Podosome Formation and Development in Monocytes Restricted by

Letter pubs.acs.org/NanoLett

Podosome Formation and Development in Monocytes Restricted by the Nanoscale Spatial Distribution of ICAM1 Andreas S. Andersen,† Hüsnü Aslan,† Mingdong Dong,† Xingyu Jiang,‡ and Duncan S. Sutherland*,† †

Interdisciplinary Nanoscience Center (iNANO), Århus University, Århus 8000, Denmark National Center for Nanoscience and Technology (NCNST), Chinese Academy of Sciences (CAS), Beijing, China

‡

S Supporting Information *

ABSTRACT: We studied podosome formation and development in activated monocytes (THP1) at ICAM1 (intercellular adhesion molecule 1) nanopatterns of circular and ring-shaped domains and show that cellular binding to a preclustered ICAM1 nanopattern requires ligand patches of at least 200 nm (corresponding to 14 or more integrins). Podosome-like adhesion formation depends on the structure of the ligand pattern under the developing podosome with larger single domains promoting adhesion in a single patch and multiple smaller domains allowing podosome formation by integration of at least 2 smaller domains on either side of the podosome core. Maturation to rosette structures and recruitment of proteases were only observed with macroscopic ICAM1 presentation. KEYWORDS: ICAM1, nanopattern, THP1, adhesion, podosome

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as clear evidence of transcellular diapedesis in vitro has only relatively recently been shown.3 Failure of the process of immune cell recruitment can lead to severe pathological conditions and even death.1,4,5 Monocytes, a subset of leukocytes and a critical part of our immune system, are circulatory cells found in blood. They function as precursors to macrophages6 and will differentiate if they are recruited to a site of infection. Understanding the molecules and mechanisms involved in the formation of strong cellular adhesion has been an area of intense research interest.7−11 Multiple cellular adhesion complexes have been identified in vivo and in vitro regulating cell−cell and cell− extracellular matrix (ECM) contacts that are characterized by specific cell-receptor mediated interactions with the local environment (e.g., integrins or cadherins) and a selection of adapter proteins from a pool shared by multiple adhesion types linking the intracellular side of the adhesion complexes to the cellular architecture (e.g., the actin matrix or fiber or intermediate filaments). Focal complexes, focal adhesions, fibrillar adhesions, and hemidesmosomes are implicated in cell−ECM contacts, while adherence junctions and desmosomes link cells together. Podosomes represent a form of adhesion complex that are linked to both cell−cell and cell− ECM contacts depending on the cellular context. Podosomes identified in transformed cells, often termed invadasomes, are linked to directed protease digestion of the ECM and cell migration and investigated in the context of cancer metastasis.

ecruitment of leukocytes at the endothelium of inflamed tissue is a critical and central mechanism in a broad set of inflammatory processes.1,2 The arrest and extravasation of neutrophils provide the majority of the first response. Then monocytes and inflammatory lymphocytes, in response to signals presented at the endothelium, leads to infiltration of inflammatory cells to the local tissue environment in a spatially specific manner. This is achieved by the presentation of different specific biomolecules at, sites of infection by endothelial cells lining the blood vessels, altered from normal endothelium. These molecules, for example, CAMs, and chemokines bound to the membrane, all play specific roles in the recruitment of leukocytes, which can be illustrated by the classical recruitment pathway of rolling, firm adhesion, diapedesis, and migration.2 The initial interaction between P- and E-selectins on the endothelial surface with sialyl-lewis X saccharides on leukocytes is characterized by weak bonds, constantly breaking and reforming, but causing the cells to roll on the surface. Mediated by this rolling, a close contact is formed between integrins on the leukocytes and signaling molecules at the endothelial surface, allowing chemokine recognition and induced inside-out activation of integrins. Subsequent firm integrin-mediated adhesion formation and associated motility allow the identification of appropriate diapedesis or transmigration sites within the endothelium either paracellularly at endothelial junctions or transcellularly through the endothelial cells followed by onward migration toward the inflammatory site. Whereas both routes are observed in vivo, the paracellular has traditionally been regarded as the most significant mechanism © XXXX American Chemical Society

