Publications

Cell mechanical phenotype, or mechanotype, is emerging as a label-free biomarker for cancer and pluripotent stem cells. Here, we demonstrate a method for rapid, single cell, mechanotype measurements, termed quantitative deformability cytometry (qDC). We track changes cell shape as they deform into microfluidic constrictions, and observe that the time-dependent strain follows power law rheology; this enables us to obtain single cell measurements of apparent elastic modulus, Ea, and power law exponent, beta, in addition to standard DC measurements of cell size, maximum strain, and transit time. To validate our method, we measure E_a for HL-60 cells as 1.4 ± 0.6 kPa, and confirm qDC is sensitive to pharmacological perturbations of the cytoskeleton. We then characterize the mechanotype of MCF7 and MDA-MB-231 breast cancer cells (E_a = 8.1 ± 0.3 and 4.3 ± 0.7 kPa) and pancreatic adenocarcinoma cells (E_a = 3.1 ± 0.2 to 7.3 ± 0.5 kPa for HPDE, MiaPaCa-2, PANC1, AsPC-1, and Hs766T). Additionally, we observe E_a increases with cell size. By contrast, E_a does not scale with size for silicone oil droplets. Our results suggest that cells undergo strain stiffening when subjected to large deformations. Ea may also increase with larger deformation depths, as the nucleus is typically stiffer than the cytoplasmic region. To determine the value of qDC compared to standard DC outputs, we identify unique and redundant parameters using Pearson's correlation coefficients. We also employ a nearest neighbor machine learning algorithm to test the predictive power of the qDC parameters. Using our algorithm, we identify a minimal set of parameters containing cell size, transit time, and E_a, which confers highly accurate classification of our cancer cell lines. Taken together, the standardized measurements provided by qDC should advance applications of cell mechanotype in basic research and clinical settings.

This study reports, for the first time, development of tyrosine kinase inhibitor-loaded, thermosensitive liposomes (TKI/TSLs) and their efficacy for treatment of renal cell carcinoma when triggered by focused ultrasound (FUS). Uptake of these nanoparticles into renal cancer cells was visualized with confocal and fluorescent imaging of rhodamine B-loaded liposomes. The combination of TKI/TSLs and focused ultrasound was tested in an in vitro tumor model of renal cell carcinoma. According to MTT cytotoxic assay and flow cytometric analysis the combined treatment led to the least viability (23.4 ± 2.49%, p<0.001), significantly lower than that observed from treatment with FUS (97.6 ± 0.67%, n.s.) or TKI/TSL (71.0 ± 3.65%, p < 0.001) at 96 hours compared to control. The importance of this unique, synergistic combination was demonstrated in viability experiments with non-thermosensitive liposomes (TKI/NTSL+FUS: 58.8 ± 1.5% vs TKI/TSL+FUS: 36.2 ± 1.4%, p<0.001) and heated water immersion (TKI/TSL+WB43°: 59.3 ± 2.91% vs TKI/TSL+FUS: 36.4 ± 1.55%, p < 0.001). Our findings coupled with the existing use of focused ultrasound in clinical practice make the proposed combination of targeted chemotherapy, nanotechnology, and focused ultrasound a promising platform for enhanced drug delivery and cancer treatment.

Background: Thromboelastography is widely used as a tool to assess the coagulation status of surgical patients. It allows observation of changes in material properties of whole blood, beginning with early stages of clot formation and ending with clot lysis. However, the contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks.
Objectives: To develop acoustic tweezing thromboelastometry as a noncontact method for perioperative assessment of whole blood coagulation.
Methods: Acoustic tweezing is used to levitate microliter drops of biopolymer and whole blood samples. By quasi-statically changing the acoustic pressure we control the sample drop location and deformation. Sample size, deformation and location are determined by digital imaging at each pressure.
Results: Simple Newtonian liquid solutions maintain a constant, reversible location vs deformation curve. In contrast, the deformation/location curves for gelatin, alginate and whole blood uniquely change as the samples solidify. Increasing elasticity causes the sample to deform less, leading to steeper stress/strain curves. By extracting a linear regime slope, we show that whole blood exhibits a unique slope profile as it begins to clot. By exposing blood samples to pro- or anti-coagulants, the slope profile changes, allowing detection of hyper- or hypo-coagulable states.

