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Pore shape defines paths of metastatic cell migration Brenda J. Green, Magdalini Panagiotakopoulou, Francesca Michela Pramotton, Georgios Stefopoulos, Shana O. Kelley, Dimos Poulikakos, and Aldo Ferrari Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b00431 • Publication Date (Web): 26 Feb 2018 Downloaded from http://pubs.acs.org on February 28, 2018

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Nano Letters

Pore shape defines paths of metastatic cell migration

Brenda J. Green†‡γ, Magdalini Panagiotakopoulou‡γ, Francesca Michela Pramotton ‡, Georgios Stefopoulos‡, Shana O. Kelley†, Dimos Poulikakos‡*, and Aldo Ferrari‡* ‡

B.J. Green, M. Panagiotakopoulou, Francesca Michela Pramotton, G. Stefopoulos, Prof. D. Poulikakos, Dr. A. Ferrari Laboratory of Thermodynamics in Emerging Technologies, Department of Mechanical and Process Engineering, ETH Zurich. Sonneggstrasse 3, CH-8092 Zurich, Switzerland †

B.J. Green, Prof. S.O. Kelley

†Institute of Biomaterials and Biomedical Engineering, University of Toronto, 144 College Street, Toronto M5S 3M2, Canada γ

These authors contributed equally to the work.

E-mail: [email protected]; [email protected]

ACS Paragon Plus Environment

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Abstract Invasion of dense tissues by cancer cells involves the interplay between the penetration resistance offered by interstitial pores and the deformability of cells. Metastatic cancer cells find optimal paths of minimal resistance through an adaptive path-finding process, which leads to successful dissemination. The physical limit of nuclear deformation is related to the minimal cross section of pores that can be successfully penetrated. However, this single biophysical parameter does not fully describe the architectural complexity of tissues featuring pores of variable area and shape. Here, employing laser nanolithography, we fabricate pore microenvironment models with well-controlled pore shapes, through which human breast cells (MCF10A) and their metastatic offspring (MCF10CA1a.cl1) could pervade. In these experimental settings we demonstrate that the pore actual shape, and not only the cross section, is a major and independent determinant of cancer penetration efficiency. In complex architectures containing pores demanding large deformations from invading cells, tall and narrow rectangular openings facilitate cancer migration. In addition, we highlight the characteristic traits of the explorative behavior enabling metastatic cells to identify and select such pore shapes in a complex multi-shape pore environment, pinpointing paths of least resistance to invasion.

KEYWORDS: cancer migration, metastasis, pore shape, Golgi polarization, path finding, microfabrication


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Interstitial migration of tumor cells constitutes the pathological link between a primary lesion and its metastatic progression in a distant body location 1. The dissemination of cancer seeds occurs through interstitial microenvironments with complex architectures generated by extracellular matrix fibers, adhesion proteins, proteoglycans, and stromal cells 2. Beyond biological signaling, the physical resistance offered by inherent obstacles encountered along the migration path is therefore a critical determinant of the tissue infiltration performance 3. Highly invasive cells can optimize their migration strategy to select paths of least resistance in the interstitium 4. Invasive cancer embedded within a dense 3D extracellular matrix (ECM) must overcome the surrounding physical constraints to allow tumor expansion

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. Metastatic cells detect matrix

elasticity and adapt their migration mode to enable efficient dissemination 7. For proteasedependent advancement they recruit proteolytic systems to sever collagen fibrils and enlarge matrix pores to a comfortable size 5. Alternatively, protease-independent migration modes can be adopted. In this case the cell exploits its deformability to fit through narrow openings while the ECM is not remodeled. Adaptive interconversion of these two migratory phenotypes is a hallmark of invasive tumor cells. Directional migration is the result of cell polarization, which generates a propulsive front-torear imbalance of cellular tractions 8. In addition, the cell spreading sustains an apico-basal distribution of the components establishing adhesion to the substrate 9. The resulting asymmetric distribution of the cytoskeleton contributes to shape the cell and the nucleus

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. Lateral

compressive forces generated by actin filaments are the main actuator of nuclear deformations typical of anisotropic spreading and polarized migration. Normal compressive forces contribute to reshape the nucleus to a much lesser extent 11.