Received: February 5, 2016

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Nano Letters In neutrophils and monocytes, podosomes mediate adhesion to the endothelial cell surface by LFA1 (lymphocyte function antigen 1) on the leukocyte cell membrane interacting with ICAM1 presented at the endothelium.1,12 Here, podosomes are implicated in the process of transcellular diapedesis with evidence suggesting that while Mac-1 (Macrophage antigen 1) may promote the paracellular route in neutrophils, LFA1 promotes transcellular diapedesis. Neutrophils lacking Mac-1 showed significantly higher rates of trans- versus paracellular diapedesis.13 Technological advances in nanoscale methodologies through nanofabrication14−17 and super resolution optics8,18,19 have been successfully applied to exploring adhesion complex formation and its role in signaling.20 Nanoscale organization of ligands via, for example, integrinligand clustering, is thought to be important in cells before the formation of strong bonds to the surface.21,22 Integrins are known to cluster together to form adhesion complexes, varying between cells.21,23−26 Generally, the valency is used to describe the effect of increased binding strength from clustering of the integrin−ligand interaction, and the avidity is the total strength of the adhesion complex. Avidity is not to be confused with the individual integrins affinity for its ligand, as the avidity of an adhesion is the combined effect of valency and affinity.27,28 Nanoscale fabrication was used to study the importance of ligand organization in the formation of strong adhesions between leukocytes and endothelium. We studied the integrindependent podosomal adhesion to mimics of activated endothelium with THP1 monocytes. We show that the formation of adhesions critically depends on the form of the ICAM1 nanopatterns with single contiguous domains as circles or rings promoting adhesion and multiple separated smaller domains restricting podosome formation. The maturation of the podosomes to form rosette structures or recruit MMP14 (matrix metalloproteinase 14) was impaired by the nanopattern compared to homogeneously presented ICAM1. Nanofabrication in the form of sparse colloidal lithography29 combined with chemical and biochemical functionalization was applied to pattern the shape and size of domains of ICAM1 for culture substrates. Nanopatterns consisting of silica chemistry background with short-range ordered arrays of gold circular domains 100−800 nm in diameter or ring shapes (outer diameter 800 and 500 nm) (Figures S1 and S2) was sequentially site specifically functionalized with hydrophobic thiols to the gold regions and PLL-g-PEG to the silica parts of the surface to allow the assembly of proteins on the gold (Au) domains, as is shown in Figure 1a. The protein structures used in these experiments were sequentially built up with irreversible nonspecific binding of a streptavidin layer followed by biotinylated protein A and a subsequent immobilization of an engineered FC-domain containing the ectodomain of ICAM1 to achieve an orientated ICAM1 pattern (Figure 1b/c). Further, the specific protein localization to the Au domains was validated by atomic force microscopy (AFM) data on ring nanopatterns showing specific recruitment to the Au ring (Figure S3). We selected THP1 cells as a model system because they are representative of monocytes and often used to study the function of these in vitro.30−33 In vivo, monocytes recruited to the endothelium will be activated by chemokines presented by the endothelial surface.34,35 This activation causes LFA1 and MAC1 to change conformation, which induces a high affinity for its ligands. This will result in a strong binding to the endothelial surface presenting the ligands. The common system

Figure 1. Immunofluorescent images of proteins immobilized on the nanostructured surfaces. (a) Protein nanopattern visualized with an immunostain immobilized at protein-A via FC binding. Images represent from left to right show 200−800 nm in diameter patterns. (b) Schematic of an ICAM1 biofunctionalized 100 nm Au hole seen in cross section. (c) Fluorescently tagged ICAM1 800 nm nanopattern, imaged after a cell study to illustrate the stability of the protein nanopattern. Scalebar 5 μm.

