Glycocalyx-mediated Cell Adhesion and Migration

Cell migration is a force-dependent adaptive process mediated by integrin-dependent adhesion as well as other yet poorly defined interactions to the extracellular matrix. Using enzymatic multi-targeted digestion of sugar moieties on the surface of mesenchymal cells and leukocytes after interference with integrin function, we demonstrate that the surface glycocalyx represents an independent adhesion system. The glycocalyx mediates cell attachment to ECM ligand in the 100-500 pN force range and amoeboid migration in 3D environments in vitro and in vivo. Glycan-based adhesions consist of actin-rich membrane deformations and appositions associated with bleb-like and other protrusions forming complex-shaped sub-micron contact sites to ECM fibrils. These data implicate the glycocalyx in mediating generic stickiness to support nanoscale interactions (nanogrips) between the cell surface and ECM, mechano-coupling, and migration.


Introduction
Cell shape, polarity and anchorage, as well as migration across surfaces depend upon the function of integrin adhesion receptors, which form transient focalized actincontaining adhesion complexes that control cytoskeletal organization, mechanotransduction and intracellular signaling 1,2 . In cancer metastasis, integrins mediate cell invasion and represent candidate targets for pharmacological interference [3][4][5] . In contrast to 2D migration models, interference with β1 integrin function in 3D environments, where cells migrate through substrate of complex geometry and along confining interfaces, provides only incomplete or no inhibition of migration [6][7][8][9] . The mechanisms mediating cell-substrate coupling and migration when integrin availability is low or absent, mediated by "friction" 9,10 , physical intercalation 11 , or alternative adhesion systems 12 remains elusive. Our aim was therefore to identify the cellular and molecular mechanisms of integrin-independent cell-matrix interaction, force generation, and migration within collagen-rich interstitial tissue in vitro and in vivo when integrin-mediated adhesion is marginalized or absent.

Mesenchymal-to-amoeboid transition after interference with integrins
Fibrillar collagen is the predominant extracellular matrix (ECM) structure in mammalian tissues and recognized with high affinity by integrins a1β1, a2β1, and a11β1, and weakly by aVβ3 13 . Invasive MV3 melanoma cells, which express β1 and β3, but lack all other integrin β-chains (Extended Data Fig. 1a), utilize a2β1 integrins for efficient migration within in vitro reconstituted 3D collagen matrix 14 and collagen remodeling 15 . During migration through 3D fibrillar collagen, up to 90% of MV3 cells adopted an elongated, spindle-shaped morphology and migrated at speeds similar to the movement of mesenchymal cells in situ 16 (Fig. 1a). Graded interference with β1 integrins was induced either by adhesion-perturbing mAb 4B4 at concentrations saturating the epitope up to 90% (Extended Data Fig. 1a and 1c) or by stable shRNAbased β1/β3 integrin downregulation (Extended Data Fig. 1d) combined with additional anti-β1 antibody based interference which achieved >99% epitope reduction for β1 without detectable β3 integrin at the cell surface (Extended Data Fig. 1e, f). Individualand dual-integrin interference strategies reduced migration speed by >80% but failed to achieve immobilization with residual slow migration speed of 0.02-0.15 µm/min (Fig.   1a). As consequence of interference with integrins, the spindle-shaped, elongated morphology converted to an ellipsoid cell shape with multiple dynamic blebs and occasional filopodia in contact with collagen fibrils and focalizations of β1 integrin and filamentous actin at contact sites to collagen fibers converted to diffuse distribution ( Fig. 1b, arrowheads). These data show for metastatic melanoma cells a transition from mesenchymal to amoeboid migration when integrin availability is limited.
To test whether β1 integrins are dispensable for cell migration in non-cancer cells, β1-deficient murine embryonic fibroblasts (MEFs) were tested (Extended Data Fig. 2a Together, these findings support the concept that in mammalian mesenchymal cells the availability of integrins determines the motility programs 17 . Mesenchymal-like migration of fibroblasts and some cancer cell types results from elongated spindleshaped morphology, focalized adhesion sites, and high traction force towards the ECM whereas low integrin availability generates weak mechanotransduction by means of poorly focalized adhesion sites and actin cytoskeleton, similar to leukocytes and otherwise adhesive cells moving in non-adhesive 3D environments 9,10 . As confirmatory model, integrin-expressing (Extended Data Fig. 3a We explored alternative collagen receptors 12 that might compensate the loss of integrin-mediated adhesion and migration in MV3 cells, including syndecan-1, discoidin domain receptors (DDR) 1 and 2, and proteoglycan CD44. DDR-1 and -2 or syndecan-1 were not expressed by MV3 cells after culture in 3D collagen matrix and interference with integrins (Extended Data Fig. 3e, f). CD44 was expressed, however CD44 perturbing antibody Hermes-1 did not affect the migration of MV3 cells after β1/ β3 integrin knockdown in 3D fibrillar collagen (Extended Data Fig. 3g, h). These data argue against an important role of these receptors in mediating amoeboid movement in MV3 cells.

