Whole blood PT/aPTT assay based on non-contact drop-of-sample acoustic tweezing spectroscopy.

Most coagulation tests are photo-optical turbidimetric assays that require the removal of cellular components from whole blood for optical clearing. If the resulting blood plasma samples are hemolyzed, they may become unsuitable for turbidimetric analysis. To resolve this issue, whole-blood analogs to plasma turbidimetric assays need to be developed. Using samples collected from non-smokers (normal group), smokers (thrombotic group), and hemophilia A (bleeding group) patients, we demonstrate that the reaction time assessed from whole blood viscosity data of the drop-of-blood acoustic tweezing spectroscopy (ATS) technique strongly correlates (Rp ≥ 0.95) with PT/aPTT values obtained from plasma turbidimetric data. Linear correlation (Rp ≥ 0.88) was also obtained between the viscous and elastic outputs of the ATS technique and the fibrinogen concentration. The integration of ATS data enabled the assessment of the functional level of fibrin cross-linkers such as factor XIII. Overall, ATS allows comprehensive sample-sparing analysis of whole blood coagulation for reliable and safe diagnosis of bleeding/thrombosis risks.

Diabetes is accompanied by secretion of pro-atherosclerotic exosomes from vascular smooth muscle cells.

BACKGROUND: Atherosclerosis is a common co-morbidity of type 2 diabetes mellitus. Monocyte recruitment by an activated endothelium and the pro-inflammatory activity of the resulting macrophages are critical components of atherosclerosis. Exosomal transfer of microRNAs has emerged as a paracrine signaling mechanism regulating atherosclerotic plaque development. MicroRNAs-221 and -222 (miR-221/222) are elevated in vascular smooth muscle cells (VSMCs) of diabetic patients. We hypothesized that the transfer of miR-221/222 via VSMC-derived exosomes from diabetic sources (DVEs) promotes increased vascular inflammation and atherosclerotic plaque development. METHODS: Exosomes were obtained from VSMCs, following exposure to non-targeting or miR-221/-222 siRNA (-KD), isolated from diabetic (DVEs) and non-diabetic (NVEs) sources and their miR-221/-222 content was measured using droplet digital PCR (ddPCR). Expression of adhesion molecules and the adhesion of monocytes was measured following exposure to DVEs and NVEs. Macrophage phenotype following exposure to DVEs was determined by measuring mRNA markers and secreted cytokines. Age-matched apolipoprotein-E-deficient mice null (ApoE-/-) mice were maintained on Western diet for 6 weeks and received injections of saline, NVEs, NVE-KDs, DVEs or DVE-KDs every other day. Atherosclerotic plaque formation was measured using Oil Red Oil staining. RESULTS: Exposure of human umbilical vein and coronary artery endothelial cells to DVEs, but not NVEs, NVE-KDs, or DVE-KDs promoted increased intercellular adhesion molecule-1 expression and monocyte adhesion. DVEs but not NVEs, NVE-KDs, or DVE-KDs also promoted pro-inflammatory polarization of human monocytes in a miR-221/222 dependent manner. Finally, intravenous administration of DVEs, but not NVEs, resulted in a significant increase in atherosclerotic plaque development. CONCLUSION: These data identify a novel paracrine signaling pathway that promotes the cardiovascular complications of diabetes mellitus.

Drop-of-blood acoustic tweezing technique for integrative turbidimetric and elastometric measurement of blood coagulation.

Many patients develop coagulation abnormalities due to chronic and hereditary disorders, infectious disease, blood loss, extracorporeal circulation, and oral anticoagulant misuse. These abnormalities lead to bleeding or thrombotic complications, the risk of which is assessed by coagulation analysis. Current coagulation tests pose safety concerns for neonates and small children due to large sample volume requirement and may be unreliable for patients with coagulopathy. This study introduces a containerless drop-of-blood method for coagulation analysis, termed "integrated quasi-static acoustic tweezing thromboelastometry" (i-QATT™), that addresses these needs. In i-QATT™, a single drop of blood is forced to levitate and deform by the acoustic radiation force. Coagulation-induced changes in drop turbidity and firmness are measured simultaneously at different instants. The parameters describing early, intermediate, and late stages of the coagulation process are evaluated from the resulting graphical outputs. i-QATT™ rapidly (<10 min) detected hyper- and hypo-coagulable states and identified single deficiency in coagulation factors VII, VIII, IX, X, and XIII. The linear relationship (r2 > 0.9) was established between fibrinogen concentration and two i-QATT™ parameters: maximum clot firmness and maximum fibrin level. Factor XIII activity was uniquely measured by the fibrin network formation time (r2 = 0.9). Reaction time, fibrin formation rate, and time to firm clot formation were linearly correlated with heparin concentration (r2 > 0.7). tPA-induced hyperfibrinolysis was detected in the clot firmness output at 10 min. i-QATT™ provides comprehensive coagulation analysis in point-of-care or laboratory settings, well suited to the needs of neonatal and pediatric patients and adult patients with anemia or blood collection issues.

