Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Lanctot, P. M., Gage, F. H. & Varki, A. P. The glycans of stem cells. Curr. Opin. Chem. Biol. 11, 373–380 (2007)
Haltiwanger, R. S. & Lowe, J. B. Role of glycosylation in development. Annu. Rev. Biochem. 73, 491–537 (2004)
Varki, A., Kannagi, R. & Toole, B. P. in Essentials Glycobiol. ( Varki, A. et al.) http://www.ncbi.nlm.nih.gov/books/NBK1963/ (Cold Spring Harbor Laboratory Press, 2009)
Paszek, M. J., Boettiger, D., Weaver, V. M. & Hammer, D. A. Integrin clustering is driven by mechanical resistance from the glycocalyx and the substrate. PLOS Comput. Biol. 5, e1000604 (2009)
Walker, J. L., Fournier, A. K. & Assoian, R. K. Regulation of growth factor signaling and cell cycle progression by cell adhesion and adhesion-dependent changes in cellular tension. Cytokine Growth Factor Rev. 16, 395–405 (2005)
Desgrosellier, J. S. & Cheresh, D. A. Integrins in cancer: biological implications and therapeutic opportunities. Nature Rev. Cancer 10, 9–22 (2010)
Shibue, T. & Weinberg, R. A. Integrin β1-focal adhesion kinase signaling directs the proliferation of metastatic cancer cells disseminated in the lungs. Proc. Natl Acad. Sci. USA 106, 10290–10295 (2009)
Godula, K. et al. Control of the molecular orientation of membrane-anchored biomimetic glycopolymers. J. Am. Chem. Soc. 131, 10263–10268 (2009)
Godula, K., Rabuka, D., Nam, K. T. & Bertozzi, C. R. Synthesis and microcontact printing of dual end-functionalized mucin-like glycopolymers for microarray applications. Angew. Chem. Int. Edn Engl. 48, 4973–4976 (2009)
Eng, E. T., Smagghe, B. J., Walz, T. & Springer, T. A. Intact αIIbβ3 integrin is extended after activation as measured by solution X-ray scattering and electron microscopy. J. Biol. Chem. 286, 35218–35226 (2011)
Paszek, M. J. et al. Scanning angle interference microscopy reveals cell dynamics at the nanoscale. Nature Methods 9, 825–827 (2012)
Hattrup, C. L. & Gendler, S. J. Structure and function of the cell surface (tethered) mucins. Annu. Rev. Physiol. 70, 431–457 (2008)
Kufe, D. W. Mucins in cancer: function, prognosis and therapy. Nature Rev. Cancer 9, 874–885 (2009)
Manley, S. et al. High-density mapping of single-molecule trajectories with photoactivated localization microscopy. Nature Methods 5, 155–157 (2008)
Rossier, O. et al. Integrins β1 and β3 exhibit distinct dynamic nanoscale organizations inside focal adhesions. Nature Cell Biol. 14, 1057–1067 (2012)
Chen, W., Lou, J., Evans, E. A. & Zhu, C. Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells. J. Cell Biol. 199, 497–512 (2012)
Friedland, J. C., Lee, M. H. & Boettiger, D. Mechanically activated integrin switch controls alpha5beta1 function. Science 323, 642–644 (2009)
Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010)
Nguyen, D. X., Bos, P. D. & Massagué, J. Metastasis: from dissemination to organ-specific colonization. Nature Rev. Cancer 9, 274–284 (2009)
Toole, B. P. Hyaluronan: from extracellular glue to pericellular cue. Nature Rev. Cancer 4, 528–539 (2004)
Bono, P., Rubin, K., Higgins, J. M. & Hynes, R. O. Layilin, a novel integral membrane protein, is a hyaluronan receptor. Mol. Biol. Cell 12, 891–900 (2001)
Dustin, M. L. Signaling at neuro/immune synapses. J. Clin. Invest. 122, 1149–1155 (2012)
Coombs, D., Dembo, M., Wofsy, C. & Goldstein, B. Equilibrium thermodynamics of cell-cell adhesion mediated by multiple ligand-receptor pairs. Biophys. J. 86, 1408–1423 (2004)
