such channels revealed that they were 2 µm wide (Fig. 3c). The surface roughness of a single polymer layer is similar to that of the glass surface, allowing good adhesion of the polymer to glass (Fig. 3d). Additional data regarding surface evaluation are presented in the Supplementary Information. Also, we confirmed very good adhesion between the organic polymer and the inorganic glass at the interface. The panpipe-shape scaffold with six channels (6, 8, 11, 14, 18 and 21 µm long) were fabricated on the coverslip surface. SEM evaluation of the structure confirmed good adhesion and stability on the glass surface, even with a surrounding polymeric wall (Figs. 3e and f).

Figure 3: 3D confined spaces generated using TPP – SEM observation: a) Polymeric ridges grown on the supporting scaffold; b) array of polymeric channels with a polymeric rooftop; c) details of the 2 µm-wide channels; d) a single polymeric layer grown in an open glass channel; e) panpipeshape scaffold with six channels (6, 8, 11, 14, 18 and 21 µm long); f) polymeric wall surrounding the panpipe-shape scaffold (tilt view). Although 2 µm-wide polymeric channels can be fabricated inside the glass microfluidic channels, it is difficult to create narrower polymeric channels due to spherical aberration related to laser propagation in the three different refractive index media. Specifically, the laser beam must traverse



through immersion oil, glass, and polymer for in-channel fabrication, which significantly decreases fabrication resolution. To overcome this problem, we employed an SLM to correct the wave front of the laser beam 37. In simulated experiments, TPP combined with SLM was performed by irradiating SU-8 on a glass substrate with the laser beam from the glass side. In this configuration, the laser beam traverses through the immersion oil, glass, and SU-8, which is equivalent to TPP inside the glass channel. Using this approach, we could fabricate channels 1.25 µm wide and 4.5 µm high (Figs. 4a and d), 1.13 µm wide and 3.4 µm high (Figs. 4b and e), and even channels 0.7 µm wide and 2.4 µm high (Fig. 4c). In addition, a polymeric wall 76 µm wide and 50 µm high standing on the panpipe-shape scaffold was constructed on the glass surface (Fig. 4f). Finally, the panpipe-shape scaffold with the same structure as shown in Fig. 4c together with the polymeric wall was integrated into the closed glass microfluidic channels. SEM cannot be used to observe the inside of a microfluidic channel, and thus only optical microscopy is available to evaluate the width of the integrated polymeric microchannels. Optical microscope observation roughly estimated the width as ~900 nm. Enlargement of the width may be due to the slightly curved surface of the glass microchannel ceiling, which can also distort the wave front.

Figure 4: 3D confined spaces fabricated using TPP coupled with SLM – SEM evaluation of the panpipe-shape scaffold with six channels (6, 8, 11, 14, 18 and 21 µm long): a) and d) channels 1.25 µm wide and 4.5 µm high; b) and e) channels 1.13 µm wide and 3.4 µm high; c) the narrowest channels, 0.7 µm wide and 2.4 µm high; f) the polymeric wall surrounding the panpipe-shape scaffold (cross-sectional view). Nanoscale diffusion-based gradient generator Once the biochemical attractant is introduced to GCh2, the panpipe-shape scaffold surrounded by the separating wall acts as a diffusion-based gradient generator. A gradient of biochemical attractant is thus formed along the sub-micron-scale polymeric channels after diffusion by smooth, continuous flow from µR4 to µR3. In order to evaluate the gradient over time, a qualitative diffusion test was carried out by dye tracing using fluorescein, a fluorophore with absorption at 494 nm and emission at 512 nm (in water). Specifically, after polymeric scaffold integration into the glass microfluidic platform (Fig.


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5a), the integrated biochip was filled with water. The biochip was then mounted in a Petri dish inside a mini-incubator attached to a time-lapse microscope (Fig. 5c). After immobilization, 100 nl of fluorescein was added in µR 4 (Figs. 5b, d, f). We observed that the fluorescein reached the panpipe-shape scaffold at the entrance of the sub-micrometric channels in about 5 minutes (Fig. 5e).

