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Macromolecular crowding tunes 3D collagen architecture and cell morphogenesis.

ABSTRACT: Collagen I is the primary extracellular matrix component of most solid tumors and influences metastatic progression. Collagen matrix engineering techniques are useful for understanding how this complex biomaterial regulates cancer cell behavior and for improving in vitro cancer models. Here, we establish an approach to tune collagen fibril architecture using PEG as an inert molecular crowding agent during gelation and cell embedding. We find that crowding produces matrices with tighter fibril networks that are less susceptible to proteinase mediated degradation, but does not significantly alter matrix stiffness. The resulting matrices have the effect of preventing cell spreading, confining cells, and reducing cell contractility. Matrix degradability and fibril length are identified as strong predictors of cell confinement. Further, the degree of confinement predicts whether breast cancer cells will ultimately undergo individual or collective behaviors. Highly confined breast cancer cells undergo morphogenesis to form either invasive networks reminiscent of aggressive tumors or gland and lobule structures reminiscent of normal breast epithelia. This morphological transition is accompanied by expression of cell-cell adhesion genes, including PECAM1 and ICAM1. Our study suggests that cell confinement, mediated by matrix architecture, is a design feature that tunes the transcriptional and morphogenic state of breast cancer cells.

SUBMITTER: Ranamukhaarachchi SK

PROVIDER: S-EPMC6375559 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Macromolecular Crowding Tunes Protein Stability by Manipulating Solvent Accessibility.

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Cell-free protein expression under macromolecular crowding conditions.

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Macromolecular crowding directs extracellular matrix organization and mesenchymal stem cell behavior.

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3D collagen architecture regulates cell adhesion through degradability, thereby controlling metabolic and oxidative stress.

Project description:The collagen-rich tumor microenvironment plays a critical role in directing the migration behavior of cancer cells. 3D collagen architectures with small pores have been shown to confine cells and induce aggressive collective migration, irrespective of matrix stiffness and density. However, it remains unclear how cells sense collagen architecture and transduce this information to initiate collective migration. Here, we tune collagen architecture and analyze its effect on four core cell-ECM interactions: cytoskeletal polymerization, adhesion, contractility, and matrix degradation. From this comprehensive analysis, we deduce that matrix architecture initially modulates cancer cell adhesion strength, and that this results from architecture-induced changes to matrix degradability. That is, architectures with smaller pores are less degradable, and degradability is required for cancer cell adhesion to 3D fibrilar collagen. The biochemical consequences of this 3D low-attachment state are similar to those induced by suspension culture, including metabolic and oxidative stress. One distinction from suspension culture is the induction of collagen catabolism that occurs in 3D low-attachment conditions. Cells also upregulate Snail1 and Notch signaling in response to 3D low-attachment, which suggests a mechanism for the emergence of collective behaviors.

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Molecular Crowding Tunes Material States of Ribonucleoprotein Condensates.

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Fibrillar fibronectin plays a key role as nucleator of collagen I polymerization during macromolecular crowding-enhanced matrix assembly.

Project description:Macromolecular crowding is used by tissue engineers to accelerate extracellular matrix assembly in vitro, however, most mechanistic studies focus on the impact of crowding on collagen fiber assembly and largely ignore the highly abundant provisional matrix protein fibronectin. We show that the accelerated collagen I assembly as induced by the neutral crowding molecule Ficoll is regulated by cell access to fibronectin. Ficoll treatment leads to significant increases in the amount of surface adherent fibronectin, which can readily be harvested by cells to speed up fibrillogenesis. FRET studies reveal that Ficoll crowding also upregulates the total amount of fibronectin fibers in a low-tension state through upregulating fibronectin assembly. Since un-stretched fibronectin fibers have more collagen binding sites to nucleate the onset of collagen fibrillogenesis, our data suggest that the Ficoll-induced upregulation of low-tension fibronectin fibers contributes to enhanced collagen assembly in crowded conditions. In contrast, chemical cross-linking of fibronectin to the glass substrate prior to cell seeding prevents early force mediated fibronectin harvesting from the substrate and suppresses upregulation of collagen I assembly in the presence of Ficoll, even though the crowded environment is known to drive enzymatic cleavage of procollagen and collagen fiber formation. To show that our findings can be exploited for tissue engineering applications, we demonstrate that the addition of supplemental fibronectin in the form of an adsorbed coating markedly improves the speed of tissue formation under crowding conditions.

| S-EPMC6810780 | biostudies-literature

An upper limit for macromolecular crowding effects.

Project description:BACKGROUND:Solutions containing high macromolecule concentrations are predicted to affect a number of protein properties compared to those properties in dilute solution. In cells, these macromolecular crowders have a large range of sizes and can occupy 30% or more of the available volume. We chose to study the stability and ps-ns internal dynamics of a globular protein whose radius is ~2 nm when crowded by a synthetic microgel composed of poly(N-isopropylacrylamide-co-acrylic acid) with particle radii of ~300 nm. RESULTS:Our studies revealed no change in protein rotational or ps-ns backbone dynamics and only mild (~0.5 kcal/mol at 37°C, pH 5.4) stabilization at a volume occupancy of 70%, which approaches the occupancy of closely packing spheres. The lack of change in rotational dynamics indicates the absence of strong crowder-protein interactions. CONCLUSIONS:Our observations are explained by the large size discrepancy between the protein and crowders and by the internal structure of the microgels, which provide interstitial spaces and internal pores where the protein can exist in a dilute solution-like environment. In summary, microgels that interact weakly with proteins do not strongly influence protein dynamics or stability because these large microgels constitute an upper size limit on crowding effects.

| S-EPMC3120801 | biostudies-literature

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