Kandice Tanner

Probing cellular mechanobiology in three-dimensional culture with collagen-agarose matrices

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Coherent angular motion in the establishment of multicellular architecture of glandular tissues

Glandular tissues form ducts (tubes) and acini (spheres) in multicellular organisms. This process is best demonstrated in the organization of the ductal tree of the mammary gland and in 3D models of morphogenesis in culture. Here, we asked a fundamental question: How do single adult epithelial cells generate polarized acini when placed in a surrogate basement membrane 3D gel? Using human breast epithelial cells from either reduction mammoplasty or nonmalignant breast cell lines, we observed a unique cellular movement where single cells undergo multiple rotations and then maintain it cohesively as they divide to assemble into acini. This coherent angular motion (CAMo) was observed in both primary cells and breast cell lines. If CAMo was disrupted, the final geometry was not a sphere. The malignant counterparts of the human breast cell lines in 3D were randomly motile, did not display CAMo, and did not form spheres. Upon “phenotypic reversion” of malignant cells, both CAMo and spherical architecture were restored. We show that cell-cell adhesion and tissue polarity are essential for the formation of acini and link the functional relevance of CAMo to the establishment of spherical architecture rather than to multicellular aggregation or growth. We propose that CAMo is an integral step in the formation of the tissue architecture and that its disruption is involved in malignant transformation.

Dissecting Regional Variations in Stress Fiber Mechanics in Living Cells with Laser Nanosurgery

The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquely triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.

Independent Control of Topography for 3D Patterning of the ECM Microenvironment.

Biomimetic extracellular matrix (ECM) topographies driven by the magnetic‐field‐directed self‐assembly of ECM protein‐coated magnetic beads are fabricated. This novel bottom‐up method allows us to program isotropic, anisotropic, and diverse hybrid ECM patterns without changing other physicochemical properties of the scaffold material. It is demonstrated that this 3D anisotropic matrix is able to guide the dendritic protrusion of cells.

Coherent Movement of Cell Layers during Wound Healing by Image Correlation Spectroscopy

We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume ( approximately 37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury.

Spectrally resolved neurophotonics: a case report of hemodynamics and vascular components in the mammalian brain

We developed a spectral technique that is independent of the light transport modality (diffusive or nondiffusive) to separate optical changes in scattering and absorption in the cats brain due to the hemodynamic signal following visual stimulation. We observe changes in oxyhemoglobin and deoxyhemoglobin concentration signals during visual stimulation reminiscent of the functional magnetic resonance imaging (fMRI) blood oxygenation level dependence (BOLD) effect. Repeated measurements at different locations show that the observed changes are local rather than global. We also determine that there is an apparent large decrease in the water concentration and scattering coefficient during stimulation. We model the apparent change in water concentration on the separation of the optical signal from two tissue compartments. One opaque compartment is featureless (black), due to relatively large blood vessels. The other compartment is the rest of the tissue. When blood flow increases due to stimulation, the opaque compartment increases in volume, resulting in an overall decrease of tissue transmission. This increase in baseline absorption changes the apparent relative proportion of all tissue components. However, due to physiological effects, the deoxyhemoglobin is exchanged with oxyhemoglobin resulting in an overall increase in the oxyhemoglobin signal, which is the only component that shows an apparent increase during stimulation.

In vivo tissue has non-linear rheological behavior distinct from 3D biomimetic hydrogels, as determined by AMOTIV microscopy.