Elongation Index as a Sensitive Measure of Cell Deformation in High-Throughput Microfluidic Systems.

One of the promising approaches for high-throughput screening of cell mechanotype is microfluidic deformability cytometry (mDC), in which the apparent deformation index (DI) of the cells stretched by extensional flow at the stagnation point of a cross-slot microchannel is measured. The DI is subject to substantial measurement errors due to cell offset from the flow centerline and velocity fluctuations in inlet channels, leading to artificial widening of DI versus cell size plots. Here, we simulated an mDC experiment using a custom computational algorithm for viscoelastic cell migration. Cell motion and deformation in a cross-slot channel was modeled for fixed or randomized values of cellular mechanical properties (diameter, shear elasticity, cortical tension) and initial cell placement, with or without sinusoidal fluctuations between the inlet velocities. Our numerical simulation indicates that mDC loses sensitivity to changes in shear elasticity when the offset distance exceeds 5 µm, and just 1% velocity fluctuation causes an 11.7% drop in the DI. The obtained relationships between the cell diameter, shear elasticity, and offset distance were used to establish a new measure of cell deformation, referred to as the "elongation index" (EI). In the randomized study, the EI scatter plots were visibly separated for the low- and high-elasticity populations of cells, with a mean of 300 and 3500 Pa, whereas the standard DI output was unable to distinguish between these two groups of cells. The successful suppression of the offset artifacts with a narrower data distribution was shown for the EI output of MCF-7 cells.

Quantitative Deformability Cytometry: Rapid, Calibrated Measurements of Cell Mechanical Properties.

Advances in methods that determine cell mechanical phenotype, or mechanotype, have demonstrated the utility of biophysical markers in clinical and research applications ranging from cancer diagnosis to stem cell enrichment. Here, we introduce quantitative deformability cytometry (q-DC), a method for rapid, calibrated, single-cell mechanotyping. We track changes in cell shape as cells deform into microfluidic constrictions, and we calibrate the mechanical stresses using gel beads. We observe that time-dependent strain follows power-law rheology, enabling single-cell measurements of apparent elastic modulus, Ea, and power-law exponent, ß. To validate our method, we mechanotype human promyelocytic leukemia (HL-60) cells and thereby confirm q-DC measurements of Ea = 0.53 ± 0.04 kPa. We also demonstrate that q-DC is sensitive to pharmacological perturbations of the cytoskeleton as well as differences in the mechanotype of human breast cancer cell lines (Ea = 2.1 ± 0.1 and 0.80 ± 0.19 kPa for MCF-7 and MDA-MB-231 cells). To establish an operational framework for q-DC, we investigate the effects of applied stress and cell/pore-size ratio on mechanotype measurements. We show that Ea increases with applied stress, which is consistent with stress stiffening behavior of cells. We also find that Ea increases for larger cell/pore-size ratios, even when the same applied stress is maintained; these results indicate strain stiffening and/or dependence of mechanotype on deformation depth. Taken together, the calibrated measurements enabled by q-DC should advance applications of cell mechanotype in basic research and clinical settings.

Ablative Focused Ultrasound Synergistically Enhances Thermally Triggered Chemotherapy for Prostate Cancer in Vitro.

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. Nonablative, 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 nontemperature 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.

IL-17 and insulin/IGF1 enhance adhesion of prostate cancer cells to vascular endothelial cells through CD44-VCAM-1 interaction.

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 flow cytometry 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 ß1 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.