of PMA activation36,37 was used to induce the high affinity LFA1 and MAC1 conformation, and the activation was validated via flow cytometry using the conformationally specific antibodies (m24 and CBRM1/5 for LFA1 and MAC1 respectibely)38 (Figure S4). We applied this system as representative of leukocytes for studying their interaction with ICAM1 (Figure S4). It is known that preclustering of integrins causes an increased cell binding compared to that via separated integrins through increased valency. ICAM1 nanopatterned surfaces were applied to change the overall valency by controlling the maximum cluster size or shape. Multiple surface types were tested together to explore different aspects of the cellular adhesion process and these are discussed as three sample sets (A, B, and C), which will be analyzed sequentially. The negative control (PEG) showed that cells were not able to bind to PEG functionalized silica regions ensuring cell-surface interactions are localized to the ICAM1 functionalized Au regions. Sample set A was composed of six different sized nanopatterns (100, 160, 200, 300/15%, 500, and 800 nm), and a homogeneously coated ICAM1 surface (homogen). Activated THP1 cells were incubated for 2 h on the surfaces, fixed in situ with minimal washing, stained, and imaged to give quantitative cell counts. Figure S5 shows representative images of cells binding to the different surfaces and it is immediately clear that the amount of cell binding was significantly altered depending on the surface pattern. The quantified adherent cell density data in Figure 2a shows that the homogeneous sample supported a large number of bound cells, statistically different from all other samples investigated, although no statistical difference was observed between 800 nm and the homogeneous sample in another round of experiments (Figure S6). The homogeneous sample had a much higher available area of ICAM1 (100% global coverage) compared to the other samples (Figure S2), which might explain the comparably high cell numbers observed (Figure S7). For all experiments, the 800 and 500 nm were statistically identical, while the 200 and 300 nm surface showed a moderate and significantly lower binding than the larger patterned and homogeneous samples, respectively. Below 200 nm pattern size (the 160 and 100 nm patterns), cell adhesion B

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Figure 2. THP1 adhesion to ICAM1 nanopatterned surfaces. (a) Quantified cell counts, n = 6. The overall experiment is divided into three sample sets A, B, and C together with a negative control (PEG). Homogen is an Au sample completely covered with ICAM1 while PEG is a pegylated silica sample. Some samples are presented with percentages, which represent the global coverage of Au on the surface. The samples ending with Ring correspond to a Au ring with outer/inner diameter in nanometers. See Figure S1 for SEM images of each sample. Error bars represent SD between samples. For statistical significances see Figure S8. (b) Confocal images of THP1 cells on surfaces. THP1 cells were stained for vinculin (green) and actin (red). The merged images show the merge of vinculin and actin. Scalebar 5 μm. (c) THP1 cells were stained for MMP14 (green) and vinculin (red). The merged images show the merge of MMP14 and vinculin. Scalebar 5 μm.

colocalized with the vinculin and was also more intensely stained in this belt region compared to the rest of the cell. By close examination, it was clear that the belt was composed of several smaller dot structures, which were seen in both the vinculin/actin and paxillin/actin stain (arrows in Figures S10 and S11). Monocyte adhesion to ICAM presenting endothelial cells typically occurs via podosomal adhesions through MAC1 and to a lesser extent via LFA1. The structure observed at the homogeneously presented ICAM1 was similar to podosome rosettes, which have been observed before, in cells of the monocytic linage.39,40 The rosette-like structure is composed of closely spaced individual podosomes. Podosomes are linked to extravasation of leukocytes through the endothelium after the formation of strong adhesion.41,42 In particular, LFA1-ICAM1 interactions are linked to transcellular diapedesis while MAC1ICAM1 interactions are linked to paracellular diapedesis (observed in neutrophils in vivo). Both para- and transcellular diapedesis have been observed in monocytes in vitro.43 In this context, cells on the homogeneous ICAM1 surfaces are proposed to show the beginning stage of diapedesis, though clearly it cannot progress on this model surface. To further validate this theory, we showed that the rosette structure specifically recruits a membrane bound metalloprotease often found in connection with podosomes (and those termed invadasomes in transformed cells) in the process of degrading the surroundings, Figure 2c.44 THP1 cells adherent at the patterned surfaces showed clear adhesive dot like structures with associated vinculin and actin with a distribution and size expected for isolated podosomes, Figure 3a−e, with full cell images shown in Figure S12. Paxillin

