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Cancer cell glycocalyx is a major line of defence against immune surveillance. However, how specific physical properties of the glycocalyx are regulated on a molecular level, contribute to immune evasion and may be overcome through immunoengineering must be resolved. Here we report how cancer-associated mucins and their glycosylation contribute to the nanoscale material thickness of the glycocalyx and consequently modulate the functional interactions with cytotoxic immune cells. Natural-killer-cell-mediated cytotoxicity is inversely correlated with the glycocalyx thickness of the target cells. Changes in glycocalyx thickness of approximately 10 nm can alter the susceptibility to immune cell attack. Enhanced stimulation of natural killer and T cells through equipment with chimeric antigen receptors can improve the cytotoxicity against mucin-bearing target cells. Alternatively, cytotoxicity can be enhanced through engineering effector cells to display glycocalyx-editing enzymes, including mucinases and sialidases. Together, our results motivate the development of immunoengineering strategies that overcome the glycocalyx armour of cancer cells.

Genetic, colocalization, and biochemical studies suggest that the ankyrin repeat-containing proteins Inversin (INVS) and ANKS6 function with the NEK8 kinase to control tissue patterning and maintain organ physiology. It is unknown whether these three proteins assemble into a static “Inversin complex” or one that adopts multiple bioactive forms. Through the characterization of hyperactive alleles in C. elegans, we discovered that the Inversin complex is activated by dimerization. Genome engineering of an RFP tag onto the nematode homologues of INVS (MLT-4) and NEK8 (NEKL-2) induced a gain-of-function, cyst-like phenotype that was suppressed by monomerization of the fluorescent tag. Stimulated dimerization of MLT-4 or NEKL-2 using optogenetics was sufficient to recapitulate the phenotype of a constitutively active Inversin complex. Further, dimerization of NEKL-2 bypassed a lethal MLT-4 mutant, demonstrating that the dimeric form is required for function. We propose that dynamic switching between at least two functionally distinct states – an active dimer and an inactive monomer – gates the output of the Inversin complex.

The glycocalyx is a coating of protein and sugar on the surface of all living cells. Dramatic perturbations to the composition and structure of the glycocalyx are frequently observed in aggressive cancers. However, tools to experimentally mimic and model the cancer-specific glycocalyx remain limited. Here, we develop a genetically encoded toolkit to engineer the chemical and physical structure of the cellular glycocalyx. By manipulating the glycocalyx structure, we are able to switch the adhesive state of cells from strongly adherent to fully detached. Surprisingly, we find that a thick and dense glycocalyx with high O-glycan content promotes cell survival even in a suspended state, characteristic of circulating tumor cells during metastatic dissemination. Our data suggest that glycocalyx-mediated survival is largely independent of receptor tyrosine kinase and mitogen activated kinase signaling. While anchorage is still required for proliferation, we find that cells with a thick glycocalyx can dynamically attach to a matrix scaffold, undergo cellular division, and quickly disassociate again into a suspended state. Together, our technology provides a needed toolkit for engineering the glycocalyx in glycobiology and cancer research.

The glycocalyx is a thick coat of proteins and carbohydrates on the outer surface of all eukaryotic cells. Overproduction of large, flexible or rod-like biopolymers, including hyaluronic acid and mucins, in the glycocalyx strongly correlates with the aggression of many cancer types. However, theoretical frameworks to predict the effects of these changes on cancer cell adhesion and other biophysical processes remain limited. Here, we propose a detailed modeling framework for the glycocalyx incorporating important physical effects of biopolymer flexibility, excluded volume, counterion mobility, and coupled membrane deformations. Because mucin and hyaluronic biopolymers are proposed to extend and rigidify depending on the extent of their decoration with side chains, we propose and consider two limiting cases for the structural elements of the glycocalyx: stiff beams and flexible chains. Simulations predict the mechanical response of the glycocalyx to compressive loads, which are imposed on cells residing in the highly confined spaces of the solid tumor or invaded tissues. Notably, the shape of the mechanical response transitions from hyperbolic to sigmoidal for more rod-like glycocalyx elements. These mechanical responses, along with the corresponding equilibrium protein organizations and membrane topographies, are summarized to aid in hypothesis generation and the evaluation of future experimental measurements. Overall, the modeling framework developed provides a theoretical basis for understanding the physical biology of the glycocalyx in human health.

The glycocalyx coating the outside of most cells is a polymer meshwork comprising proteins and complex sugar chains called glycans. From a physical perspective, the glycocalyx has long been considered a simple ‘slime’ that protects cells from mechanical disruption or against pathogen interactions, but the great complexity of the structure argues for the evolution of more advanced functionality: the glycocalyx serves as the complex physical environment within which cell-surface receptors reside and operate. Recent studies have demonstrated that the glycocalyx can exert thermodynamic and kinetic control over cell signalling by serving as the local medium within which receptors diffuse, assemble and function. The composition and structure of the glycocalyx change markedly with changes in cell state, including transformation. Notably, cancer-specific changes fuel the synthesis of monomeric building blocks and machinery for production of long-chain polymers that alter the physical and chemical structure of the glycocalyx. In this Review, we discuss these changes and their physical consequences on receptor function and emergent cell behaviours.

Background: TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties. Methods: HA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively. Results: TNF-α concentrations were greater in OA synovial fluid, and TSG6expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity. Conclusions: Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.