Figure 5: Dye tracing in the biochip: a) integration using TPP of the panpipe-shape scaffold surrounded by a wall in GCh2; b) fluorescein loading in µR4; c) immobilizing a Petri-dishcontaining biochip inside a mini-incubator attached to a time-lapse microscope; d) fluorescence image of fluorescein flow from µR4 to GCh3; e) fluorescence image of fluorescein flow from µR3 to GCh2, reaching the submicron-scale polymeric channels, and f) zoom-in photo of the microchip 5 minutes after fluorescence loading, showing that µR4 and µR3 contain fluorescein (in green). The gradient was monitored by time-lapse microscopy for more than 9 hours. Figure 6a shows the initial moment when we started flow monitoring (t = 0 min). In Figs. 6b and c we show two frame images at t = 200 and 320 minutes, respectively. There is little difference between the two figures, confirming the stability of the gradient and the smooth flow. A time lapse movie (Movie 1) spanning 200 minutes to 320 minutes is provided in the Supplementary Information. Further, little change in the gradient was observed over the period of the evaluation. We therefore conclude that a stable gradient can be maintained for more than 9 hours between the channel entrance and exit. This is important for the study of cancer cell migration since such studies typically require continuous observation for several hours, as shown below.



Figure 6: Diffusion-based gradient in sub-micrometer-scale channels: fluorescence images of fluorescein a) at the moment when the dye reaches the panpipe-shape scaffold; b) 200 and c) 320 minutes after loading the fluorescein, respectively. Cancer cell migration through nanochannels The first tests with PC3 cells were undertaken using EGF as a chemoattractant. Our intent was to stimulate cell migration through the sub-micrometric channels and thus we developed a protocol for in vitro tests. First, PC3 cells were seeded in µR1 and µR2 and cultured almost to confluency in GCh1. In this configuration, they approached the entrance of the confined channels in the panpipe-shape scaffold (Figs. 7). Then, the cells were starved overnight (8 to 13 hours) by replacing the cell medium containing fetal bovine serum (FBS) (FBS+) with cell medium without FBS (FBS-). The biochip containing the starved PC3 cells was then mounted in a Petri dish and immobilized in the mini-incubator attached to the time lapse microscope (similar to the fluorescein test conducted in Fig. 5). Next, we added 100 nl of an EGF stock solution in µR4. The experimental conditions used are summarized in Table 1. EGF was diluted with FBS+ medium for some experiments and with FBS- medium for others. The EGF concentration was also varied from tens to hundreds of ng/ml. To obtain the results presented in Table 1 we have used different biochips (duplicate for each experiment) in order to preserve experimental controls. Table 1: Experimental conditions for cell chemotaxis experiments

*Response time is the time required for the cell to begin to orientate and migrate to the channel entrance (not the time required to transmigrate through the channel). **Migration is the capacity of the cell to move towards the chemoattractant up to the entrance of the channels. ***Incomplete means that the cell did not transmigrate through the channels.


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Below, we present the best results obtained using the conditions presented in the last row of Table 1. In the experiment, images were acquired every 20 seconds for 6 hours. Time t = 0 (Fig. 7a) is when the EGF should reach the panpipe-shape scaffold (about 5 minutes after loading in µR4). The first PC3 cell movements were observed at the exit of the 11 µm-long channel (3rd channel from the left, see Figs. 1c, d). Due to the very narrow 3D confined space, it appears that it is difficult for the cell nucleus to pass through this channel, but passage may be possible if the cell suffers severe deformation and its nuclear envelope disintegrates. This cell clearly fragmented to generate several entities, as observed at t = 25 min. (Fig. 7b). However, 42 minutes later (t = 67 min.), the cell could fuse the divided parts back into a single cell body following successful migration through the channel (red arrow in Fig. 7d) and further exhibiting reduced motile activity (red arrow in Figs. 7d-f). In the same experiment, we also focused on the migration of a second PC3 cell (yellow arrow), as shown in Fig. 7c. Cell activity was evident after 53 minutes at the exit of the 14 µm-long channel (4th channel from the left, see Fig. 1c, d). The same behavior as observed with the first cell (red arrow) was evidenced but in a much shorter time interval. A third PC3 cell (blue arrow) appeared at the exit of the longest (21 µm) channel (6th channel from the left, see Fig. 1c, d). The initial movement of this cell was visible after 101 minutes (Fig. 7e) and then the cell exhibited similar behavior, i.e., splitting and fusion, after 120 minutes. More interestingly, this cell appeared to fuse with parts of the second PC3 cell (Figs. 7e and f). A movie showing the entire process is available in the Supplementary information (Movie 2). In Movie 3 in the Supplementary information, we present the sequences for cells grown in the biochip without the addition of a chemo-attractant. Neither migration nor invasion is observed.