was abrogated giving very low cell attachment at the background control level. These results were confirmed in five independent repeats (Figures S7 and S6). The binding of activated THP1 monocytes to surfaces presenting ICAM1 showed a clear dependence on the preclustering of ICAM1 with a minimum ICAM1 patch size for binding of 200 nm corresponding to ∼14 integrins. This value was calculated from the minimum integrin spacing for adhesion observed between 58 and 73 nm by Arnold et al. for focal adhesion-based cell attachment.15 Large pattern sizes (500 and 800 nm) showed a high-binding level while intermediate pattern sizes (200 and 300 nm) showed reduced cell attachment, which may indicate an altered binding mechanism for cells on these surfaces, compared to cells on the 500−800 nm surfaces. Blocking experiments showed no reduction of adhesion with anti-CD11b (MAC1) but significant reduction with anti-CD11a (LFA1), and some reduction with anti CD18 that should block both MAC1 and LFA1. No cell attachment was observed in the absence of ICAM1 (Figure S9). Becasue blocking of LFA1 did not completely remove cell binding it cannot be ruled out that MAC1 is involved in the adhesion, possibly as a backup binding mechanism taking over if LFA1 fails to attach. To investigate the mechanism of adhesion confocal microscopy was utilized and the cells were stained for the general adhesion protein vinculin and actin, which forms part of the cytoskeleton in cells. The homogeneous ICAM1 surface showed a very distinct structure on many of the cells (Figure 2b and Figure S10). It was composed of a bright, large vinculin belt up to and sometimes above 10 μm in diameter. The actin C

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Figure 3. Close-up confocal images of podosomes. (a−l) THP1 cells were stained for vinculin (green) and actin (red). The merged images show the merge of vinculin and actin. Scalebar 1 μm. (m) Diameter of the podosome core and associated vinculin patches measured on the different nanostructured surfaces in μm. Offset represents the center-to-center distance from the podosome core to the center of the vinculin patch. Error bars represent the standard deviation.

with an actin podosome core was ∼600 nm, compatible with the reported size of standard podosomal adhesions.45 At this size the podosome would be able to span over multiple patches for the 200 and 300 nm surface and potentially integrate multiple adhesive patches into a single cellular adhesion. The local coverage of ICAM1 around a forming podosome as well as the pattern of ICAM1 distribution (as large continuous ICAM1 domains as on the 800 nm pattern or as multiple discrete smaller ICAM1 domains) may play a role in determining the cell attachment. To explore the role of ICAM1 coverage, we prepared the sample set B with the same domain size (300 nm) and increasing area of ICAM1 coverage (with global coverage varying from 8% to 31% for samples 300/8% to 300/31%) and showed an increase in cell binding as the patches were spaced closer (Figure 2a). Both for cells adherent at the 300/24% and 300/31% the cell numbers were not statistically different from the 800 nm sample showing that the cell binding can be increased to levels similar to these “continuous” ICAM1 patches. As the 300/31% patterns have a higher global coverage

is also observed colocalized within the podosomes (Figure S11). The size of the core of the podosomal adhesions as seen by the diameter of the dotlike actin regions were ∼450 nm with no dependence on the ICAM1 pattern size/shape (Figure 3m and Figure S13). The vinculin-stained images showed that the actin podosome core is often associated with one or more vinculin patches of variable size (Figure 3m and Figure S13). The vinculin patch center was in many cases offset from the center of the actin podosome core. We have quantified the diameter of the actin cores, the nearest associated vinculin patch, and the offset between their centers. Both the homogeneous sample and 800 nm had larger vinculin patches than the podosome cores with a small offset meaning the podosome core and vinculin patch colocalized; however, for the 500−200 nm patterns the offset plus actin podosome diameter was larger than the vinculin patch, meaning that the center of the podosome cores were not directly above vinculin domains. This indicates that for samples below 800 nm the podosomes were able to span multiple patches of ICAM1. For the homogeneous sample, the average vinculin patch associated D

