Novel 3-D Macrophage Spheroid Model Reveals Reciprocal Regulation of Immunomechanical Stress and Mechano-Immunological Response

Abstract

Purpose In many diseases, an overabundance of macrophages contributes to adverse outcomes. While numerous studies have compared macrophage phenotype after mechanical stimulation or with varying local stiffness, it is unclear if and how macrophages directly contribute to mechanical forces in their microenvironment. Methods Raw 264.7 murine macrophages were embedded in a confining agarose gel, and proliferated to form spheroids over days/weeks. Gels were synthesized at various concentrations to tune stiffness and were shown to support cell viability and spheroid growth. These cell-agarose constructs were treated with media supplements to promote macrophage polarization. Spheroid geometries were used to computationally model the strain generated in the agarose by macrophage spheroid growth. Agarose-embedded macrophages were analyzed for viability, spheroid size, stress generation, and gene expression. Results Macrophages form spheroids and generate growth-induced mechanical forces (i.e., solid stress) within confining agarose gels, which can be maintained for at least 16 days in culture. Increasing agarose concentration increases gel stiffness, restricts spheroid expansion, limits gel deformation, and causes a decrease in Ki67 expression. Lipopolysaccharide (LPS) stimulation increases spheroid growth, though this effect is reversed with the addition of IFNγ. The mechanosensitive ion channels Piezo1 and TRPV4 have reduced expression with increased stiffness, externally applied compression, LPS stimulation, and M1-like polarization. Conclusions Macrophages alone both respond to and generate solid stress. Understanding how macrophage generation of growth-induced solid stress responds to different environmental conditions will help to inform treatment strategies for the plethora of diseases that involve macrophage accumulation and inflammation.