Figure 7: PC3 cell migration through 3D sub-micron-scale confined spaces: a) red arrow indicates the exit of the 3rd channel from the left from which the first cell will appear; b) the first PC3 cell is disintegrating after migration; c) first PC3 cell (red arrow) is reintegrating after migration while a second cell (yellow arrow) appears at the exit of the 4th channel; d) first PC3 cell (red arrow) is still reintegrating after migration while the second cell (yellow arrow) is spreading and disintegrating; e) first PC3 cell (red arrow) is reducing motility, second cell (yellow arrow) is reintegrating after migration while the third cell (blue arrow) appears at the exit of the 6th channel; f) the second PC3 cell (yellow arrow) fusing with third cell (blue arrow).



Discussion There have been several reports of the design and fabrication of microfluidic systems for the study of cancer cell migration in confined spaces. For example, using photo- and soft-lithography microfabrication techniques, PDMS was cast on SU-8 masters to fabricate a chamber consisting of two channels separated by a vertical barrier with multiple bays of pores with widths varying from 6 μm to 16 μm and lengths varying from 25 μm to 50 μm 38. Improved imaging potential relative to Boyden chambers was achieved by monitoring the migration rate and how the pore size affects cell morphology under the influence of chemotaxis. Another study proposed a sequence of microfluidic devices fabricated by applying the same PDMS casting technology that could mechanically constrain cancer cells to migrate inside microchannels with cross-sections comparable to the cell size, e.g., rectangular cross-sections with heights of either 3 or 12 µm, and widths of 6, 10, 12, 15, 18, 25, 30, 50, 75 or 100 µm 39. It has been demonstrated that cancer cells of different tissue origin exhibit motility that occurs spontaneously (in the absence of any external gradient) and that the cells show fast and persistent movement in one direction for several hours. In another study, a microfluidic device consisting of two open chambers each with a volume of 70 μL were connected by ten micro-channels 10 μm high and 600 μm long which tapered in width from 20 to 5 μm over a transition length of 50 μm 40. Chemoattractant-driven cell migration through the narrow tapered channels showed that the physical stress on cell migration might be cell-type specific. A microfluidic device consisting of an open access reservoir connected to a large channel by several transverse micro-channels 150 µm long, 5 µm high, and widths ranging from 2 to 20 µm, was fabricated by PDMS casting 41. The obtained results proved that adhering cancer cells could protrude their cytoplasm through a channel regardless of its width to migrate in response to a chemoattractant, although the nucleus acts as a limiting factor for migration when the channel cross-section is below 7 × 5 µm2. More recent studies, however, succeeded in demonstrating that due to the substantial physical stress, cell migration through confined spaces of 2 × 5 µm2 resulted in nuclear envelope rupture, followed by nuclear envelope reassembly and DNA damage repair, allowing survival of the cell 27. The microfluidic device in that study consisted of parallel channels formed by a series of PDMS pillars, through which cells could migrate along the chemotactic gradient over a period of several hours 24. In the present study, we have proposed the use of even smaller, unique, sub-micrometer-size channels with lengths from 6 to 21 µm built in a closed glass microfluidic device which can be also be applied to the identification of novel therapeutic targets. We believe that channel length dimensions approching a single cell size are similar to actual lenghts of in vivo transendothelial migration of cancer cell, while the narrowest diameter allows elucidacing new cell characteristics. We demonstrated that PC3 cells could migrate very fast (one hour only) in 3D confined spaces of 0.9 × 2.4 µm2. The closed microfluidic structure is advantageous for preventing evaporation of the liquid medium during extended observation. More importantly, the closed structure provides a safer environment for handling biohazardous samples, including tumor-derived cancer cells. In addition, the glass chip allows improved optical interrogation, reusability, and user-friendliness for the development of future assays. We hypothesize that 3D space confinement inside the reservoir provides a potential physical stimulus capable of initiating and modulating cell migration and invasion mechanisms through the microchannel, as well as regulating cell signaling events important for cancer cell fate. Acquiring a deeper understanding regarding migration in a 3D confined space using such biochips will play


