Sangwon Byun

Characterizing deformability and surface friction of cancer cells

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell’s passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cells size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constrictions walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.

Deformability of Tumor Cells versus Blood Cells

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.

Sustained delivery of bioactive TGF-β1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells

Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor β1 (TGF-β1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-β1, fivefold higher retention was found in peptide. Excess unlabeled TGF-β1 minimally displaced retained radiolabeled TGF-β1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly threefold lower than agarose hydrogels, at 18% of loaded TGF-β1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-β1 was 32-44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-β1 degradation and release of small labeled species. TGF-β1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.

Transport and Equilibrium Uptake of a Peptide Inhibitor of PACE4 into Articular Cartilage is Dominated by Electrostatic Interactions

The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be approximately 1 x 10(-6) cm(2)/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.

Characterizing Cellular Biophysical Responses to Stress by Relating Density, Deformability, and Size

Cellular physical properties are important indicators of specific cell states. Although changes in individual biophysical parameters, such as cell size, density, and deformability, during cellular processes have been investigated in great detail, relatively little is known about how they are related. Here, we use a suspended microchannel resonator (SMR) to measure single-cell density, volume, and passage time through a narrow constriction of populations of cells subjected to a variety of environmental stresses. Osmotic stress significantly affects density and volume, as previously shown. In contrast to density and volume, the effect of an osmotic challenge on passage time is relatively small. Deformability, as determined by comparing passage times for cells with similar volume, exhibits a strong dependence on osmolarity, indicating that passage time alone does not always provide a meaningful proxy for deformability. Finally, we find that protein synthesis inhibition, cell-cycle arrest, protein kinase inhibition, and cytoskeletal disruption result in unexpected relationships among deformability, density, and volume. Taken together, our results suggest that by measuring multiple biophysical parameters, one can detect unique characteristics that more specifically reflect cellular behaviors.

Transport and Binding of Tumor Necrosis Factor-α in Articular Cartilage Depend on its Quaternary Structure

The effect of tumor necrosis factor-α (TNFα) on cartilage matrix degradation is mediated by its transport and binding within the extracellular matrix (ECM) of the tissue, which mediates availability to cell receptors. Since the bioactive form of TNFα is a homotrimer of monomeric subunits, conversion between trimeric and monomeric forms during intratissue transport may affect binding to ECM and, thereby, bioactivity within cartilage. We studied the transport and binding of TNFα in cartilage, considering the quaternary structure of this cytokine. Competitive binding assays showed significant binding of TNFα in cartilage tissue, leading to an enhanced uptake. However, studies in which TNFα was cross-linked to remain in the trimeric form revealed that the binding of trimeric TNFα was negligible. Thus, binding of TNFα to ECM was associated with the monomeric form. Binding of TNFα was not disrupted by pre-treating cartilage tissue with trypsin, which removes proteoglycans and glycoproteins but leaves the collagen network intact. Therefore, proteoglycan loss during osteoarthritis should only alter the passive diffusion of TNFα but not its binding interaction with the remaining matrix. Our results suggest that matrix binding and trimer-monomer conversion of TNFα both play crucial roles in regulating the accessibility of bioactive TNFα within cartilage.

Attogram mass sensing based on silicon microbeam resonators

Using doubly-clamped silicon (Si) microbeam resonators, we demonstrate sub-attogram per Hertz (ag/Hz) mass sensitivity, which is extremely high sensitivity achieved by micro-scale MEMS mass sensors. We also characterize unusual buckling phenomena of the resonators. The thin-film based resonator is composed of a Si microbeam surrounded by silicon nitride (SiN) anchors, which significantly improve performance by providing fixation on the microbeam and stabilizing oscillating motion. Here, we introduce two fabrication techniques to further improve the mass sensitivity. First, we minimize surface stress by depositing a sacrificial SiN layer, which prevents damage on the Si microbeam. Second, we modify anchor structure to find optimal design that allows the microbeam to oscillate in quasi-one dimensional mode while achieving high quality factor. Mass loading is conducted by depositing Au/Ti thin films on the local area of the microbeam surface. Using sequential mass loading, we test effects of changing beam dimensions, position of mass loading, and distribution of a metal film on the mass sensitivity. In addition, we demonstrate that microbeams suffer local micro-buckling and global buckling by excessive mass loading, which are induced by two different mechanisms. We also find that the critical buckling length is increased by additional support from the anchors.