Salaita, K. et al. Restriction of receptor movement alters cellular response: physical force sensing by EphA2. Science 327, 1380–1385 (2010)
Groves, J. T. Bending mechanics and molecular organization in biological membranes. Annu. Rev. Phys. Chem. 58, 697–717 (2007)
Lundmark, R. & Carlsson, S. R. Driving membrane curvature in clathrin-dependent and clathrin-independent endocytosis. Semin. Cell Dev. Biol. 21, 363–370 (2010)
Comoletti, D. et al. Synaptic arrangement of the neuroligin/β-neurexin complex revealed by X-ray and neutron scattering. Structure 15, 693–705 (2007)
Hildebrandt, H., Mühlenhoff, M., Weinhold, B. & Gerardy-Schahn, R. Dissecting polysialic acid and NCAM functions in brain development. J. Neurochem. 103 (Suppl. 1). 56–64 (2007)
Levental, K. R. et al. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139, 891–906 (2009)
Paszek, M. J. et al. Tensional homeostasis and the malignant phenotype. Cancer Cell 8, 241–254 (2005)
Lakins, J. N., Chin, A. R. & Weaver, V. M. Exploring the link between human embryonic stem cell organization and fate using tension-calibrated extracellular matrix functionalized polyacrylamide gels. Methods Mol. Biol. 916, 317–350 (2012)
Bruns, N., Pustelny, K., Bergeron, L. M., Whitehead, T. A. & Clark, D. S. Mechanical nanosensor based on FRET within a thermosome: damage-reporting polymeric materials. Angew. Chem. Int. Ed. 48, 5666–5669 (2009)
Friedrichs, J., Helenius, J. & Muller, D. J. Quantifying cellular adhesion to extracellular matrix components by single-cell force spectroscopy. Nature Protocols 5, 1353–1361 (2010)
Pankov, R. & Momchilova, A. Fluorescent labeling techniques for investigation of fibronectin fibrillogenesis (labeling fibronectin fibrillogenesis). Methods Mol. Biol. 522, 261–274 (2009)
Magbanua, M. J. M. et al. Genomic profiling of isolated circulating tumor cells from metastatic breast cancer patients. Cancer Res. 73, 30–40 (2013)
We thank S. Gendler, J. Goedhart and M. McMahon for cDNAs, as indicated in the Methods section. We thank A. Walker for bioinformatics support, L. Hauranieh for assistance in CTC analysis, H. Aaron for assistance with FLIM, J. B. Sibarita and M. Lagardère for support in sptPALM analysis, B. Hoffman in design of the FRET sensor, and T. Wittmann in assistance with pbFRET measurements. Image acquisition was partly performed at the Nikon Imaging Center and Biological Imaging Development Center at UCSF and the Berkeley Molecular Imaging Center. This work was supported by the Kavli Institute and UCSF Program for Biomedical Breakthrough postdoctoral fellowships to M.J.P.; DoD NDSEG Fellowship to M.G.R.; NIH GM59907 to C.R.B.; NIH Pathway to Independence Award K99 EB013446-02 to K.G.; French Ministry of Research, CNRS, ANR grant Nanomotility, INSERM, Fondation ARC pour la Recherche sur le Cancer, France BioImaging ANR-10-INBS-04-01, and Conseil Régional Aquitaine to O.R. and G.G.; NIH AI082292-03A1 to D.A.H.; The Breast Cancer Research Foundation to M.J.M., H.S.R. and J.W.P.; NIH 2R01GM059907-13 to C.R.B. and V.M.W.; and BCRP DOD Era of Hope Scholar Expansion grant BC122990, and NIH NCI grants U54CA163155-01, U54CA143836-01, 1U01 ES019458-01, and CA138818-01A1 to V.M.W.
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Large-scale gene expression analysis reveals increased expression of genes encoding bulky glycoproteins and glycan-modifying enzymes in primary tumours of patients with disseminated disease.