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an important role in furthering our understanding of the mechanisms underlying cancer metastasis, and in facilitating high-throughput, physiologically relevant drug and cell line screening 42. It is noteworthy that our hybrid fabrication technique can create true 3D structures with sub-micronsized features, and the technique is highly flexible, which enables us to control the structures of the confined narrow spaces in a 3D tumor tissue-like architecture. Specifically, cross sectional shapes, including their width and height, can be changed, even for a single channel. 3D curved channels can also be fabricated. These distinct features allow the fabrication of structures with increased biomimetic features, which will further advance research into cancer cell metastasis, cell response in the presence of test drugs during screening on cell lines, as well as research using patient-derived material. In the present study, we used SU8 photoresist, since we had used it to explore and optimize the technology in previous works 34-36. Of course, the material to be integrated is not limited to it, and other biocompatible photoresists like Ormocers and IP-Ls can be used. More interestingly, we recently succeeded in integrated 3D protein microstructures inside the closed glass microfluidic channels 43-44, which would provide a more biomimetic environment. Conclusions We proposed with this study a new type of microfluidic chip based on a glass platform integrated with biomimetic 3D polymer structures with unprecedentedly sub-micron-sized features was fabricated by “ship-in-a-bottle” laser processing combined with the wavefront correction using SLM. Specifically, by two photonic processes, TPP and FLAE, six sub-micron polymeric channels of different lengths in a panpipe architecture were grown at the bottom of a closed microfluidic glass channel. By surrounding the panpipe-shape polymer scaffold in the closed glass channel with a polymer wall, the channels in the scaffold could act as a diffusion-based RGF gradient generator. A fluorescein dye tracing test confirmed that the gradient inside the polymer microchannels remained stable for over 9 hours. The sub-micrometer channels were proposed to act as confined biomimetic environments suitable for studying the motility of metastatic cancer cells. The migration and invasion potential of metastatic PC3 cells through 3D sub-micrometric spaces 900 nm wide was tested. We demonstrated in the same experiment that three PC3 cells could penetrate and migrate through three different lengths of channels. The first PC3 cell could migrate very fast (1 h) through a channel 11 µm long, the second cell migrated through a channel 14 µm long in 1 h and 10 minutes, while the third PC3 cell reached the exit of the longest channel (21 µm in length) in 2 hours. Interestingly, we found that in all cases, the cells first split their bodies into vesicular fragments after migrating through the channels and then fused the divided parts back into single bodies. We believe that the new “ship-in-a-bottle” laser technology can be used now to fabricate highly functional biochips integrated with shape-free 3D nanocomponents, specifically designed to support rapid in vitro tests, and will open new avenues into research not only into the mechanism underlying cancer metastasis, but also for drug screening and the discovery or testing of personalized therapies using patient-derived tissue/cells. Experimental Procedures Fabricaton of 3D Biomimetic Chips The two major steps involved in “ship-in-a-bottle” FLAE-TPP laser processing can be carried out using the same set-up (Figure S4). The second harmonic of a Yb-fiber laser beam (532 nm; 360