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Penetration of a cell through dense tissues and narrow openings is connected to the nuclear stiffness 12. Nuclear deformability thus defines the physical limits of interstitial pore penetration 13, 14

and subtends to different performances in distinct cell types and cell cycle phases

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. It is

therefore logical to hypothesize that the penetration efficiency of a cell when interfacing an obstacle that requires high nuclear deformation, is influenced not only by the pore area but also by the pore shape confronted by the cell 16, 17. Based on this assumption, the pore geometry may significantly contribute to define paths of least resistance that can be hijacked by metastatic cells. The influence of pore cross-section on interstitial penetration has been established by detailed studies, which exploited reconstituted collagen matrixes channels

14, 19, 20, 21

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, artificial filters

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, or microfluidic

to constrain the advancement of migrating cells. However, while interstitial

fissures of human tissues feature heterogeneous cross-sections and geometries,

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these

methods do not adequately provide the flexibility to explore the mutual interplay between the size and shape of constrictions challenging cell invasion. In this work, 3D laser nanolithography

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of a host of basic rectangular pore designs was

exploited to distinguish the effects of pore cross-section and shape on interstitial cancer migration. Insurmountable vertical walls were fabricated on optically transparent substrates to obstruct the progression of migrating cells and to force them to explore pore gates imposing large nuclear deformations. The flexibility of the nanofabrication method allowed complete freedom in the spatial arrangement and geometry of the openings, whose well-defined perimeter corresponds to the values reported for interstitial pores of human tissues

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. This platform therefore provides a

model of protease-independent pore penetration during interstitial migration. The direct observation of cell interaction with openings displaying variable shape allowed the establishment


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of these topographic parameters as independent determinants of interstitial migration. Based on this paradigm, complex spatial arrangements of pores with identical area and variable shape were generated to reveal the pathfinding capability and polarization versatility of migrating metastatic human breast cancer cells.


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Vertical walls with basal pores of defined area and shape (Fig. 1A) were designed to halt the broad migration of human breast cells, and enable the focusing of their passage through the basal openings. The 3D nanolithography structures were fabricated on glass coverslips, in block arrays (Fig. S1). The unit of the array comprised a 22 µm tall, 112 µm in length and 2.5 µm thick vertical wall. These dimensions were comfortably realizable by laser nanolithography. The unit wall height was selected to generate an impassable obstacle to cell migration while the wall length ensured its structural stability. A single unit contained 3 pores of a given cross-sectional area and aspect ratio (i.e. the width to height ratio; Fig. 1A, Table S1). The openings were spaced out by 20 µm to enable the cells to explore multiple passages at once (Fig. S2). The block array consisted of 2 units per side; and 8 units total per block. The structures were printed in block fashion to allow cells to interact with 24 pores over a period of 24 h. The large number of passages per block increased the probability of pore engagement. The cross-sectional area of the openings was selected in a range encompassing the values reported for pores in the human dermis 22. Specifically, four distinct values ranging from 16 to 49 µm2 were included in order to impose large nuclear deformations on penetrating cells (60% or more

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). Pores with the same cross-sectional area were designed in four aspect ratio (a.r.)

variations, yielding either squared (a.r. = 1), tall and narrow (a.r. = 0.1, 0.3) or flat and wide rectangular (a.r. = 3) openings. Therefore, the resulting parametric matrix included 16 pores of variable shape and/or size which could be freely arranged in the migration study (Fig. 1A-C). Human breast epithelial cells (MCF10A) and corresponding highly metastatic offspring (MCF10CA1a.cl1) expressing histone-2B-eGFP were selected as their migratory behavior and metastatic potential is well established

24, 25

. The two cell lines feature similar nuclear size,

therefore setting the same nuclear deformation to penetrate identical pores (Fig. S3A). Migration


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of MCF10CA1a.cl1 cells on gratings (2 µm period, 50% duty cycle, 1 µm grove depth;