Effects of microfluidic channel geometry on leukocyte rolling assays.

Microfluidic cell adhesion assays have emerged as a means to increase throughput as well as reduce the amount of costly reagents. However as dimensions of the flow chamber are reduced and approach the diameter of a cell (D(c)), theoretical models have predicted that mechanical stress, force, and torque on a cell will be amplified. We fabricated a series of microfluidic devices that have a constant width:height ratio (10:1) but with varying heights. The smallest microfluidic device (200 µm ×20 µm) requires perfusion rates as low as 40 nL/min to generate wall shear stresses of 0.5 dynes/cm(2). When neutrophils were perfused through P-selectin coated chambers at equivalent wall shear stress, rolling velocities decreased by approximately 70 % as the ratio of cell diameter to chamber height (D(c)/H) increased from 0.08 (H = 100 µm) to 0.40 (H = 20 µm). Three-dimensional numerical simulations of neutrophil rolling in channels of different heights showed a similar trend. Complementary studies with PSGL-1 coated microspheres and paraformaldehyde-fixed neutrophils suggested that changes in rolling velocity were related to cell deformability. Using interference reflection microscopy, we observed increases in neutrophil contact area with increasing chamber height (9-33 %) and increasing wall shear stress (28-56 %). Our results suggest that rolling velocity is dependent not only on wall shear stress but also on the shear stress gradient experienced by the rolling cell. These results point to the D(c)/H ratio as an important design parameter of leukocyte microfluidic assays, and should be applicable to rolling assays that involve other cell types such as platelets or cancer cells.

Vessel Rupture Thresholds for Vessel-Bubble Interactions Using an Earthworm Vasculature Model.

OBJECTIVE: Intravenous microbubble oscillation in the presence of ultrasound has the potential to yield a wide range of therapeutic benefits. However, the likelihood of vessel damage caused by mechanical effects has not been quantified as a function of the numerous important parameters in therapeutic ultrasound procedures. In this study, we examined the effects of microbubbles injected into the vasculature of the earthworm. It was found that the elastic properties of earthworm blood vessels are similar to those of arteries in older humans, and that earthworms are well suited to the large number of experiments necessary to investigate safety of procedures involving microbubble oscillation in sonicated vessels. METHODS: Microbubbles were infused into earthworm vessels, and the rupture time during sonication was recorded as a function of ultrasound frequency, pulse repetition frequency and acoustic pressure. DISCUSSION: A modified mechanical index (MMI) was defined that successfully captured the trends in rupture probability and rupture time for the different parameter values, creating a database of vessel rupture thresholds. In the absence of bubbles, the product of MMI squared and rupture time was approximately constant, indicating a possible radiation-force effect. CONCLUSION: The MMI was an effective correlating parameter in the presence of bubbles, though the mathematical dependence is not yet apparent. The results of the study are expected to be valuable in designing more refined studies in vertebrate models, as well as informing computational models.

Leukocyte rolling on P-selectin: a three-dimensional numerical study of the effect of cytoplasmic viscosity.

Rolling leukocytes deform and show a large area of contact with endothelium under physiological flow conditions. We studied the effect of cytoplasmic viscosity on leukocyte rolling using our three-dimensional numerical algorithm that treats leukocyte as a compound droplet in which the core phase (nucleus) and the shell phase (cytoplasm) are viscoelastic fluids. The algorithm includes the mechanical properties of the cell cortex by cortical tension and considers leukocyte microvilli that deform viscoelastically and form viscous tethers at supercritical force. Stochastic binding kinetics describes binding of adhesion molecules. The leukocyte cytoplasmic viscosity plays a critical role in leukocyte rolling on an adhesive substrate. High-viscosity cells are characterized by high mean rolling velocities, increased temporal fluctuations in the instantaneous velocity, and a high probability for detachment from the substrate. A decrease in the rolling velocity, drag, and torque with the formation of a large, flat contact area in low-viscosity cells leads to a dramatic decrease in the bond force and stable rolling. Using values of viscosity consistent with step aspiration studies of human neutrophils (5-30 Pa·s), our computational model predicts the velocities and shape changes of rolling leukocytes as observed in vitro and in vivo.

Drop-of-sample rheometry of biological fluids by noncontact acoustic tweezing spectroscopy.