fs, 200 kHz) at a power of 5 mW was used for irradiation. Two lenses, L1 (f = 200 cm) and L2 (f = 50 cm), were used to reduce the diameter of the fundamental IR beam for efficient coupling with the second-harmonic generation (SHG) crystal. The power was adjusted using an attenuator (A). The power settings for FLAE were 3-5 mW, and below 0.1 mW for 2PP. Other two lenses, L3 (f = 30 cm) and L4 (f = 300 cm), were used to expand the diameter of the generated green laser beam to match the NA of the appropriate objective lens (OL). One dielectric mirror (DM) of ultrahigh reflectivity at 522 nm allowed white light to pass through for in situ process monitoring. The sample was placed on a 3D stage with two stepping motor controllers which allowed light to also pass through from beneath. A 3D stage controller, shutter, and CCD camera were controlled by PC programs. The process started with FLAE of Foturan glass in order to fabricate closed 3D microchannels, followed by TPP of SU-8 photoresist inside the fabricated microchannels. The hybrid process is schematically shown in Figure S5. Foturan from Mikroglas, Germany, was used as it provided suitable properties such as high Young’s modulus, low absorption, chemical stability, and biocompatibility. Foturan consists of lithium aluminosilicate doped with small amounts of sodium, antimony, silver and cerium. Glass substrates 10 mm × 10 mm × 2 mm were cleaned with acetone, alcohol, and deionized water, then immobilized on a 3D translation stage. For the FLAE process, irradiation was conducted through a 20× magnification objective lens with a NA of 0.46 to focus the laser beam into the sample. A stage controller moved the sample during irradiation at 1000 μm/s, according to the pre-programmed 3D patterns. Next, the sample was annealed with a twostep treatment at 500 ºC for 1 hour followed by 605 ºC for one hour. A subsequent chemical etching procedure in 10% HF solution was used to selectively remove the laser-modified regions, resulting in an embedded microchannel. A second annealing at 500 ºC for 1 hour, followed by another hour at 645 ºC, was needed to smooth the surface inside the glass microchannel. Further information regarding the procedures and mechanism is available elsewhere 34, 45-46. After fabrication, the channel was filled with a negative-tone photoresist, SU-8, chosen for its mechanical strength, aspect-ratio, chemical resistance, and biocompatibility. SU-8 was first diluted with acetone at a ratio of 1:1.2, followed by prebaking at 90-95 ºC for up to 24 hours to evaporate the solvent from the SU-8 resin and improve its adherence to the surface. The same laser set-up can be employed for the TPP process to integrate the 3D polymer nano- and micro-structures inside the glass microchannel. The Yb-fiber laser beam at a power setting of 100 μW was focused through a 100× magnification objective lens of NA 1.4 into the sample through oil immersion. The sample was translated using the stage controller at 400 μm/s to polymerize the SU-8 along the laser beam trajectory, followed by post-baking at 95 °C for 10 min. Unpolymerized resin was removed using SU-8 developer (1-methoxy-2-propylacetate) and then the polymeric patterns were developed inside the glass channels. The procedure was completed by drying for 5 min at 65 ºC. SLM was employed for wave front correction to improve the fabrication resolution for TPP inside the glass channel. In vitro experiments. Cell cultures In vitro on-chip experiments were designed to study the chemotaxis of metastatic prostate cells in response to the chemo-mechanical stress induced by EGF in sub-micrometer-size channels. The chemical gradient generated by the diffusion of tens of ng/mL EGF within the channels of the microfluidic device stimulated the migration. The concentration of EGF was chosen after conducting optimization tests.


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The microfluidic devices can be repeatedly used. They were sterilized with ethanol after every experiment and then mounted on a glass bottom Petri dish. If the polymer scaffold is affected by clogging after several experiments, it can be completely removed by Piranha solution and then another scaffold is fabricated in the same glass platform within one day. The human cell line PC3, derived from bone metastasis of a 62 year old male, was purchased from RIKEN BRC Cell Bank and cultured inside the chip. The cells were grown in RPMI 1640 supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin and maintained under a 5% CO2 atmosphere at 37 °C. Approximately 20-40 cells in the growth medium were seeded from µR1 into GCh1 and cultured to confluence in GCh1. After 72-h incubation (confluency), the medium was replaced with FBS-. After starvation for 8 to 13 hours, 100 nl EGF (20-200 ng/mL) was added to the appropriate chemoattractant chambers (in particular µR4). Live cell migration was recorded using time-lapse microscopy. The microscope was equipped with a mini-incubator with a CO2 supply and the temperature was controlled at 37 ºC (Figure S6). Images of the cells within the channels were captured at 40× to show their relative location along the microchannels at 20 second frame intervals for up to 15 hours. The frame images were compiled into a movie (SI). Acknowledgements Felix Sima is grateful to JSPS for a fellowship and support for this research. This work was partially supported by fundings received from the CONCERT-Japan Photonic Manufacturing Joint Call (FEASIBLE project) and the AMADA Foundation Research & Development grant (AF2017201). This project has also received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No. 654148 Laserlab-Europe. This work was further supported by UEFISCDI grant TE07/2018. Conflicts of interest “There are no conflicts to declare”. 1. Vashist, S. K.; Luppa, P. B.; Yeo, L. Y.; Ozcan, A.; Luong, J. H., Emerging Technologies for Next-Generation Point-of-Care Testing. Trends Biotechnol. 2015, 33 (11), 692-705. 2. Yazdi, A. A.; Popma, A.; Wong, W.; Nguyen, T.; Pan, Y.; Xu, J., 3D Printing: an Emerging Tool for Novel Microfluidics and Lab-on-a-Chip Applications. Microfluid. Nanofluid. 2016, 20 (3), 50, 1-18. 3. Alvarez, M. M.; Aizenberg, J.; Analoui, M.; Andrews, A. M.; Bisker, G.; Boyden, E. S.; Kamm, R. D.; Karp, J. M.; Mooney, D. J.; Oklu, R., Emerging Trends in Micro-and Nanoscale Technologies in Medicine: From Basic Discoveries to Translation. ACS Nano 2017, 11 (6), 5195– 5214. 4. Mark, D.; Haeberle, S.; Roth, G.; Von Stetten, F.; Zengerle, R., Microfluidic Lab-on-aChip Platforms: Requirements, Characteristics and Applications. In Microfluidics Based Microsystems, Springer: 2010, pp 305-376. 5. Whitesides, G. M., The Origins and the Future of Microfluidics. Nature 2006, 442 (7101), 368–373. 6. Hong, J.; Edel, J. B., Micro-and Nanofluidic Systems for High-Throughput Biological Screening. Drug Discovery Today 2009, 14 (3), 134-146.