Multi-enzyme removal of surface glycans
Besides cell surface receptors, the surface glycocalyx can interact with proteins and other materials, through carbohydrate-binding domains 18 or unclassified ionic and non-ionic bonds providing generic stickiness 19 . We therefore hypothesized that nonspecific low-affinity interaction of mammalian cells with collagen fibers could be mediated by the glycocalyx [20][21][22] and/or even non-adhesive 3D cell intercalation 11,23 .
In mammalian cells, the glycocalyx forms a thick, polar layer, which is composed of various classes of glycoconjugates, that are directly or indirectly coupled to the cell membrane 24  To address the role of surface glycans in cell migration, protein-and lipid-linked glycoconjugates were enzymatically removed from the surface of live cells by a twostep glycosidase treatment. The enzymatic digestion combined hyaluronidase, heparitinase, chondroitinase and neuraminidase with galactosidase, the latter hydrolyzing β-1,4 coupled galactose residues in N-and O-linked glycans and glycolipids, lowered cell surface heparan-and chondroitin sulfate by ~98% and dermatan sulfate by ~95% (Extended Data Fig. 4 c). When combined with sialic acid removal by neuraminidase, this treatment resulted in reduction of sialic acid residues by ~80% in MV3 control and β1/β3KD cells, by ~95% in Molt-4 cells (Extended Data Likewise, during the recovery, the cells showed consistent viability (Extended Data Fig.   4h).
To verify that the digestion procedure was non-toxic and further did not compromise the basic migration ability through integrins, MV3 control cells expressing β1/β3 integrin after enzymatic digestion (P/N/G) together with non-inhibitory cRAD showed normal baseline migration and mesenchymal phenotype (Fig. 3a-c). Likewise, β1-/-but β3 integrin-expressing MEFs developed similar morphological phenotypes and unperturbed migration speeds after enzymatic digestion compared with β1+/+(fl/fl) MEFs ( Fig. 3d versus 1c). These data indicate unperturbed migration and, hence, cell viability and cytoskeletal activity after glycosidase treatment.

Surface-glycan dependent cell migration
While mesenchymal migration of integrin-competent control cells was unaffected, combining glycan removal and interference with integrin expression severely compromised cell migration and persistence in 3D collagen lattices across cell models.
Glycan removal immobilized all cells, despite ongoing cytoskeletal activity ("running on the spot"), irrespective of whether integrin-independent baseline migration was slow in We next sought to address whether the glycan-dependence of cell migration is relevant in collagen-rich interstitial tissue in vivo. MV3 β1/β3KD cells were additionally pretreated with integrin-blocking mAbs 4B4 and 17E6, injected into the deep dermis of nude mice and monitored by intravital microscopy for up to 6h (Fig. 3f). Whereas MV3 vector control cells developed spindle-shaped morphology (Fig. 3g, Extended Data  29 , fibrillar collagen 8 as well as interstitial tissue in vivo. However, when surface glycans were removed before injection into the mouse dermis, MV3 β1/β3KD cells underwent near-complete migration arrest (Fig. 3g, h), but maintained oscillatory shape change as an indication for unperturbed viability (Extended Data Fig. 5g, E, F, Supplementary Video 6). Thus, an intact surface glycocalyx is required to maintain amoeboid migration in collagen-rich tissue in vitro and in vivo when integrin functions are perturbed.