a, Bioinformatics pipeline to estimate the extracellular bulkiness of a protein from its corresponding amino acid sequence. For each isoform sequence, the transmembrane and extramembrane domains were identified using a hidden Markov model (TMHMM). A combination of motif searches and neural network prediction then identified likely N- and O-glycosylation sites within each sequence. Isoform-level bulkiness estimates were generated by summing the number of predicted N- and O-glycosylation sites located within the extramembrane regions of the isoform. b, Heat map depicting the pairwise spearman correlation coefficients calculated by comparing all per-gene estimates of the total number of extra-membrane amino acids (AAoutside), N-glycosylation sites (Nglyc), O-glycosylation sites (Oglyc), and the overall bulkiness measure (total sites; for example, the sum of extra-membrane N- and O- glycosylation sites). Correlation coefficients relating the corresponding gene-wise measures are listed in the corresponding cells and depicted on a colour scale, where white corresponds to perfect correlation (rho = 1), and the dendrograms indicate the overall relationship between the parameters, estimated by Euclidean distance. High correlation coefficients indicate that gene-wise estimates of the compared parameters are similarly ranked (for example, genes with high values of X also tend to have high values of Y). The data indicate that the number of extracellular N-glycosylation sites and O-glycosylation sites identified within a gene are only weakly correlated, and neither dominates the total number of sites estimated per gene. c, Violin plots contrasting the distributions of gene-wise one-sided P values (y axis) quantifying evidence for transcriptional upregulation of glycosidases and glycosyltransferases, and subsets of glycosyltransferases (sialyltransferases and N-acetylgalactosaminyltransferases) with the full distribution. White dots and thick black lines indicate the median and interquartile range of the gene-wise P-value distribution among category members, and the width of the violin along the y axis indicates the density of the corresponding values. P values are derived from comparisons of expression levels in primary tumours of patients with or without distant metastases using a t-test. Indicated P values were estimated using a one-sided Kolmogorov–Smirnov test. d, Violin plots quantifying transcriptional upregulation of glycan-modifying enzymes in primary tumours of patients presenting with circulating tumour cells compared to tumours without detectable circulating tumour cells. e, Table of bulky glycoproteins and potential bulk-adding glycosyltransferases whose expression is upregulated in tumours that present with circulating tumour cells.
Schematic of an integrated model that describes how the physical properties of the glycocalyx influence integrin–ECM interactions. The cell surface is modelled as a three-dimensional elastic plate; the ECM as a rigid substrate underneath the cell surface; and the glycocalyx as a repulsive potential between the plate and substrate. To compute stress–strain behaviour, the model is discretized using the three-dimensional lattice spring method, the cross-section of which is depicted above. Integrins are tethered to the cell surface and their distance-dependent binding to the ECM–substrate is calculated according to the Bell model. To calculate integrin-binding rate as a function of lateral distance from an adhesion cluster, an adhesion cluster is first constructed by assembling a 3 × 3 bond structure. The rates for additional integrin–ECM bonds then are computed at various distances from the cluster.
a, Scheme for synthesis of lipid-terminated mucin mimetics labelled with Alexa Fluor 488 (AF488). b, Reagents and yields for the synthesis of polymers 3a–c. c, Characteristics of polymers 6a–c based on 1H NMR spectra. Glycoprotien mimetics were engineered to have minimal biochemical interactivity with cell surface lectins. d, Flow cytometry results quantifying incorporation of polymer on the surface of mammary epithelial cells (left) and binding with recombinant Alexa568-labelled galectin-3 with or without competitive inhibitor, β-lactose (right). Although a weak affinity between galectin-3 and the pendant N-acetylgalactosimes has previously been reported, the results suggest that incorporation of polymer does not significantly change the affinity of the cell surface for lectins.
a, Schematic of MUC1 expression constructs. Full-length MUC1 consists of a large ectodomain with 42 mucin-type tandem repeats, a transmembrane domain, and short cytoplasmic tail. The tandem repeats and cytoplasmic tail are deleted in MUC1(ΔTR) and MUC1(ΔCT), respectively. For fluorescent protein fusions, mEmerald (GFP) and mEOS2 are fused to the C terminus of full-length MUC1 or MUC1(ΔCT). b, Schematic of MUC1 strain sensor and control constructs. Cysteine-free mTurqoiuse2 (CFP), Venus (YFP), or a FRET module consisting of the fluorescent proteins separated by an elastic linker (8 repeats of GPGGA) are inserted into the MUC1 ectodomain adjacent to the MUC1 tandem repeats. The mucin tandem repeats are deleted in ectodomain-truncated variants (ΔTR).
