Cells continuously adapt to changing conditions through coordinated molecular and mechanical responses. This adaptation requires the transport of molecules and signaling through intracellular regions with differing material properties, such as variations in viscosity or elasticity. To determine the impact of regional variations on cell structure and physiology, an approach, termed bio-microrheology, or the study of deformation and flow of biological materials at small length scales has emerged. By tracking the thermal and driven motion of probe particles, organelles, or molecules, the local physical environment in distinct subcellular regions can be explored. On the surface or inside cells, tracking the motion of particles can reveal the rheological properties that influence cell features, such as shape and metastatic potential. Cellular microrheology promises to improve our concepts of regional and integrated properties, structures, and transport in live cells. Since bio-microrheology is an evolving methodology, many specific details, such as how to interpret complex combinations of thermally mediated and directed probe transport, remain to be fully explained. This work reviews the current state of the field and discusses the utility and challenges of this emerging approach.

We evaluated the transport of polymeric particles internalized into living cancer cells. The mean-square displacement demonstrates superdiffusion with a scaling exponent of 1.25. Scaling exponents of a range of displacement moments are bilinear with moment order, exhibiting slopes of 0.6 and 0.8. Thus, we present experimental evidence of strong anomalous diffusion. Bilinearity indicates that particle motion is composed of subdiffusive regimes separated by active yet nonballistic flights. We discuss the results in terms of particle interactions with their microenvironment.

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Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.

We present here the first evidence of mechanical penetration by a metastatic cancer cell. During metastasis, the invasive cancer-cell penetrates tissue and extracellular matrix, changes shape and applies force. These applied forces, in turn, depend on substrate stiffness and degradability. The initial stage of metastatic penetration comprises substrate indentation, which, however, has not yet been studied. Hence, we evaluate the evolution of indentation, focusing on differences relating to the metastatic potential (MP) of the cells and substrate stiffness. We found that metastatic cells attain a mushroom-like morphology and then, over several hours, repeatedly indent the substrate in a manner suggestive of a special role for the nucleus. Cells with higher MP have previously been shown to be softer internally and externally than those with lower MP yet, paradoxically, applied stronger forces. Cells of higher MP develop stronger forces on gels stiff enough to provide grip handles yet soft enough to indent, whereas benign cells did not indent substrates at all. These findings provide insight into the central role of physical forces in the initial stages of metastatic penetration and reveal new targets for treatment.

Cholesterol crystals are the building blocks of cholesterol gallstones. The exact structure of early-forming crystals is still controversial. We combined cryogenic-temperature transmission electron microscopy with cryogenic-temperature electron diffraction to sequentially study crystal development and structure in nucleating model and native gallbladder biles. The growth and long-term stability of classic cholesterol monohydrate (ChM) crystals in native and model biles was determined. In solutions of model bile with low phospholipid-to-cholesterol ratio, electron diffraction provided direct proof of a novel transient polymorph that had an elongated habit and unit cell parameters differing from those of classic triclinic ChM. This crystal is exactly the monoclinic ChM phase described by Solomonov and coworkers ( Biophysical J. , In press) in cholesterol monolayers compressed on the air-water interface. We observed no evidence of anhydrous cholesterol crystallization in any of the biles studied. In conclusion, classic ChM is the predominant and stable form in native and model biles.

Therapeutic ultrasound is widely employed in clinical applications but its mechanism of action remains unclear. Here we report prompt fluidization of a cell and dramatic acceleration of its remodeling dynamics when exposed to low intensity ultrasound. These physical changes are caused by very small strains (10−5) at ultrasonic frequencies (106 Hz), but are closely analogous to those caused by relatively large strains (10−1) at physiological frequencies (100 Hz). Moreover, these changes are reminiscent of rejuvenation and aging phenomena that are well-established in certain soft inert materials. As such, we suggest cytoskeletal fluidization together with resulting acceleration of cytoskeletal remodeling events as a mechanism contributing to the salutary effects of low intensity therapeutic ultrasound.

We show that metastatic breast cancer cells are quantitatively identifiable from benign cells during adherence onto soft, elastic gels. We identify differences in time-dependent morphology and strength of adherence of single breast cells that are likely related to their malignancy and metastatic potential (MP). Specifically, we compare high and low MP breast cancer cells with benign cells as a control on collagen-coated, polyacrylamide gels with Young's modulus in the physiological range of 2.4-10.6 kPa. We observe that the evaluated metastatic breast cancer cells remain rounded, with small contact area, up to 6.5 h following seeding. In contrast, the benign cells spread and become more elongated on stiffer gels. We identify measurable differences in the two-dimensional, lateral, traction forces exerted by the cells, where the rounded, metastatic cells apply significantly larger, traction forces, as compared to the benign cells, on gels stiffer than 2.4 kPa. The metastatic cell lines exhibited gel-stiffness-dependent differences in traction forces, strain energies, and morphologies during the initial stages of adhesion, which may relate to their MP or invasiveness.