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)

showed comparable alignment and slightly increased persistence, indicating a similar behavior in response to topographic contact guidance (Fig. S4). As expected, transformed cells were characterized by a shorter cell cycle, faster migration velocity, and higher invasiveness in matrigel invasion assays (Fig. S3B-D). Protein expression profiles further confirmed the different phenotype of MCF10CA1a.cl1 consistent with their metastatic transition

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(Fig. S5, Fig. S6). MCF10CA1a.cl1 cells showed elevated protein levels

of HRas, vimentin, Talin1 and neural cell adhesion molecule relative to MCF10A cells. In penetration experiments, cells were seeded on substrates featuring multiple topographic elements (Fig. 1C, Fig. S2). Cells migrating along the 3D structures required nuclear deformation for successful penetration through the basal openings. This penetration process was monitored through live cell microscopy over 24 h. The nuclear deformation and position relative to the pore was used to define three different outcomes of a penetration attempt (Fig. 1D). After introducing the nucleus under the pore, the cell may either complete its translocation on the opposite side (successful penetration), remain blocked (impasse), or disengage on the same side (disengagement). Engagement events encompass any of penetration, disengagement or impasse. Long-term observation allowed the capture of multiple engagement events. Each penetration attempt was fully resolved for the corresponding size and a.r. of the engaged pore, as well as for its temporal dynamics. This set of data provided a quantitative fingerprint for the behavior of the two cell lines under investigation. Neither cell line could successfully penetrate pores featuring the smallest cross-section (i.e. 16 µm2) and very few attempts were recorded for these openings (Fig. 2A, 2C, and Fig. S7A, B). This result is in line with previous reports setting the limit of nuclear deformation to 80-90%

13, 15

. Attempts to penetrate larger passages (27 µm2) were


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recorded for tall and narrow (vertically-oriented) pores (a.r. = 0.1 and 0.3) but rarely for isotropic (a.r. = 1) or wide and flat (horizontally oriented) ones (a.r. = 3; Fig. 2A, 2C, Fig. S7C,D). Few penetration attempts were observed for flat pores of all tested cross-sections, indicating that the necessary nuclear deformations are highly disfavored (Fig. S7). These results indicate that the pore shape and orientation are major determinants of the outcome for penetrations requiring a large deformation of the cell nucleus (in the range between 70-90%). In addition, they demonstrate that penetration attempts requiring lateral nuclear compression can be accomplished more efficiently that those requiring a normal (to the cell surface) compression. The ability of cells to better penetrate pores featuring low a.r. may be related to the architecture of the actin cytoskeleton which actuates the required nuclear deformation

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. Lateral

compressions enabling the penetration of tall and narrow pores (low a.r.) are generated by actin filaments flanking the nucleus. Normal deformations, necessary for the penetration of flat and wide openings (high a.r.), entail a contractile structure overhanging the nucleus (i.e. the actin cap;

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). Both MCF10A and MCF10CA1a.cl1 cells displayed prominent actin fibers on their

basal and dorsal sides (Fig. 2E, Fig. S15A). However, no organized actin structure was detected at the apical side above the nucleus. The absence of an actin cap is typical of transformed cells and has been associated to increased nuclear deformability upon migration through narrow constrictions

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. In our experimental settings, pervading MCF10A and MCF10CA1a.cl1 cells

displayed a characteristic actin meshwork organized at the lateral sides of the nucleus in correspondence to the region withstanding a large deformation to fit the opening11; (Fig. 2F, G, S15B). Based on these observations, we speculate that low a.r. (tall and narrow) pores, which display greater lateral surface area, may offer an easier access to the required actin-mediated nuclear deformation, leading to faster and more successful penetration.