a, Quantification of the average number of large adhesions, greater than 1 μm2, per area of cell in control epithelial cells (Control) and those ectopically expressing ectodomain-truncated MUC1 (+MUC1(ΔTR)), wild-type MUC1 (+MUC1), or cytoplasmic-tail-deleted MUC1 (+MUC1(ΔCT)). Results are the mean ± s.e.m. of three separate experiments. b, Fluorescence micrographs showing immuno-labelled MUC1 and fluorescently labelled fibronectin fibrils in control and MUC1-expressing epithelial cells. Soluble, labelled fibronectin in the growth media was deposited by cells at sites of cell–matrix adhesion. Binding of soluble fibronectin to MUC1 was not detected. Scale bar, 10 μm. c, Time lapse images of MUC1–YFP and vinculin–mCherry, showing the dynamics of adhesion assembly (Vinc.) and MUC1 patterning (MUC1). Scale bar, 1 μm. d, Rate of adhesion measured with single cell force spectroscopy of control (Cont.), α5 integrin-blocked (anti-α5), and MUC1-expressing cells (+MUC1) to fibronectin-coated surfaces and control cells to BSA-coated surfaces (BSA). Results are the mean ± s.e.m. of at least 15 cell measurements. Statistical significance is given by *P < 0.05; **P < 0.01; ***P < 0.001.
a, Molecular diffusivity and adhesion enrichment measured with sptPALM in mouse embryonic fibroblasts (MEFs). Adhesion enrichment is reported as the ratio of the number of molecules detected inside focal adhesions per unit area to the number of molecules detected outside focal adhesions per unit area. b, Mean diffusion coefficients measured for freely diffusive β3 integrin tracks outside of adhesive contacts in control (Cont.) and MUC1-transfected (+MUC1) MEFs with and without Mn2+ to activate β3. c, Mean diffusion coefficients measured for confined β3 integrin tracks outside of adhesive contacts in MEFs with and without Mn2+. d, Mean radius of confinement measured for confined β3 integrin tracks outside of adhesive contacts in MEFs with and without Mn2+. e, Fraction of immobilized (Imm.), confined (Conf.), and freely diffusive (Free) β3 integrins inside of adhesive contacts in control and MUC1-transfected MEFs with and without Mn2+ treatment. f, From left to right, panels show GFP-tagged wild-type MUC1 (red) and positions of individual β3 integrins (green) in MEFs without Mn2+ treatment (left panel) and individual integrin trajectories recorded with sptPALM within MUC1-rich regions, outside MUC1-rich regions, and that cross MUC1 boundaries (scale bar, 2 μm). The ratio of integrins crossing out versus crossing in the MUC1 boundaries per cell is close to one (1.0 ± 0.1, n = 9 cells, 4,145 trajectories) showing that the flux of free diffusing integrins crossing in or out the mucin region is the same. g, From left to right, panels show integrin trajectories within an arbitrary region drawn in a MUC1-rich area (dashed white circles), outside of the circled region, and that cross the circled region (scale bar, 2 μm). The ratio of integrins crossing the MUC1-rich boundaries versus the fictive boundaries per cell is close to one (1.2 ± 0.2, n = 9 cells, 9,321 trajectories), showing that the MUC1–adhesive zone boundary does not affect the diffusive crossing of integrins. For all bar graphs, results are the mean ± s.e.m.
a, Western blot of indicated construct expressed in HEK 293T cells and probed with anti-GFP family antibody, or full-length MUC1 construct expressed in HEK 293T cells and probed with an antibody against the MUC1 tandem repeats. b, Pseudo-coloured images showing similar FRET efficiencies measured by the photobleaching FRET method for mammary epithelial cells (MECs) expressing low (Low) and high (High) levels of the sensor construct. Scale bar, 5 μm. c, Plot showing the level of CFP bleaching per CFP imaging cycle in MECs. d, Control images showing minimal intermolecular FRET in MECs expressing similar levels of both MUC1 CFP and MUC1 YFP. e, Micrographs showing the emitted photons from CFP and their fluorescence lifetimes in MECs expressing ectodomain-truncated (MUC1(ΔTR) sensor) or full-length MUC1 strain sensors (MUC1 sensor). Shorter lifetimes are indicative of higher energy transfer between the CFP donor and YFP acceptor, and thus closer spatial proximity of the donor and acceptor (scale bar, 10 μm). f, Representative profile of CFP lifetimes and emitted photons of the full-length MUC1 sensor along the red line in panel e. Pixels 0 and 40 correspond to the base and tip of the arrow, respectively. A drop in fluorescence lifetime (Lifetime) is often observed before the drop in MUC1 molecular density (Photons) as an adhesive zone is approached.