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Figure 4. Close-up SEM images with a 600 nm ring overlay to illustrate podosome size on the different nanopatterns. The ring has been placed at representative high-density sites between and on structures. The red percentages show the average local coverage calculated to be present within such sites. Each image measures 2 × 2 μm with a 500 nm scalebar. Original images can be seen in Figure S1.

patterns. Eight hundred and 500 nm outer diameter ring patterns were fabricated with various inner ring dimensions, effectively patterning ICAM1 in rings with various wall thicknesses (Figures S1 and S3). The 800 nm ring samples with a wall thickness of 173 and 133 nm both showed a good level of cell binding (Figure 2a), although it was only the 800/ 173Ring that was statistically identical to the 800 and 500 nm circular samples, while the 800/67Ring and 800/31Ring did not show any significant cell binding (Figure 2a). Similarly, the 500/125Ring showed an intermediate cell binding, while the 500/97Ring and 500/56Ring did not show cell binding (Figure 2a). The cell binding correlates well with the wall thickness of available ICAM1 with a threshold apparently at ∼100 nm, below which no cell adhesion was observed. Close examination of the podosome structures reveal that there is a shift in the adhesion location from centered on the patches for 800 nm samples (Figure 3b) to both centered at and between the rings for the 800/173Ring (Figure 3j) and almost only between rings for the 800/133Ring (Figure 3k). This is supported by the measured offset values for the central actin domain and the associated vinculin domains that showed an increasing offset from 800 nm to 800/133Ring (Figure 3m). Similarly the 500/ 125Ring showed podosomes between vinculin patches (Figure 3l) resulting in a high offset. As the offset in podosome location was higher for these ring patterns compared to the 500 nm, it could indicate that while on the 500 nm pattern podosomes can form centered on and between patches. Podosomes predominantly form between rings on the 500/125Ring pattern. The size of the podosomal structures observed for homogeneous was ∼600 nm. We present illustrative images superimposing this size of such podosomal structures onto the nanostructured surfaces to show how the podosome might attach to the different-sized patterns (Figure 4). Such overlays allowed an estimate of the local coverage of Au and thereby ICAM1 exposed to a single podosomal adhesion (Figure S2). Taken together with the observed locations of the podosome seen in the confocal images potential locations can be marked. Going from 100 to 800 nm patches it is clear that the

compared to the 800 nm (24%) but less cell binding, the global coverage does not seem to be the main determinant of cell binding. A sample with a very low global coverage but large patch size 950/10% was also shown to have a significant amount of cell binding (Figure S6), again illustrating that the global coverage does not seem to directly control the level of cell binding. Minimal cell attachment was observed to the 300/ 8% samples, which would not allow the podosome to span over multiple patches due to the high nearest neighbor distance (NND ∼ 900 nm), indicating that a single 300 patch did not allow the assembly of a strong podosomal attachment (Figure 2a). At the more densely packed 300 nm/15% samples, intermediate cell attachment was observed with a podosome size of ∼450 nm. Here smaller vinculin patches associated with the actin core with an offset that indicated that the podosome cores were not situated directly above any patches (Figure 3d,g−i,m) but likely spanned multiple 300 nm patches. Interestingly, confocal images of cells attached to the 500 nm sample revealed that the actin podosome core was only sometimes centered on a vinculin patch (Figure 3c). To investigate whether the podosomes could bridge between multiple 500 nm patches a similarly sized pattern 470/11% with a large NND ∼ 1200 nm was shown to have a very low cell binding (Figure 2a). This indicates that podosomes formed at single 470 nm patches were too weak to support cell attachment and that on the 500 nm sample the podosomes were able to bridge between two patches of ICAM1. Two other structures were investigated, a 420 nm diameter patch with 8% global coverage (420/8%) and a crescent structure also with 8% global coverage (Crescent/8%). Both these showed a very low cell binding (Figure 2a). Because these samples had a large NND (∼1200 nm), it was likely that this distance was too large for the podosome to span between patches, while a single patch was too small to support cell attachment. In sample set C, a ring pattern of ICAM1 was used to reduce the local distribution of ligand (the available area of ICAM1) while retaining a contiguous ICAM1 domain as a comparison to multiple discrete patches of ICAM1 at the smaller circular E