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MCF10A cells could engage all vertically-oriented (tall and narrow) or square pores. Most penetration attempts led to either a successful penetration or to an impasse (~75% and ~15% of the events; respectively. Fig. 2A and 2B). Therefore, only very rare disengagement events (~10%) ensued an initial nuclear engagement (Fig. 2A-B, Fig. S7). When exposed to corresponding pores MCF10CA1a.cl1 cells showed a surprisingly different behavior characterized by an almost complete absence of impasse (~4%) and a high disengagement frequency (~50%; Fig. 2C-D, Fig. S7). This was further supported by the dynamics of cell disengagement, which was accomplished three times faster by these metastatic cells (Fig. 2H). Penetration times showed lower differences between the two cell types (Fig. 2I). These results indicate that, while their non-transformed counterpart mostly remain committed to the engagement of a pore, metastatic cells have the ability to temporarily explore an opening and quickly retract to continue searching. This exploratory behavior exhibited with specific pore shapes defines a typical migration strategy of MCF10CA1a.cl1 cells, which we defined as ‘pathfinding’ (see Experimental Section; Fig. 2A and 2C). Over the course of an experiment, cells underwent division or migrated to a sufficient extent to form clusters. Therefore, the cell density increased both globally (as a result of proliferation) and locally (as a result of cell clustering). MCF10CA1a.cl1 cells did not show any notable change in the pore penetration performance as a function of the local density. The same frequency of penetrations, disengagements, or impasse was recorded for a given pore geometry at low (i.e. individual cells engaging the pore) or high (cells in contact with one or more neighbors) densities. Differently, MCF10A cells exhibited fewer disengagement events and increased impasse at higher cell densities. The majority (80%) of engagement events for MCF10A cells took place at low cell density, yet these results suggest that the establishment of cell-to-cell


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junctions between epithelial cells restricts migration and thus demotes the pervasion of pores. Mesenchymal transition in metastatic cancer relaxes contact inhibition and may thus allow pore penetration despite local crowding 29. Directional migration requires key intracellular structures, including the actin cytoskeleton, the mitochondria, the Golgi apparatus (Golgi), the microtubule organizing center, and the plasma membrane, to assume a typical front-to-rear position relative to the nucleus in a process sustained by the coordinated activity of small GTPases

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. The relocation of the Golgi at the

front edge provides membrane and associated proteins required for the generation of cell protrusions 32. Instability of this polarization mechanism is associated with cancer cell plasticity and results from the dysregulation of controlling factors such as Cdc42 29. To decipher the role of directional migration in the navigation through complex environments, we analyzed the polarity of MCF10A and MCF10CA1a.cl1 cells attempting to penetrate the 3D microstructures (Fig. 3A). The front-to-rear polarity of cells interacting with pores was visualized by a double fluorescent staining, reporting the mutual position of the Golgi (Cell Light Golgi RFP) and the cell nucleus (histone 2B GFP; Fig. 3C-F). In the absence of directional signals, neither cell type displayed a preferential positioning of the Golgi, indicative of a random, unpolarized migration modality (Fig. 3A). The formation of small clusters (less than 10 cells) did not influence cell polarity (Fig. S12). During the experiments, nuclear engagement of the basal pores (Fig. 2) was accomplished by the cells either in a polarized or unpolarized manner. In the majority of cases for polarized engagement, (65% for MCF10A and 66% for MCF10CA1a.cl1 cells; respectively) the Golgi was the first compartment to be inserted in the opening (Fig. 3B, Fig. S9A,B, Fig. S10A,B, Fig. S11A,B). The Golgi’s rapid penetration was followed by the nuclear engagement of the same passage (Fig. 3C,