High-intensity focused ultrasound (HIFU) can locally ablate biological tissues such as tumors, i.e., induce their rapid heating and coagulative necrosis without causing damage to surrounding healthy structures. It is widely used in clinical practice for minimally invasive treatment of prostate cancer. Non-ablative, low-power HIFU was established as a promising tool for triggering the release of chemotherapeutic drugs from temperature-sensitive liposomes (TSLs). In this study, we combine ablative HIFU and thermally triggered chemotherapy to address the lack of safe and effective treatment options for elderly patients with high-risk localized prostate cancer. DU145 prostate cancer cells were exposed to chemotherapy (free and liposomal Sorafenib) and ablative HIFU, alone or in combination. Prior to cell viability assessment by trypan blue exclusion and flow cytometry, the uptake of TSLs by DU145 cells was verified by confocal microscopy and cryogenic scanning electron microscopy (cryo-SEM). The combination of TSLs encapsulating 10 μM Sorafenib and 8.7W HIFU resulted in a viability of less than 10% at 72 h post treatment, which was significant less than the viability of the cells treated with free Sorafenib (76%), Sorafenib-loaded TSLs (63%), or HIFU alone (44%). This synergy was not observed on cells treated with Sorafenib-loaded non-temperature sensitive liposomes and HIFU. According to cryo-SEM analysis, cells exposed to ablative HIFU exhibited significant mechanical disruption. Water bath immersion experiments also showed an important role of mechanical effects in the synergistic enhancement of TSL-mediated chemotherapy by ablative HIFU. This combination therapy can be an effective strategy for treatment of geriatric prostate cancer patients.

BACKGROUND: Extravasation is a critical step in cancer metastasis, in which adhesion of intravascular cancer cells to the vascular endothelial cells is controlled by cell surface adhesion molecules. The role of interleukin-17 (IL-17), insulin, and insulin-like growth factor 1 (IGF1) in adhesion of prostate cancer cells to the vascular endothelial cells is unknown, which is the subject of the present study.
METHODS: Human umbilical vein endothelial cells (HUVECs) and human prostate cancer cell lines (PC-3, DU-145, LNCaP, and C4-2B) were analyzed for expression of vascular cell adhesion molecule 1 (VCAM-1), integrins, and cluster of differentiation 44 (CD44) using flowcytometry and Western blot analysis. The effects of IL-17, insulin and IGF1 on VCAM-1 expression and adhesion of prostate cancer cells to HUVECs were examined. The interaction of VCAM-1 and CD44 was assessed using immunoprecipitation assays.
RESULTS: Insulin and IGF1 acted with IL-17 to increase VCAM-1 expression in HUVECs. PC-3, DU-145, LNCaP, and C4-2B cells expressed β1 integrin but not α4 integrin. CD44 was expressed by PC-3 and DU-145 cells but not by LNCaP or C4-2B cells. When HUVECs were treated with IL-17, insulin or IGF1, particularly with a combination of IL-17 and insulin (or IGF1), adhesion of PC-3 and DU-145 cells to HUVECs was significantly increased. In contrast, adhesion of LNCaP and C4-2B cells to HUVECs was not affected by treatment of HUVECs with IL-17 and/or insulin/IGF1. CD44 expressed in PC-3 cells physically bound to VCAM-1 expressed in HUVECs.
CONCLUSIONS: CD44-VCAM-1 interaction mediates the adhesion between prostate cancer cells and HUVECs. IL-17 and insulin/IGF1 enhance adhesion of prostate cancer cells to vascular endothelial cells through increasing VCAM-1 expression in the vascular endothelial cells. These findings suggest that IL-17 may act with insulin/IGF1 to promote prostate cancer metastasis.

Microvascular remodeling is a common denominator for multiple pathologies and involves both angiogenesis, defined as the sprouting of new capillaries, and network patterning associated with the organization and connectivity of existing vessels. Much of what we know about microvascular remodeling at the network, cellular, and molecular scales has been derived from reductionist biological experiments, yet what happens when the experiments provide incomplete (or only qualitative) information? This review will emphasize the value of applying computational approaches to advance our understanding of the underlying mechanisms andeffects of microvascular remodeling. Examples of individual computational models applied to each of the scales will highlight the potential of answering specific questions that cannot be answered using typical biological experimentation alone. Looking into the future, we will also identify the needs and challenges associated with integrating computational models across scales.