7. Legendre, L. A.; Bienvenue, J. M.; Roper, M. G.; Ferrance, J. P.; Landers, J. P., A Simple, Valveless Microfluidic Sample Preparation Device for Extraction and Amplification of DNA from Nanoliter-Volume Samples. Anal. Chem. 2006, 78 (5), 1444-1451. 8. Dixon, C.; Lamanna, J.; Wheeler, A. R., Printed Microfluidics. Adv. Funct. Mater. 2017, 27 (11), 1604824-n/a. 9. Sackmann, E. K.; Fulton, A. L.; Beebe, D. J., The Present and Future Role of Microfluidics in Biomedical Research. Nature 2014, 507 (7491), 181–189. 10. El-Ali, J.; Sorger, P. K.; Jensen, K. F., Cells on Chips. Nature 2006, 442 (7101), 403–411. 11. Yi, C.; Li, C.-W.; Ji, S.; Yang, M., Microfluidics Technology for Manipulation and Analysis of Biological Cells. Anal. Chim. Acta 2006, 560 (1), 1-23. 12. Sia, S. K.; Whitesides, G. M., Microfluidic Devices Fabricated in Poly (dimethylsiloxane) for Biological Studies. Electrophoresis 2003, 24 (21), 3563-3576. 13. Li, C.; Boban, M.; Snyder, S. A.; Kobaku, S. P. R.; Kwon, G.; Mehta, G.; Tuteja, A., PaperBased Surfaces with Extreme Wettabilities for Novel, Open-Channel Microfluidic Devices. Adv. Funct. Mater. 2016, 26 (33), 6121-6131. 14. Chen, J.; Li, J.; Sun, Y., Microfluidic Approaches for Cancer Cell Detection, Characterization, and Separation. Lab Chip 2012, 12 (10), 1753-1767. 15. Hsiao, A. Y.; Torisawa, Y.-s.; Tung, Y.-C.; Sud, S.; Taichman, R. S.; Pienta, K. J.; Takayama, S., Microfluidic System for Formation of PC-3 Prostate Cancer Co-Culture Spheroids. Biomaterials 2009, 30 (16), 3020-3027. 16. McGregor, A. L.; Hsia, C.-R.; Lammerding, J., Squish and Squeeze—the Nucleus as a Physical Barrier during Migration in Confined Environments. Curr. Opin. Cell Biol. 2016, 40, 3240. 17. Albini, A.; Iwamoto, Y.; Kleinman, H.; Martin, G.; Aaronson, S.; Kozlowski, J.; McEwan, R., A Rapid In Vitro Assay for Quantitating the Invasive Potential of Tumor Cells. Cancer Res. 1987, 47 (12), 3239-3245. 18. Kramer, N.; Walzl, A.; Unger, C.; Rosner, M.; Krupitza, G.; Hengstschläger, M.; Dolznig, H., In Vitro Cell Migration and Invasion Assays. Mutat. Res., Rev. Mutat. Res. 2013, 752 (1), 1024. 19. Boyden, S., The Chemotactic Effect of Mixtures of Antibody and Antigen on Polymorphonuclear Leucocytes. J. Exp. Med. 1962, 115 (3), 453-466. 20. Zicha, D.; Dunn, G. A.; Brown, A. F., A New Direct-Viewing Chemotaxis Chamber. J. Cell Sci. 1991, 99 (4), 769-775. 21. Wells, A., Tumor Invasion: Role of Growth Factor-Induced Cell Motility. Adv. Cancer Res. 1999, 78, 31-101. 22. Laskin, J. J.; Sandler, A. B., Epidermal Growth Factor Receptor: a Promising Target in Solid Tumours. Cancer Treat. Rev. 2004, 30 (1), 1-17. 23. Wang, S.-J.; Saadi, W.; Lin, F.; Nguyen, C. M.-C.; Jeon, N. L., Differential Effects of EGF Gradient Profiles on MDA-MB-231 Breast Cancer Cell Chemotaxis. Exp. Cell Res. 2004, 300 (1), 180-189. 24. Davidson, P. M.; Sliz, J.; Isermann, P.; Denais, C.; Lammerding, J., Design of a Microfluidic Device to Quantify Dynamic Intra-Nuclear Deformation during Cell Migration through Confining Environments. Integr. Biol. 2015, 7 (12), 1534-1546. 25. Saadi, W.; Wang, S.-J.; Lin, F.; Jeon, N. L., A Parallel-Gradient Microfluidic Chamber for Quantitative Analysis of Breast Cancer Cell Chemotaxis. Biomed. Microdevices 2006, 8 (2), 109118.