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D). Such configuration, resulted in successful nuclear penetration in the majority of cases (p = 0.75 and p = 0.81; for MCF10A and MCF10CA1a.cl1 cells: respectively). After penetration, the cell continued to migrate away from the pore. A consistent fraction of polarized MCF10A cells that entered the opening remained blocked into a non-evolving nuclear engagement, yielding a significant impasse probability (p = 0.17). Nuclear disengagement from the pore was rare, and was only observed with very small frequency (p = 0.03; Fig. 3B). On the contrary, MCF10CA1a.cl1cells attempting a penetration through a polarized engagement did not linger at impasses, but instead showed a high disengagement frequency (p = 0.19), which allowed them to release from the pore. During unpolarized engagement events (35% for MCF10A and 34% for MCF10CA1a.cl1 cells; respectively, Fig. 3B), nuclear engagement was accomplished by the cell when the Golgi was lagging behind (Fig. 3E, F). These engagements were generally unproductive, yielding a low penetration rate (p = 0.31 and p = 0.23 for MCF10A and MCF10CA1a.cl1 cells; respectively) and mostly resulting in disengagement (p = 0.62 and p = 0.65 for MCF10A and MCF10CA1a.cl1 cells; respectively, Fig. 3B, Fig. S9C,D, Fig. S10C,D; S11C,D). Impasse events were not observed for unpolarized cells (Fig. 3B). Finally, relocation of the Golgi from the rear to the front was possible upon nuclear engagement (p = 0.08 and p = 0.12 for MCF10A and MCF10CA1a.cl1 cells; respectively). For such transition the Golgi had to squeeze and penetrate a passage engulfed by the nucleus. In summary, the frequency of disengagement events recorded for MCF10CA1a.cl1 cells, combined with their faster dynamics (Fig. 2E) demonstrate a higher versatility during penetrative migration of these cells. This result is supported by the observation that activity levels of Cdc42 are upregulated in MCF10CA1a.cl1 cells as compared to MCF10A (Fig. 3G). The Rac1 and RhoA levels between the two cells lines are similar (Fig. S13);


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highlighting the observation that the polarization of the cells is a function mainly regulated by Cdc42. We next investigated whether the engagement instability of metastatic cells is a key driving force for selective navigation in a complex porous environment. A dedicated experiment was designed to test whether MCF10CA1a.cl1 cells can find a preferential path (Fig. 4). Four barriers, each composed of a repetition of vertical walls for a total width of 1.5 mm, were printed in a parallel arrangement (Fig. 4A,B). Individual vertical walls featured 5 basal pores with identical cross section (36 µm2) but variable a.r. While the central pore was vertically-oriented (tall and narrow, a.r.= 0.3) to offer a low-resistance passage, the remaining 4 pores had a horizontal orientation (flat and wide, a.r. = 3) thus producing a majority of high-resistance openings (Fig. 2 and Fig. 4A). Finally, the periodic assembly of vertical walls was out of phase between subsequent parallel barriers, yielding a staggered distribution of low-resistance pores (Fig. 4B). MCF10A or MCF10CA1a.cl1 cells were seeded on one side of the array and were allowed to migrate towards the obstacles. While approaching the parallel barriers, cells tended to proliferate and aggregate forming multicellular clusters progressing in the same direction

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. A cell image

velocimetry (CIV)34 analysis of motility in these clusters revealed that MCF10A cells moved with high correlation typical of connected epithelial cells, prior to making contact with the barriers (Fig. S14).35-37 The correlation length indicates that groups of up to 10 cells tended to move coherently within these clusters. The vertical barriers hindered the advancement of MCF10A cells, and the movement lost coherence displaying a correlation length close to a single cell diameter (Fig. 4C, D). In contrast, MCF10CA1a.cl1 collectives showed an initial low coordination (2-3 cells; Fig. S14), which was however not affected by the interaction with the


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array (Fig. 4D). Specifically, the correlation lengths indicates that small cell clusters (Fig. S14) were able to navigate coherently in the complex porous environment. These results suggest that the ability of metastatic cells to retain some degree of coordination in the presence of physical barriers may contribute to pathfinding and increase their pervasion efficiency. The migration of MCF10A and MCF10CA1a.cl1 cells interacting with basal pores in the barrier array was monitored to capture multiple penetration events. Selective navigation of cells was evaluated by measuring the overall frequency of penetration through low resistance pores. A random pore selection (identified by the selection index in Fig 4E) was expected to approach 0.2, a value imposed by the design (Fig. 4E). Any preferential pervasion of more favorable openings would render higher values with a maximum of 1 if only central pores are penetrated. The analysis of MCF10A cells navigating through the barrier array showed that for these cells, the penetration of high-resistance pores was almost 4 times more frequent than for the lowresistance counterparts. The resulting selection index of 0.29 ± 0.05 (Fig. 4E) is indicative of a poorly selective advancement. Pervading MCF10CA1a.cl1 cells showed a markedly different behavior, and were able to preferentially penetrate low-resistance pores. The selection index = 0.52 ± 0.06 was correspondingly much higher (Fig. 4E). This adaptive pathfinding scheme was further supported by an increased explorative movement in the direction parallel to the barriers (Fig. 4F), which was less pronounced in MCF10A cells. These results demonstrate that MCF10CA1a.cl1 cells are able to navigate through a complex arrangement of pores and select openings with a vertical orientation. Cell migration in interstitial tissues is an adaptive process resulting from the interplay between advancing cells and the surrounding mechanical and molecular extracellular environment. This mutual exchange, or reciprocity38 involves ECM stiffness and dimension as well as cell


