Mechanical Properties of the Tumor Stromal Microenvironment Probed In Vitro and Ex Vivo by In Situ-Calibrated Optical Trap-Based Active Microrheology

Abstract

One of the hallmarks of the malignant transformation of epithelial tissue is the modulation of stromal components of the microenvironment. In particular, aberrant extracellular matrix (ECM) remodeling and stiffening enhances tumor growth and survival and promotes metastasis. Type I collagen is one of the major ECM components. It serves as a scaffold protein in the stroma contributing to the tissue’s mechanical properties, imparting tensile strength and rigidity to tissues such as those of the skin, tendons, and lungs. Here we investigate the effects of intrinsic spatial heterogeneities due to fibrillar architecture, pore size and ligand density on the microscale and bulk mechanical properties of the ECM. Type I collagen hydrogels with topologies tuned by polymerization temperature and concentration to mimic physico-chemical properties of a normal tissue and tumor microenvironment were measured by in situ-calibrated Active Microrheology by Optical Trapping revealing significantly different microscale complex shear moduli at Hz-kHz frequencies and two orders of magnitude of strain amplitude that we compared to data from bulk rheology measurements. Access to higher frequencies enabled observation of transitions from elastic to viscous behavior that occur at ~200Hz to 2750Hz, which largely was dependent on tissue architecture well outside the dynamic range of instrument acquisition possible with SAOS bulk rheology. We determined that mouse melanoma tumors and human breast tumors displayed complex moduli ~5–1000 Pa, increasing with frequency and displaying a nonlinear stress-strain response. Thus, we show the feasibility of a mechanical biopsy in efforts to provide a diagnostic tool to aid in the design of therapeutics complementary to those based on standard histopathology.

High-frequency microrheology in 3D reveals mismatch between cytoskeletal and extracellular matrix mechanics

“…Drawing on polymer physics, we can further classify the cell rheological properties by examining the power law frequency dependence jG*j ∝ ω b . Flexible polymer networks are predicted to have complex moduli with b = 0.5, whereas b = 0.75 for semiflexible polymers (16). We observed that for high frequencies (400 to 15,000 Hz), the magnitudes jG*j of cells, near-ECM and far-ECM each fit to power laws with different exponents (i.e., slopes) b ranging from 0.46 to 0.70 (SI Appendix, Fig.…”

“…Formulaic explanations allow us to draw from our understanding of non-Newtonian fluids to model rheological properties of tissue (12)(13)(14). One property of tissues that emerges from non-Newtonian fluid mechanics is that viscoelasticity (complex moduli [G′*]) obeys frequency-dependent power laws, jG*(ω)j = Aω B , where the dependence B varies for different frequency regimes and different cell types (12)(13)(14)(15)(16). Gaining a mechanistic understanding of how these dynamics influence physiological and pathological mechanobiology thus requires quantitative measurements of cells and ECM in context.…”

Probing cellular response to topography in three dimensions

“…In addition to differences in the magnitude of the complex modulus, we observed a difference in frequency dependence between the aligned gels and unaligned gels. Power law dependence of the complex modulus on frequency has been previously observed in a number of biomaterials, and various models have been proposed to explain this behavior according to the underlying dynamics of the constituent polymers (47)(48)(49)(50). In fibrillar collagen gels polymerized at 2 mg/ml or 6 mg/ml at 4°C or 37°C, we previously observed power law exponents ranging from 0.66 -0.74 (48), in the range predicted for a semi-flexible polymer network.…”

“…For complete experimental details, see (48,59,60). Our home-built setup consists of a 1064 nm trapping beam steered by an acousto-optic deflector to oscillate the trap and a stationary 975 nm detection beam that is coupled into and colocated with the trap with a dichroic before being sent into the backport of an inverted microscope with a long working distance water objective and a high NA condenser.…”

Optical elastography and tissue biomechanics

“…In the invited talk for this session, Dr. Kandice Tanner from the U.S. National Institutes of Health discussed the applications of local viscoelastic properties of tumors and extracellular matrix surrounding those tumors. [36][37][38] Her group is searching for the understanding of mechanisms of metastasis, which were linked to local mechanical properties of cells and tissues. To quantify those small mechanical forces, they use optical tweezers and fluctuation correlation analysis.…”