Measuring mechanical cues for modeling the stromal matrix in 3D cell cultures.

A breast-cancer tumor develops within a stroma, a tissue where a complex extracellular matrix surrounds cells, mediating the cancer progression through biomechanical and -chemical cues. Current materials partially mimic the stromal matrix in 3D cell cultures but methods for measuring the mechanical properties of the matrix at cell-relevant-length scales and stromal-stiffness levels are lacking. Here, to address this gap, we developed a characterization approach that employs probe-based microrheometry and Bayesian modeling to quantify length-scale-dependent mechanics and mechanical heterogeneity as in the stromal matrix. We examined the interpenetrating network (IPN) composed of alginate scaffolds (for adjusting mechanics) and type-1 collagen (a stromal-matrix constituent). We analyzed viscoelasticity: absolute-shear moduli (stiffness/elasticity) and phase angles (viscous and elastic characteristics). We determined the relationship between microrheometry and rheometry information. Microrheometry reveals lower stiffness at cell-relevant scales, compared to macroscale rheometry, with dependency on the length scale (10 to 100 µm). These data show increasing IPN stiffness with crosslinking until saturation (≃15 mM of Ca2+). Furthermore, we report that IPN stiffness can be adjusted by modulating collagen concentration and interconnectivity (by polymerization temperature). The IPNs are heterogeneous structurally (in SEM) and mechanically. Interestingly, increased alginate crosslinking changes IPN heterogeneity in stiffness but not in phase angle, until the saturation. In contrast, such changes are undetectable in alginate scaffolds. Our nonlinear viscoelasticity analysis at tumor-cell-exerted strains shows that only the softer IPNs stiffen with strain, like the stromal-collagen constituent. In summary, our approach can quantify the stromal-matrix-related viscoelasticity and is likely applicable to other materials in 3D culture.

Fibrin Stiffness Regulates Phenotypic Plasticity of Metastatic Breast Cancer Cells.

The extracellular matrix (ECM)-regulated phenotypic plasticity is crucial for metastatic progression of triple negative breast cancer (TNBC). While ECM faithful cell-based models are available for in situ and invasive tumors, such as cell aggregate cultures in reconstituted basement membrane and in collagenous gels, there are no ECM faithful models for metastatic circulating tumor cells (CTCs). Such models are essential to represent the stage of metastasis where clinical relevance and therapeutic opportunities are significant. Here, CTC-like DU4475 TNBC cells are cultured in mechanically tunable 3D fibrin hydrogels. This is motivated, as in circulation fibrin aids CTC survival by forming a protective coating reducing shear stress and immune cell-mediated cytotoxicity and promotes several stages of late metastatic processes at the interface between circulation and tissue. This work shows that fibrin hydrogels support DU4475 cell growth, resulting in spheroid formation. Furthermore, increasing fibrin stiffness from 57 to 175 Pa leads to highly motile, actin and tubulin containing cellular protrusions, which are associated with specific cell morphology and gene expression patterns that markedly differ from basement membrane or suspension cultures. Thus, mechanically tunable fibrin gels reveal specific matrix-based regulation of TNBC cell phenotype and offer scaffolds for CTC-like cells with better mechano-biological properties than liquid.

Magnetic microrheometry of tumor-relevant stiffness levels and probabilistic quantification of viscoelasticity differences inside 3D cell culture matrices.

The progression of breast cancer involves cancer-cell invasions of extracellular matrices. To investigate the progression, 3D cell cultures are widely used along with different types of matrices. Currently, the matrices are often characterized using parallel-plate rheometry for matrix viscoelasticity, or liquid-like viscous and stiffness-related elastic characteristics. The characterization reveals averaged information and sample-to-sample variation, yet, it neglects internal heterogeneity within matrices, experienced by cancer cells in 3D culture. Techniques using optical tweezers and magnetic microrheometry have measured heterogeneity in viscoelasticity in 3D culture. However, there is a lack of probabilistic heterogeneity quantification and cell-size-relevant, microscale-viscoelasticity measurements at breast-tumor tissue stiffness up to ≃10 kPa in Young's modulus. Here, we have advanced methods, for the purpose, which use a magnetic microrheometer that applies forces on magnetic spheres within matrices, and detects the spheres displacements. We present probabilistic heterogeneity quantification using microscale-viscoelasticity measurements in 3D culture matrices at breast-tumor-relevant stiffness levels. Bayesian multilevel modeling was employed to distinguish heterogeneity in viscoelasticity from the effects of experimental design and measurement errors. We report about the heterogeneity of breast-tumor-relevant agarose, GrowDex, GrowDex-collagen and fibrin matrices. The degree of heterogeneity differs for stiffness, and phase angle (i.e. ratio between viscous and elastic characteristics). Concerning stiffness, agarose and GrowDex show the lowest and highest heterogeneity, respectively. Concerning phase angle, fibrin and GrowDex-collagen present the lowest and the highest heterogeneity, respectively. While this heterogeneity information involves softer matrices, probed by ≃30 µm magnetic spheres, we employ larger ≃100 µm spheres to increase magnetic forces and acquire a sufficient displacement signal-to-noise ratio in stiffer matrices. Thus, we show pointwise microscale viscoelasticity measurements within agarose matrices up to Young's moduli of 10 kPa. These results establish methods that combine magnetic microrheometry and Bayesian multilevel modeling for enhanced heterogeneity analysis within 3D culture matrices.