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deformability. In dense matrixes obstructing cell pervasion, the shape of pores and their spatial distribution along the direction of movement may constitute an independent physical parameter contributing to define both the path and the outcome of cell migration (Fig. 4). In this scenario, metastatic cells may tailor their migration mode to successfully navigate across the interstitium.


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In summary, this work exploited an on-demand nanolithography approach to generate complex arrangements of topographic openings offering a passage with well-defined cross section and shape to migrating cells. The resulting reductionist model of the interstitium was used to decouple the role of pore shape during the penetrative migration of normal and cancer cells (Fig. 1). The data obtained from live cell observation clearly demonstrate that the pervasion of pores demanding large nuclear deformation is pore shape dependent. In particular, elongated pore shapes oriented along the apico-basal axis of cells offer a more favorable passage as compared to identical pores with horizontal orientation (Fig. 2). Furthermore, the comparative analysis of breast epithelial cells and their metastatic offspring showed a higher versatility in the penetrative behavior of the latter, which was linked to a rapid and frequent reversal of migration directionality and to a versatile front-to-rear polarization (Fig. 3). Upon the pervasion of complex porous environments, the pathfinding behavior typical of metastatic cells is depicted as a functional preference for low-resistance pore shapes (Fig. 4). Therefore, while non-transformed cells remain committed to the pores encountered along the path of directional migration, regardless of the offered resistance, the quick engagementdisengagement turnover allows metastatic cells to dislodge from pores demanding a long-lasting interaction and only commit to penetrate favorable passages.


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Figure 1. Experimental design. (A) Schematic of cells engaging with printed pores and (B) characteristic scanning electron microscopy images (45° tilt) of the structure design with pores featuring variable aspect ratio (a.r.). (C) Overview of the pore geometries, with varying cross section area (µm2) and a.r. (D) Images extracted from a time lapse depicting the inverted fluorescence signal of the nucleus of a MCF10CA1a.cl1 cell disengaging (upper row) from a 36 µm2 pore with a.r. = 0.3, a MCF10A cell penetrating (middle row) through a 16 µm2 pore with


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a.r. = 1, and a MCF10A cell at an impasse (lower row) through a 36 µm2 pore with a.r. = 0.3; respectively. The relative engagement times are 1.7, 4.7, and 9.7 h, respectively.


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Figure 2. Effect of pore shape and geometry on cell penetration dynamics. Pore penetration and disengagement of MCF10A cells (A, B) and MCF10CA1a.cl1 cells (C, D), as a function of cross sections and a.r. The color-coding corresponds to the pathfinding index (PI), a descriptor of the cell-pore interaction outcome (see Experimental Section). Each dot in B and D represents 2 events. (E, F) Representative immunofluorescence confocal sections along the apical, equatorial, and basal surfaces of MCF10A cells stained for nucleus (green) and actin (red) on substrate without (E) and with (F) constrictions. Printed vertical barriers forming pores are reported in blue. (G) Three-dimensional reconstruction of the confocal image of panel F which reveals the basal localization of actin fibers and the lack of an actin cap. Typical disengagement (H) and penetration time (I) of MCF10A and MCF10CA1a.cl1 cells as a function of a.r. for pores featuring a cross section of 36 µm2. Error bars correspond to the standard error of the mean. The number of events (n) includes penetration, disengagement, or impasse for either cell type. Engagement events were recorded over 24 h. * p

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