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coverage. To further investigate the involvement of LFA1 and MAC1 in the ICAM1-dependent adhesion THP1 cells binding to 800 nm patterns were stained with several different antibodies against cd11a or cd11b, but the results were inconclusive, not allowing a definitive statement on whether LFA1, MAC1, or both are involved in the adhesion (Figure S16), leaving open the possibility of another ICAM1 receptor being involved. Formation of adhesions are thought to be promoted by receptor preclustering and, particularly for leukocytes, it has been shown that activated LFA1 nanoclusters, 100−150 nm in size, are found even before the contact with a surface. A proposed mechanism for adhesion is the initial attachment of these nanoclusters followed by recruitment and growth of larger anchoring structures.21,22 Interestingly 800/133Ring and 500/125Ring patterns both showed good cell adhesion despite the wall thickness being smaller than the 160 nm distance characteristic of no cell adhesion for circular patterns, suggesting that the possibility to grow the adhesion in one dimension was enough to form a stable attachment point. Together with the suggested size of the LFA1 nanoclusters, it offers an explanation for why there seemed to be a threshold for the ring wall thickness at 100 nm. For wall thickness of 125, 133, and 173, the nanocluster presumably would be able to attach and the adhesion could grow laterally into a stabile adhesion, while perhaps rings with a wall thickness below 100 nm did not allow the initial attachment of the LFA1 nanocluster thereby preventing any adhesion formation, even though the local coverage was high. Adhesion of cells of the monocytic lineage while commonly utilizing podosomal adhesions can form other adhesion types such as focal adhesions at extracellular matrix proteins. The fact that cell adhesion was observed on several ring structures is opposite to a recent result found for cells attaching by focal adhesions.46 In that study, cell attachment to similar-sized adhesion patches of ECM proteins showed attachment to 800 nm diameter domains via focal adhesions but no attachment to ring domains with 800 nm outer diameter and ∼200 nm wide ring walls. This indicates that the assembly process of podosomes is different to that of focal adhesions. Recent work utilizing superresolution microscopy to track vinculin dynamics around podosomes (for cells attaching to extracellular matrix proteins) showed the vinculin in a complex geometry forming denser dots or vertices around the actin with thin vinculin bars in between.19 Our data supports the ability to form podosomal adhesions based on discrete nanoscale dots of extracellular attachment as long as each of these dots is 200 nm or larger, allowing connection to 14 or more integrins. We also observe that above this threshold the adhesion can be strengthened by increasing the density of smaller patches, shown for the 300/15% to 300/31% samples, or by increasing the single patch size shown for the 500 and 800 nm samples. Further, the 300 nm/15%, 800/133Ring, and 500/125Ring seem to form podosomes between patches/rings, suggesting that two points of attachment to smaller contacts is sufficient for podosome formation. We do not routinely observe the concentric ring of vinculin around actin cores often reported for high-resolution imaging of podosomes, for example, Figure 3a/ b, although the ring samples do show rings due to their structure. This geometry is proposed to allow the actin core to be articulated through the vinculin ring into the surrounding matrix and thus forming a 3D adhesion complex showing a concentric ring in a 2D confocal image at specific cuts through