Three dimensional simulation of the leukocyte detachment subjected to blood flow is presented. The initially captured leukocyte is modeled as a sphere adhered to the bottom wall of a cylindrical vessel via receptor/ligand bonds (P-selectin/PSGL-1). Ansys Parametric Design Language is used to create the geometrical model and couple the Navier-stokes flow solver with structural equations and the Monte Carlo equation to define the stochastic breakage of the bonds. The assumption of equal forces on bonds has been ignored and the force on each bond is obtained from the balance between hydrodynamic forces and cellular viscoelasticity at every time step. In this model, catch-slip behavior of the P-selectin/PSGL-1 is considered by using the two-pathway dissociation model instead of the Bell model to define the rate of dissociation of each bond. Detachment time of the leukocyte is the time elapsed until all the bonds break. The effects of various values of blood inlet velocities, bond stiffness and kinetic properties of the catch bonds on the detachment time of the leukocyte are studied.

Oxidized low-density lipoprotein (OxLDL) is a risk factor for atherosclerosis, due to its role in endothelial dysfunction and foam cell formation. Tissue-resident cells such as macrophages and mast cells can release inflammatory mediators upon activation that in turn cause endothelial activation and monocyte adhesion. Two of these mediators are tumor necrosis factor (TNF)-α, produced by macrophages, and histamine, produced by mast cells. Static and microfluidic flow experiments were conducted to determine the number of adherent monocytes on vascular endothelium activated by supernatants of OxLDL-treated macrophages and mast cells or directly by OxLDL. The expression of adhesion molecules on activated endothelial cells and the concentration of TNF-α and histamine in the supernatants were measured by flow cytometry and enzyme-linked immunosorbent assay, respectively. A low dose of OxLDL (8 μg/ml), below the threshold for the clinical presentation of coronary artery disease, was sufficient to activate both macrophages and mast cells and synergistically increase monocyte-endothelium adhesion via released TNF-α and histamine. The direct exposure of endothelial cells to a much higher dose of OxLDL (80 μg/ml) had less effect on monocyte adhesion than the indirect activation via OxLDL-treated macrophages and mast cells. The results of this work indicate that the co-activation of macrophages and mast cells by OxLDL is an important mechanism for the endothelial dysfunction and atherogenesis. The observed synergistic effect suggests that both macrophages and mast cells play a significant role in early stages of atherosclerosis. Allergic patients with a lipid-rich diet may be at high risk for cardiovascular events due to high concentration of low-density lipoprotein and histamine in arterial vessel walls.

Leukocytes and other circulating cells deform and move relatively to the channel flow in the lateral and translational directions. Their migratory property is important in immune response, hemostasis, cancer progression, delivery of nutrients, and microfluidic technologies such as cell separation and enrichment, and flow cytometry. Using our three-dimensional computational algorithm for multiphase viscoelastic flow, we have investigated the effect of pairwise interaction on the lateral and translational migration of circulating cells in a microchannel. The numerical simulation data show that when two cells with the same size and small separation distance interact, repulsive interaction take place until they reach the same lateral equilibrium position. During this process, they undergo swapping or passing, depending on the initial separation distance between each other. The threshold value of this distance increases with cell deformation, indicating that the cells experiencing larger deformation are more likely to swap. When a series of closely spaced cells with the same size are considered, they generally undergo damped oscillation in both lateral and translational directions until they reach equilibrium positions where they become evenly distributed in the flow direction (self-assembly phenomenon). A series of cells with a large lateral separation distance could collide repeatedly with each other, eventually crossing the centerline and entering the other side of the channel. For a series of cells with different deformability, more deformable cells, upon impact with less deformable cells, move to an equilibrium position closer to the centerline. The results of our study show that the bulk deformation of circulating cells plays a key role in their migration in a microchannel.

We investigated the combined effect of ethanol and high-intensity focused ultrasound (HIFU), first, on heating and cavitation bubble activity in tissue-mimicking phantoms and porcine liver tissues and, second, on the viability of HepG2 liver cancer cells. Phantoms or porcine tissues were injected with ethanol and then subjected to HIFU at acoustic power ranging from 1.2 to 20.5 W (HIFU levels 1-7). Cavitation events and the tememperature around the focal zone were measured with a passive cavitation detector and embedded type K thermocouples, respectively. HepG2 cells were subjected to 4% ethanol solution in growth medium (v/v) just before the cells were exposed to HIFU at 2.7, 8.7 or 12.0 W for 30 s. Cell viability was measured 2, 24 and 72 h post-treatment. The results indicate that ethanol and HIFU have a synergistic effect on liver cancer ablation as manifested by greater temperature rise and lesion volume in liver tissues and reduced viability of liver cancer cells. This effect is likely caused by reduction of the cavitation threshold in the presence of ethanol and the increased rate of ethanol diffusion through the cell membrane caused by HIFU-induced streaming, sonoporation and heating.