Page 16 of 27

Page 17 of 27 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


26. Truong, D.; Puleo, J.; Llave, A.; Mouneimne, G.; Kamm, R. D.; Nikkhah, M., Breast Cancer Cell Invasion into a Three Dimensional Tumor-Stroma Microenvironment. Sci. Rep. 2016, 6, 34094, 1-18. 27. Denais, C. M.; Gilbert, R. M.; Isermann, P.; McGregor, A. L.; te Lindert, M.; Weigelin, B.; Davidson, P. M.; Friedl, P.; Wolf, K.; Lammerding, J., Nuclear Envelope Rupture and Repair during Cancer Cell Migration. Science 2016, 352 (6283), 353-358. 28. Yuan, B.; Li, Y.; Wang, D.; Xie, Y.; Liu, Y.; Cui, L.; Tu, F.; Li, H.; Ji, H.; Zhang, W.; Jiang, X., A General Approach for Patterning Multiple Types of Cells Using Holey PDMS Membranes and Microfluidic Channels. Adv. Funct. Mater. 2010, 20 (21), 3715-3720. 29. Bhatia, S. N.; Ingber, D. E., Microfluidic Organs-on-Chips. Nat. Biotechnol. 2014, 32 (8), 760-772. 30. Hanada, Y.; Sugioka, K.; Kawano, H.; Ishikawa, I. S.; Miyawaki, A.; Midorikawa, K., Nano-Aquarium for Dynamic Observation of Living Cells Fabricated by Femtosecond Laser Direct Writing of Photostructurable Glass. Biomed. Microdevices 2008, 10 (3), 403-410. 31. Hanada, Y.; Sugioka, K.; Shihira-Ishikawa, I.; Kawano, H.; Miyawaki, A.; Midorikawa, K., 3D Microfluidic Chips with Integrated Functional Microelements Fabricated by a Femtosecond Laser for Studying the Gliding Mechanism of Cyanobacteria. Lab Chip 2011, 11 (12), 2109-2115. 32. Xu, J.; Wu, D.; Hanada, Y.; Chen, C.; Wu, S.; Cheng, Y.; Sugioka, K.; Midorikawa, K., Electrofluidics Fabricated by Space-Selective Metallization in Glass Microfluidic Structures using Femtosecond Laser Direct Writing. Lab Chip 2013, 13 (23), 4608-4616. 33. Xu, J.; Kawano, H.; Liu, W.; Hanada, Y.; Lu, P.; Miyawaki, A.; Midorikawa, K.; Sugioka, K., Controllable Alignment of Elongated Microorganisms in 3D Microspace using Electrofluidic Devices Manufactured by Hybrid Femtosecond Laser Microfabrication. Microsyst. Nanoeng. 2017, 3, 16078, 1-9. 34. Wu, D.; Wu, S. Z.; Xu, J.; Niu, L. G.; Midorikawa, K.; Sugioka, K., Hybrid Femtosecond Laser Microfabrication to Achieve True 3D Glass/Polymer Composite Biochips with Multiscale Features and High Performance: the Concept of Ship-in-a-Bottle Biochip. Laser Photonics Rev. 2014, 8 (3), 458-467. 35. Wu, D.; Xu, J.; Niu, L.-G.; Wu, S.-Z.; Midorikawa, K.; Sugioka, K., In-Channel Integration of Designable Microoptical Devices using Flat Scaffold-Supported Femtosecond-Laser Microfabrication for Coupling-Free Optofluidic Cell Counting. Light: Sci. Appl. 2015, 4 (1), e228. 36. Wu, D.; Niu, L.-G.; Wu, S.-Z.; Xu, J.; Midorikawa, K.; Sugioka, K., Ship-in-a-Bottle Femtosecond Laser Integration of Optofluidic Microlens Arrays with Center-Pass Units Enabling Coupling-Free Parallel Cell Counting with a 100% Success Rate. Lab Chip 2015, 15 (6), 15151523. 37. Horváth, B.; Ormos, P.; Kelemen, L., Nearly Aberration-Free Multiphoton Polymerization into Thick Photoresist Layers. Micromachines 2017, 8 (7), 219, 1-9. 38. Breckenridge, M. T.; Egelhoff, T. T.; Baskaran, H., A Microfluidic Imaging Chamber for the Direct Observation of Chemotactic Transmigration. Biomed. Microdevices 2010, 12 (3), 543553. 39. Irimia, D.; Toner, M., Spontaneous Migration of Cancer Cells under Conditions of Mechanical Confinement. Integr. Biol. 2009, 1 (8-9), 506-512. 40. Rao, S.; Tata, U.; Lin, V. K.; Chiao, J.-C., The Migration of Cancer Cells in Gradually Varying Chemical Gradients and Mechanical Constraints. Micromachines 2014, 5 (1), 13-26.



