A novel 3-D immunomechanical model reveals reciprocal regulation between macrophages and solid stress

Introduction: Macrophages play a critical role in health and disease, but their response to and generation of mechanical forces is incompletely understood. From atherosclerotic plaques to tuberculosis granulomas to solid tumors, macrophages play an important role not just in immune effector function and orchestration, but also as active constituents of the mechanical microenvironment. This is especially true in cancer, as macrophages accumulate in tumors in large numbers and contribute to tumor growth and disease progression. Macrophage polarization – whether they contribute to an inflammatory or anti-inflammatory immune response – is plastic and can reverse if they are exposed to shear stress, cyclic compression or stretching, or hydrodynamic pressure changes. However, the degree to which macrophages can contribute to mechanical stress in disease, especially compressive stress generated by a growing mass, has not been studied.

This work aims to characterize the solid stress that macrophages generate through proliferation in a confining agarose gel, simulating the mechanics of the tissue microenvironment independent of confounding biological cues. We explore how matrix stiffness, inflammatory and anti-inflammatory cues, drug treatment, and hypoxia affect the generation of compressive solid stress in this novel and high-throughput macrophage spheroid model. Understanding how macrophages generate stress, and how they respond to external cues under chronic compressive stress, will inform strategies to target or reprogram macrophages in the plethora of diseases that involve macrophage accumulation. This platform also has promise for screening macrophage-targeted drugs and is highly tunable to be applicable to a range of diseases.

Materials and

Methods: Single-cell suspensions of RAW264.7 murine macrophages were mixed with low-gelling-temperature agarose to create a cell-laden agarose gel. These were immersed in growth medium and incubated in standard culture conditions. Gels were created with 0.5%, 1%, and 2% agarose to tune the gel stiffness. For external compression, a weight was placed on top of the gel. For polarization of the macrophages, the media was supplemented with IFNγ and LPS to induce an inflammatory M1 phenotype, and IL4 to produce an M2 anti-inflammatory phenotype. Media was supplemented with LPS alone to assess macrophage response to inflammatory stimuli. Brightfield images of the gels were used to quantify size. Gels were stained with calcein-AM and propidium iodide to assess viability. Gels were also fixed and stained with DAPI and CellMask Actin Tracking Stain to assess 3D morphology.

For computational modelling of the strain fields that resulted from spheroid expansion, we used a 2D model with a defined starting and ending geometry. The starting geometry was a 5 µm circle representing a single cell, and the final geometry was defined based on the size and morphology measurements from gel images. To model a discoid shape, the starting geometry was a line (representing a crack in the agarose gel), and the final geometry was based on the major and minor axes of measured spheroids. We also used ImageJ to generate 3D files representing the spheroid surface based on multiphoton imaging, which will be used for more accurate 3D computational models that are currently under development.

Results, Conclusions, and Discussions: RAW264.7 cells readily form spheroids when embedded in agarose and display excellent long-term viability (Fig. 1A). As agarose concentration decreases, the average spheroid size increases significantly (Fig. 1B). Treating the agarose-embedded macrophages with 200 ng/ml LPS resulted in increased spheroid size compared to untreated, in all three agarose concentrations. However, treatment with 20 ng/ml IFNγ in addition to 200 ng/mL LPS (M1 treatment) abrogated this effect. Increased gel stiffness and externally applied compression both caused decreased expression of the mechanosensory ion channels Piezo1 and TRPV4. There was also a trending increase in both M1 and M2 polarization markers with increasing stiffness and externally applied compression (Fig. 1C).

Because macrophages cannot alter the agarose matrix, any increase in spheroid size must cause displacement of the surrounding elastic gel, generating solid stress (Fig. 1D). Spheroids in 0.5% agarose cause the greatest maximum strain in the surrounding gel, while 2% agarose spheroids caused the lowest. However, the strain from a spheroid in 2% gel propagates further into the surrounding gel, relative to the spheroid size. 3D imaging revealed that many spheroids adopt a flattened discoid shape, rather than a spherical shape, reflecting anisotropy either in the gel properties or in cell behavior. This results in a distinct estimated strain field shape compared to the round spheroids. Ongoing work will determine whether this anisotropy is due to the mechanical environment (crack formation) or biological phenomena (differential cell proliferation). We are currently characterizing the mechanical properties of the gel and the cells in the spheroid to obtain stress values both inside and outside the spheroids to determine how macrophage-induced solid stress compares to the solid stress seen in tumors in vivo.

For the first time, these results demonstrate that macrophages alone are capable of both generating and responding to chronic solid stress. These results highlight the importance of macrophages as active players in the mechanical microenvironment, especially relevant in diseases such as cancer that harbor both immune and mechanical pathologies. The novel 3D immuno-mechanical constructs have excellent potential for short- and long-term experiments and are easily tunable to include different cell types or matrix modifications.

Acknowledgements (Optional): Imaging was performed at the Notre Dame Integrated Imaging Facility, with the assistance of Dr. Sara Cole. The authors also thank Ms. R’nld Rumbach for technical assistance.