Monocytes use protrusive forces to generate migration paths in viscoelastic collagen-based extracellular matrices

Introduction:: Circulating monocytes are recruited to tumors during which they must migrate through stromal matrix to potentiate a pro- or anti-tumor response. It is thought that these cells play an important role in the evolution of tumors as they can constitute up to 50% of the tumor mass. Despite clinically observed changes in matrix properties such as viscoelasticity, the potential impact of changes in matrix viscoelasticity on monocyte migration is not understood. One consequence of viscoelasticity is stress relaxation or decrease in stress in the matrix when it is deformed. Materials that relax stress quickly are referred to as fast-relaxing and those that relax stress more slowly are referred to as slow-relaxing. Cells have been described to migrate in three dimensions utilizing mesenchymal or amoeboid mode of migration. Mesenchymal modes of migration typically involve spread cell morphologies and relies on strong cell-matrix adhesion while amoeboid migration uses rounded morphologies with weak adhesions.


Materials and Methods:: Interpenetrating networks (IPNs) of alginate and collagen type 1 matrix were used as viscoelastic substrates for 3D monocyte cell migration studies. Stress relaxation of the IPNs was independently modulated by changing the molecular weight of the alginate.

Migration of U937 human monocyte cells line and primary human monocytes was monitored using confocal microscopy and then quantified using image analysis. The roles of various molecular players in cell migration were assessed using small molecule inhibitors, CRISPR KO experiments, live-actin imaging, and traction force microscopy experiments. Furthermore, cells embedded in the IPNs were immunophenotyped to determine the role of stress relaxation on potentiating transcriptional changes.


Results, Conclusions, and Discussions:: Previous studies developed tunable type-1 stromal-matrix mimics using a macromolecular crowding agent (polyethylene glycol) to modulate the network architecture and mechanics of type-1 collagen gels or click chemistry to covalently crosslink the alginate network in collagen-alginate interpenetrating networks (IPNs), and thereby reduce the stress relaxation rate. However, the approach described here is distinct as neither a crowding agent nor covalent bonds are involved, with IPN stiffness increased by adding more calcium ionic crosslinker and stress relaxation enhanced by lowering the molecular weight of the alginate. This allowed independent modulation of stress-relaxation rate from ~100 s for fast-relaxing IPNs to ~ 2,000 s for slow-relaxing IPNs. Furthermore, the Young’s modulus was independently modulated from ~1 kPa to ~2.5 kPa.

We find that faster stress relaxation, independently enhanced the 3D migration of monocytes (Figure 1). Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Surprisingly, monocytes migrate independent of matrix adhesion and Rho-mediated contractility but are dependent on actin polymerization and myosin contractility for migration. The adhesion-independent mode of monocyte migration observed here exhibits some key distinctions from previously reported modes of adhesion-independent migration found in immune cells in that the traction forces and protrusive and cells migrate with ROCK inhibition. Our mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that generate a path to migrate in the confining viscoelastic matrices. Taken together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal an adhesion-independent mode of migration in monocytes. More broadly, this study highlights the importance of utilizing physiologically relevant materials to study cell migration. Importantly, our data raises the possibility that changes in ECM stiffness and viscoelasticity could determine immune cell recruitment and ultimately shape the immune environment under pathological conditions.


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