41. Malboubi, M.; Jayo, A.; Parsons, M.; Charras, G., An Open Access Microfluidic Device for the Study of the Physical Limits of Cancer Cell Deformation during Migration in Confined Environments. Microelectron. Eng. 2015, 144, 42-45. 42. Paul, C. D.; Mistriotis, P.; Konstantopoulos, K., Cancer Cell Motility: Lessons from Migration in Confined Spaces. Nat. Rev. Cancer 2017, 17 (2), 131–140. 43. Serien, D.; Kawano, H.; Miyawaki, A.; Midorikawa, K.; Sugioka, K., Femtosecond Laser Direct Write Integration of Multi-Protein Patterns and 3D Microstructures into 3D Glass Microfluidic Devices. Appl. Sci. 2018, 8 (2), 147, 1-13. 44. Serien, D.; Sugioka, K., Fabrication of Three-Dimensional Proteinaceous Micro-and NanoStructures by Femtosecond Laser Cross-Linking. Opto-Electr. Adv. 2018, 1 (4), 180008. 45. Hongo, T.; Sugioka, K.; Niino, H.; Cheng, Y.; Masuda, M.; Miyamoto, I.; Takai, H.; Midorikawa, K., Investigation of Photoreaction Mechanism of Photosensitive Glass by Femtosecond Laser. J. Appl. Phys. 2005, 97 (6), 063517. 46. Sugioka, K.; Cheng, Y., Fabrication of 3D Microfluidic Structures inside Glass by Femtosecond Laser Micromachining. Appl. Phys. A: Mater. Sci. Process. 2014, 114 (1), 215-221. Supporting Information Biochip design and concept; Roughness evaluation; Experimental procedures. Movie 1: Time lapse microscopy of fluorescein flow; Movie 2: Time lapse microscopy of cancer cell migration; Movie 3: Time lapse microscopy of cancer cell migration; TOC graphic Graphic for manuscript


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