Knowledge of rheological properties, such as viscosity and elasticity, is necessary for efficient material processing and transportation as well as biological analysis. Existing rheometers operate with large sample volume and induce sample contact with container or device walls, which are inadequate for rheological analysis of sensitive fluids limited in availability. In this work, we introduce acoustic tweezing spectroscopy (ATS), a novel noncontact rheological technique that operates with a single 4-6 µl drop of fluid sample. In ATS, a sample drop is acoustically levitated and then exposed to a modulated acoustic signal to induce its forced oscillation. The time-dependent sample viscosity and elasticity are measured from the resulting drop response. The ATS measurements of polymeric solutions (dextran, xanthan gum, gelatin) agree well with previously reported data. The ATS predicts that the shear viscosity of blood plasma increases from 1.5 cP at 1.5 min of coagulation onset to 3.35 cP at 9 min, while its shear elastic modulus grows from a negligible value to 10.7 Pa between 3.5 min and 6.5 min. Coagulation increases whole blood viscosity from 5.4 cP to 20.7 cP and elasticity from 0.1 Pa to 19.2 Pa at 15 min. In summary, ATS provides the opportunity for sensitive small-volume rheological analysis in biomedical research and medical, pharmaceutical, and chemical industries.

Pre-Exposure to Stress-Inducing Agents Increase the Anticancer Efficacy of Focused Ultrasound against Aggressive Prostate Cancer Cells.

Despite the initial success in treatment of localized prostate cancer (PCa) using surgery, radiation or hormonal therapy, recurrence of aggressive tumors dictates morbidity and mortality. Focused ultrasound (FUS) is being tested as a targeted, noninvasive approach to eliminate the localized PCa foci, and strategies to enhance the anticancer potential of FUS have a high translational value. Since aggressive cancer cells utilize oxidative stress (Ox-stress) and endoplasmic reticulum stress (ER-stress) pathways for their survival and recurrence, we hypothesized that pre-treatment with drugs that disrupt stress-signaling pathways in tumor cells may increase FUS efficacy. Using four different PCa cell lines, i.e., LNCaP, C4-2B, 22Rv1 and DU145, we tested the in vitro effects of FUS, alone and in combination with two clinically tested drugs that increase Ox-stress (i.e., CDDO-me) or ER-stress (i.e., nelfinavir). As compared to standalone FUS, significant (p < 0.05) suppressions in both survival and recurrence of PCa cells were observed following pre-sensitization with low-dose CDDO-me (100 nM) and/or nelfinavir (2 µM). In drug pre-sensitized cells, significant anticancer effects were evident at a FUS intensity of as low as 0.7 kW/cm2. This combined mechanochemical disruption (MCD) approach decreased cell proliferation, migration and clonogenic ability and increased apoptosis/necrosis and reactive oxygen species (ROS) production. Furthermore, although activated in cells that survived standalone FUS, pre-sensitization with CDDO-me and/or nelfinavir suppressed both total and activated (phosphorylated) NF-κB and Akt protein levels. Thus, a combined MCD therapy may be a safe and effective approach towards the targeted elimination of aggressive PCa cells.

Earthworm, Lumbricus Terrestris: A Novel Microinjection Vasculature In vivo Invertebrate Model.

Although vertebrates are indispensable to biomedical research, studies are often limited by factors such as cost, lengthy internal review, and ethical considerations. We present the earthworm as an alternative, low-cost, invertebrate applicable to certain preliminary vasculature studies. Due to the surgical availability of the earthworm's dorsal vessels, ventral vessels, and five pairs of pseudo hearts, earthworms are readily accessible, offer low-cost maintenance, and require administration of only small doses of a given compound. The earthworm model provides a simple closed vascular circulatory system with a hemoglobin structure similar to human blood. A protocol is provided for anaesthetizing the earthworms and performing surgical incisions to expose relevant blood vessels. Micropipettes for compound administration are formed by heating and pulling glass with a pipette puller and using a beveling system to create a micron-scale fine needle tip. The tips are then used with a micropositioner and microinjector to inject arbitrary compounds into the vascular system of an earthworm, repeatably, with the availability of large sample sizes and small compound volumes. Details on the intricacies of injection procedure are provided. The small vessel size of the earthworm is challenging, particularly in the case of the ventral vessel; however, mastery of the techniques presented offers high repeatability as a low-cost solution, making studies of very large sample size practical.