Extended Data Figure 8 Tension-dependent integrin activation and focal adhesion assembly in MUC1-expressing cells.
a, Fluorescence micrographs of fibronectin-crosslinked α5 integrin in control and MUC1-expressing mammary epithelial cells (MECs) treated with solvent alone (DMSO), myosin-II inhibitor (blebbistatin; 50 μM), or Rho kinase inhibitor (Y-27632; 10 μM) for 1 h and detergent-extracted following crosslinking. Only fibronectin-bound integrins under mechanical tension are crosslinked and visualized following detergent extraction (scale bar, 15 μm). b, Fluorescence micrographs showing formation of myosin-independent adhesion complexes in MUC1-expressing MECs. Cells were pre-treated for 1 h and plated for 2 h in 50 μM blebbistatin (scale bar, 10 μm). c, Fluorescence micrographs of paxillin–mCherry and immuno-labelled activated FAK (pY397) in control and MUC1(ΔCT) expressing MECs plated on compliant fibronectin-conjugated hydrogels (E = 140 Pa; scale bar, 3 μm; ROI scale bar, 0.5 μm). d, Western blots showing phosphorylation of paxillin (pY118) in control and MUC1-expressing MECs on compliant substrates (E = 140 Pa) following overnight serum starvation and stimulation with EGF. MUC1-expressing cells treated with a pharmacological inhibitor of focal adhesion kinase (+FAKi) for 1 h before EGF stimulation did not exhibit robust paxillin phosphorylation.
a, Fluorescence micrographs showing DAPI-stained nuclei of control and MUC1(ΔCT)-expressing MECs after 24 h of plating on soft, fibronectin-conjugated hydrogels (E = 140 Pa; scale bar, 250 μm). The majority of cells plated as single cells, indicating that multi-cell colonies that formed at later time points were largely attributed to cell proliferation. b, Quantification of cell proliferation of MUC1(ΔCT)-expressing epithelial cells on soft hydrogels conjugated with bovine serum albumin (BSA) or fibronectin (Fn). Cells plated similarly on BSA– and Fn–hydrogels, but cell proliferation was significantly enhanced on Fn–hydrogels. Results are the mean ± s.e.m with statistical significance given by *P < 0.05; **P < 0.01; ***P < 0.001.
a, Quantification of hyaluronic acid (HA) cell surface levels on control (10A-Cont.), transformed (10A-v-Src, 10A-HRAS) and malignant (MCF7, T47D) mammary epithelial cells (MECs). b, Fluorescence micrographs of HA and immuno-labelled paxillin on the v-Src transformed MECs (scale bars, 3 μm). c, Quantification of the number of v-Src-transformed MECs per colony 48 h after plating on soft polyacrylamide gels (fibronectin-conjugated) and treated with vehicle (DMSO), hyaluronic acid synthesis inhibitor 4-methylumbelliferone (+4MU; 0.3 μM), or competitive inhibitor HA oligonucleotides (+Oligo; 12-mer average oligonucleotide size; 100 mg ml−1). Results are the mean ± s.e.m with statistical significance is given by *P < 0.05; **P < 0.01; ***P < 0.001.
This file contains Supplementary Notes 1-6, Supplementary Table 1, Supplementary Figures 1-6 and Supplementary References. (PDF 696 kb)
Timelapse video of Muc1-YFP (green) and vinculin-mCherry (red), showing the coupled dynamics of adhesion assembly (Vinc.) and Muc1 patterning (Muc1; scale bar - 1 μm). (AVI 850 kb)
Timelapse video of a single integrin molecule (red) recorded with single particle tracking photo-activation localization microscopy (sptPALM) and Muc1-GFP (green) in mouse embryonic fibroblasts (scale bar - 6 μm). The video illustrates integrin mobility in the Muc1-rich region, integrin crossing the Muc1-adhesion zone boundary, and rapid integrin immobilization in the adhesion zone. (MOV 2070 kb)
About this article
Cite this article
Paszek, M., DuFort, C., Rossier, O. et al. The cancer glycocalyx mechanically primes integrin-mediated growth and survival. Nature 511, 319–325 (2014). https://doi.org/10.1038/nature13535
Nature Cell Biology (2021)
Nature Metabolism (2021)
Atomic force microscopy for revealing micro/nanoscale mechanics in tumor metastasis: from single cells to microenvironmental cues
Acta Pharmacologica Sinica (2021)
The cancer glycocalyx mediates intravascular adhesion and extravasation during metastatic dissemination
Communications Biology (2021)
Molecular motion and tridimensional nanoscale localization of kindlin control integrin activation in focal adhesions
Nature Communications (2021)