distribution of ICAM1 changes from discrete patches to a single patch but also that the local coverage changes drastically from 23% on 100 nm to 100% on the 800 nm (Figure 4a−e). Interestingly, the threshold for the onset of THP1 cell adhesion observed between 160 and 200 nm samples does not obviously correlate to a change in the local coverage as that only change is from 29% to 32%. Therefore, the lack of cell adhesion to the smallest pattern sizes may indicate that the bridging of podosomes across several smaller patches required a minimum ICAM1 pattern size rather than that which resulted from limited local or global coverage of ICAM1. At the 300 nm/15% patterns a podosomal area would cover just two patches giving a local coverage of 36% (Figure 4i). The high local coverage at the 500 nm sample (69% if centered on a patch, Figure 4d) would explain the large increase in cell binding, but as it has been shown the 500 nm patterns also had a significant amount of podosomes located between patches where the local coverage would only reach 32%. In addition, cells did not attach to well-spaced patches of a similar size (470/8%). It is suggested that the possibility for the 500 nm to form adhesions both centered on and between patches gave a better chance of podosome formation and cellular attachment. Increasing the density of the 300 nm patches also increased the local coverage from 36% on 300 nm/15% to 62% local coverage on 300/31% (Figure 4i−l), which fits with the observed cell binding data and, interestingly, shows that the number of patches available increases from 2 to 4. For the 800/173Ring, the local coverages of podosomes, centered at and between rings, is similar being 39% and 31%, respectively (Figure 4f), which supports the concept that on the 800/173Ring pattern the podosomes can form in both locations. For the 800/133Ring, with local coverages of 14% and 27% for adhesions centered at and between rings, respectively (Figure 4g), the podosomes formed predominantly between patches. The podosome formation at 500/125Ring pattern, which has local coverage values of 58% and 25% for podosomes centered at and between rings, respectively (Figure 4h), showed podosomes predominantly between patches suggesting that the local coverage does not explain the whole story and that the specific distribution of ligands is also important. Podosomes typically form with a central actin core surrounded by a ring of ligand-binding integrins. This configuration may mean it is more important to have ligands distributed at the outer edge of a podosome than to have ligands distributed in the core of the podosome and here we suggest that the possibility to get two adhesion points in the outer rim of the podosome between two 500/125Ring patches outweighed the possibility to have more ICAM1 in a single patch where most of the ICAM1 would be distributed very close to a centrally placed actin core. To better illustrate this point local coverages were calculated for an overlay of a ring 600 nm in outer diameter and 400 nm inner diameter representing roughly a classical distribution of integrins in a podosome. The local coverage of the 600 ring overlay is 25% and 24% for such a podosome centered at and between rings respectively (Figure S14), showing that the local coverages become more comparable. Overall, the ring structures did not allow cell binding if the wall thickness is reduced to ∼100 nm as seen for the 500/97Ring, indicating a threshold for cellular adhesion. The local coverage of ICAM1 under a podosome is reduced as well but for the 500/97Ring pattern centrally placed podosomes is still at the level where adhesion would be expected (46%, Figure S15), further indicating that for the ring patterns the wall thickness was more influential than the local F

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endothelium as a precursor to probing the endothelial cell surface and later diapedesis. We show a clear role for nanoscale ligand organization in ICAM1 dependent adhesion. The formation of strong adhesions at discrete nanopatterned surfaces above a threshold size indicates a minimum of 14 integrins for engagement. The role of the patterns of ligands at the macromolecular length scale in immune adhesion processes may also have importance in a range of pattern recognition events (both cellular and macromolecular). In general, this should help improve the detailed knowledge and understanding of our immune system, which is vital for the battle against most pathological conditions. Further, the extravasation of cancer cells, which is the defining trait of metastasis, is thought to use processes very similar to that of leukocytes and these approaches can be applied in the context of cancer cell extravasation.48