Variation in matrix elasticity has been shown to determine cell fate in both differentiation and development of malignant phenotype. The tissue microenvironment provides complex biochemical and biophysical signals in part due to the architectural heterogeneities found in extracellular matrices (ECMs). Three dimensional cell cultures can partially mimic in vivo tissue architecture, but to truly understand the role of viscoelasticity on cell fate, we must first determine in vivo tissue mechanical properties to improve in vitro models. We employed Active Microrheology by Optical Trapping InVivo (AMOTIV), using in situ calibration to measure in vivo zebrafish tissue mechanics. Previously used trap calibration methods overestimate complex moduli by ∼ 2-20 fold compared to AMOTIV. Applying differential microscale stresses and strains showed that hyaluronic acid (HA) gels display semi-flexible polymer behavior, while laminin-rich ECM hydrogels display flexible polymer behavior. In contrast, zebrafish tissues displayed different moduli at different stresses, with higher power law exponents at lower stresses, indicating that living tissue has greater stress dependence than the 3D hydrogels examined. To our knowledge, this work is the first vertebrate tissue rheological characterization performed in vivo. Our fundamental observations are important for the development and refinement of in vitro platforms.

In situ calibration of position detection in an optical trap for active microrheology in viscous materials

In optical trapping, accurate determination of forces requires calibration of the position sensitivity relating displacements to the detector readout via the V-nm conversion factor (β). Inaccuracies in measured trap stiffness (k) and dependent calculations of forces and material properties occur if β is assumed to be constant in optically heterogeneous materials such as tissue, necessitating calibration at each probe. For solid-like samples in which probes are securely positioned, calibration can be achieved by moving the sample with a nanopositioning stage and stepping the probe through the detection beam. However, this method may be applied to samples only under select circumstances. Here, we introduce a simple method to find β in any material by steering the detection laser beam while the probe is trapped. We demonstrate the approach in the yolk of living Danio rerio (zebrafish) embryos and measure the viscoelastic properties over an order of magnitude of stress-strain amplitude.

Effects of vasodilation on intrinsic optical signals in the mammalian brain: a phantom study

Using a broadband spectral technique, we recently showed [J. Biomed. Opt. 10, 064009 (2005)] that during visual stimulation of the cat brain there were not only changes in oxy- and deoxyhemoglobin levels, reminiscent of the optical blood oxygenation level dependence (BOLD) effect reported in humans, but also the apparent water content of the tissue and the optical scattering contribution decreased during stimulation. These relatively fast changes (in seconds) in water tissue content are difficult to explain in physiological terms. We developed a simple model to explain how local vasodilation, which occurs as a result of the stimulation, could cause this apparent change in water content. We show that in a phantom model we can obtain spectral effects similar to those observed in the cat brain such as the apparent decrease of the water spectral component without changing the water content of the bath in which the phantom measurements were performed. Furthermore, using the phantom model, we show that the relative apparent changes in the spectral components due to vasodilation during stimulation are roughly comparable in magnitude to the changes in tissue chromophores due to the optical equivalent of the BOLD effect reported in the literature.

Deconstructing the Role of the Microenvironment on Drug Efficacy in a Brain-Mimetic Platform for Cutaneous Metastatic Melanoma

Although survival in patients with malignant melanoma has significantly improved due to therapeutic interventions based on the molecular basis of tumor etiology, durable responses in the face of metastatic disease are rarely realized. A “systems pharmacological” approach to uncover drug potency at the physically distinct stages of the metastatic cascade is required. We modeled disparate microenvironments in the brain; the perivascular niche and hyaluronic acid (HA) rich parenchyma, to assess contextual drug efficacy. These two microenvironments are not only differ in composition, but in dimensionality, with the perivascular niche inducing a 2D morphology in cells, while the HA-rich parenchyma leads to 3D cellular clusters. These in vitro models recapitulated in vivo morphology and motility for an isogenic, human model of melanoma metastatic progression. By independently modulating adhesion strength and ECM composition, we found that ERK inhibition decreased cell adhesion, whereas BRAF inhibition was only effective when combined with an ERK inhibitor. BRAF and ERK inhibition individually reduced cell motility in the less metastatic clone, with a lesser effect on the more metastatic clone. We observed that cells are resistant to BRAFV600E inhibition when cultured in 3D Fibronectin rich HA hydrogels, but Laminin rich HA-gels offered no protection. The opposite held true for ERK inhibition. These data reinforce that a dynamic microenvironment not only contributes to systemic metastasis, but also significantly modifies drug efficacy.