Glycan-mediated attachment forces in the pN range
To address directly whether the glycocalyx functions as adhesion system, atomic force spectroscopy (AFS) was used to probe the binding of synthesized glycan polymers and live cells to fibrillar collagen. The tip of the cantilever was functionalized with simple-structured amylose and cellulose that lack modifications such as sulfation or acetylation and the forces to disrupt the bonds upon cantilever retraction were recorded (Extended Data Fig. 6a). With increasing amylose density on the cantilever, the binding to collagen was dose-dependently strengthened (Fig. 4a, Extended Data To test, whether the glycocalyx engages with substrate other than collagen BSA was used as non-specific ligand. Whereas integrins did not favor cell binding to BSA, glycans supported interaction of MV3 control, MV3 β1/β3KD and Molt-4 cells to BSA with forces similar to collagen binding (Fig. 4g, Extended Data Fig. 6h, 7b). This implicates the glycocalyx as an adhesion system to ECM and other substrates.

Glycan-mediated interactions to collagen fibrils
We finally aimed to identify the cell contact structures mediated by surface glycans towards collagen fibers and first classified the protrusion types in control cells and cells after limiting integrin and glycan availability. Cells with polarized elongated or rounded morphology developed either (i) pointed actin-rich pseudopod-and filopodlike extensions or (ii) bleb-shaped, rounded protrusions in a cell-type dependent manner (Fig. 5a). After targeting integrins and converting from elongated to rounded morphology MV3 cells preferentially developed bleb-like protrusions ( Fig. 5a To detect the 3D topologies of glycan-mediated contacts at high resolution, beyond the diffraction limit of light microscopy, we used scanning electron microscopy to visualize the surface of cells located inside the collagen matrix, by mechanically separating the matrix at mid-level (Fig. 7a). Blebs in direct contact with a collagen fiber formed complex-shaped indentations (Fig. 7b, left panel; arrowheads in regions 1-5), whereas contact-free blebs remained spherical and without indentation (Fig. 7b, left panel; arrows in region 6). The "grip-like" tight interphase between cell surface and collagen fibrils corresponded to a contact area ranging from 0.18 up to 0.29 µm 2 , calculated from the bleb diameter, fiber caliber and indentation depth, and the length of the interactions (Fig. 7c, d). Notably, these interactions withstood mechanical processing of the collagen lattice during sample preparation in most cells ( Fig. 7e; yellow colored area). However occasional disruptions of the interaction uncovered a half-cylindrical grove matching the orientation and calibre of the displaced fibril (Fig. 7e, left panel; arrowheads), thus confirming the curved shape and otherwise tight submicron scaled apposition of the cell surface to the fibril ("nanogrips"). After enzymatic glycan removal, the nanogrips were perturbed in shape and lacked tight folding but instead showed a flattened, weakly concave area adjacent to the fiber (Fig. 7b, right panel; arrowheads in regions 1-6 and 7e, right panel; yellow colored area). This data indicates a previously unappreciated scaffold function of the glycocalyx, mediating grip-like membrane topologies towards irregular-shaped extracellular structures (Fig. 7f).

Discussion
These results identify the glycocalyx in mediating low-to-moderately adhesive mechano-coupling in the pN force range and cell migration in 3D ECM, and neither of these functions depends on integrins. Glycan-mediated substrate interactions consist of linear membrane appositions to collagen fibrils of finger-like protrusions or curved membrane indentations across or adjacent to blebs (Fig. 7g). The graded transition between migration modes after interference with integrins and surface glycans indicates that integrin-mediated high-and glycan-mediated low-adhesive interactions coexist substitute for each other during migration and support migration plasticity 3 .
To detect the adhesion forces provided by surface glycans and simultaneously minimize overlapping integrin-mediated attachment, we antagonized integrin functions

Competing interests
The authors declare no competing interests.

Additional information
Extended Data Information is available in the online version of the paper.
Correspondence and requests for materials should be addressed to P.F.   P values (all graphs), non-paired t-test, 2 tailed. See also Extended Data Fig. 6 and 7.