the adhesion. For leukocyte adhesion to endothelial cells, the podosomes are proposed to probe the underlying cellular mechanical properties through this physical action pushing actin into the endothelial cell membrane. The noncompliance of the surface likely prevents the cells from pushing the actin into the substrate forming the classical 3D geometry. The podosomal structures formed at homogeneously presented ICAM1 were often arranged in rosette structures and showed evidence of recruitment of MMP14 within the maturing adhesion structures (Figure 2c).45 At patterned surfaces, no mature rosette structures or match between MMP14 and podosomes were observed, suggesting that the nanopattern limits the maturation process. However, early stages of such organization were observed in some cells (e.g., the cells at the 300 and 500 nm patterns in Figure 2b). Rosette formation is considered to be existing podosomes grouping together and this would very likely be impaired on the structured surfaces as the ICAM1 patches are stationary compared to ICAM1 in an endothelial cell membrane.47 This work highlights the importance of the nanoscale distribution of ICAM1 in the context of leukocyte adhesion. The arrangement of ligands in patterns underneath individual adhesion complexes significantly changes the outcomes of the cells’ adhesion. A good cell binding was found when the diameter of the individual ICAM1 patches is 800 nm, matching the normal size found for podosomes. For smaller patches, THP1 cells were able to form podosomes by integrating several closely spaced patches so long as the patches were larger than 160 nm, suggesting a limit of ∼14 integrins to form an initial contact allowing this integration. A higher local availability of ICAM1 around a podosome apparently increased the potential for strong attachment. To investigate the importance of continuous versus discrete patterning of ICAM1 under a single podosome, two sets of nanopatterns were tested. First, a series of 300 nm patches with an increasing density were shown to give an increasing amount of cell binding to a level comparable to continuous adhesions. This indicates that it is not the continuity of ICAM1 that decides cell adhesion, but perhaps more the number of contact points over a specific size or the local coverage under each podosome. In the second set, ring samples with a large outer diameter and variable wall thickness were shown to have a good cell adhesion with wall thicknesses above ∼100 nm. The minimum wall thickness fits with the observation that monocytes form LFA1 nanoclusters 100−150 nm in size that would perhaps be hindered to attach to ICAM1 regions with a dimension smaller than 100 nm, which is different compared to circular patterns where 200 nm minimum dimensions are required for attachment. We suggest that after initial contact of nanoclusters strengthening of the adhesion up to an estimated 14 integrins is required to allow strong adhesion, which requires large circular patterns, but can occur via lateral growth in ring patterns. Taken together, the results indicate that while it is necessary to have a high (>∼25%) local coverage it is perhaps more important to have available ICAM1 domains with an appropriate separation that fits with the podosome size, which would allow the formation of at least two adhesion points on either side of the podosomal core. Increasing the number of contact points, over a threshold size of 200 nm, around the central core further strengthens the podosome structure and thereby cell adhesion. The role of ligand patterns in cellular attachment have been studied via ICAM1-dependent interactions that are of relevance for the formation of strong adhesions of immune cells at the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.6b00519. Materials and methods, SEM of nanostructures, quantified parameters of nanostructures, LFA1 activation, cell data repeats, cells on homogeneous surfaces, cells stained for paxillin and actin, AFM of ring nanostructure, blocking experiment, podosomes in cells, integrin numbers in patches, and different thresholding settings for MMP14 recruitment. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions

The experiments were designed by A.S.A. and D.S.S. and carried out by A.S.A. AFM data were taken and analyzed by H.A. and M.D. Analyzes was done by A.S.A. and interpretation was done by A.S.A., J.X., and D.S.S. The manuscript was written by A.S.A and D.S.S. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by grants from the Sino-Danish Center, the AUFF travel fund, and the Danish Research Council (Sags no. 12-126120)

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DOI: 10.1021/acs.nanolett.6b00519 Nano Lett. XXXX, XXX, XXX−XXX