Cell trapping in Y-junction microchannels: A numerical study of the bifurcation angle effect in inertial microfluidics.

The majority of microfluidic technologies for cell sorting and isolation involve bifurcating (e.g., Y- or T-shaped junction) microchannels to trap the cells of a specific type. However, the microfluidic trapping efficiency remains low, independently of whether the cells are separated by a passive or an active sorting method. Using a custom computational algorithm, we studied the migration of separated deformable cells in a Y-junction microchannel, with a bifurcation angle ranging from 30° to 180°. Single or two cells of initially spherical shape were considered under flow conditions corresponding to inertial microfluidics. Through the numerical simulation, we identified the effects of cell size, cytoplasmic viscoelasticity, cortical tension, flow rate, and bifurcation angle on the critical separation distance for cell trapping. The results of this study show that the trapping and isolation of blood cells, and circulating tumor cells in a Y-junction microchannel was most efficient and least dependent on the flow rate at the bifurcation angle of 120°. At this angle, the trapping efficiency for white blood cells and circulating tumor cells increased, respectively, by 46% and 43%, in comparison with the trapping efficiency at 60°. The efficiency to isolate invasive tumor cells from noninvasive ones increased by 32%. This numerical study provides important design criteria to optimize microfluidic technology for deformability-based cell sorting and isolation.

Monitoring of blood coagulation with non-contact drop oscillation rheometry.

BACKGROUND: Thromboelastography is widely used as a tool to assess the coagulation status of critical-care patients. It allows observation of changes in the material properties of whole blood brought about by clot formation and clot lysis. However, contact activation of the coagulation cascade at surfaces of thromboelastographic systems leads to inherent variability and unreliability in predicting bleeding or thrombosis risks, while also requiring large sample volumes. OBJECTIVES: To develop a non-contact drop oscillation rheometry (DOR) method to measure the viscoelastic properties of blood clots and to compare the results with current laboratory standard measurements. METHODS: Drops of human blood and plasma (5-10 µL) were acoustically levitated. Acoustic field modulation induced drop shape oscillations, and the viscoelastic properties of the sample were calculated by measuring the resonance frequency and damping ratio. RESULTS: DOR showed sensitivity to coagulation parameters. An increase in platelet count resulted in an increase in the maximum clot stiffness. An increase in the calcium ion level enhanced the coagulation rate prior to saturation. An increase in hematocrit resulted in a higher rate of clot formation and increased clot stiffness. Comparison of the results with those obtained with thromboelastography showed that coagulation started sooner with DOR, but with a lower rate and lower maximum stiffness. CONCLUSIONS: DOR can be used as a monitoring tool to assess blood coagulation status. The advantages of small sample size, the lack of contact and small strain (linear viscoelasticity) makes this technique unique for real-time monitoring of blood coagulation.

Phenotypic alterations in liver cancer cells induced by mechanochemical disruption.

Hepatocellular carcinoma (HCC) is a highly fatal disease recognized as a growing global health crisis worldwide. Currently, no curative treatment is available for early-to-intermediate stage HCC, characterized by large and/or multifocal tumors. If left untreated, HCC rapidly progresses to a lethal stage due to favorable conditions for metastatic spread. Mechanochemical disruption of cellular structures can potentially induce phenotypic alterations in surviving tumor cells that prevent HCC progression. In this paper, HCC response to mechanical vibration via high-intensity focused ultrasound and a chemical disruptive agent (ethanol) was examined in vitro and in vivo. Our analysis revealed that mechanochemical disruption caused a significant overproduction of reactive oxygen species (ROS) in multiple HCC cell lines (HepG2, PLC/PRF/5, and Hep3B). This led to a decrease in cell viability and long-term proliferation due to increased expression and activity of death receptors TNFR1 and Fas. The cells that survived mechanochemical disruption had a reduced expression of cancer stem cell markers (CD133, CD90, CD49f) and a diminished colony-forming ability. Mechanochemical disruption also impeded HCC migration and their adhesion to vascular endothelium, two critical processes in hematogenous metastasis. The HCC transformation to a non-tumorigenic phenotype post mechanochemical disruption was confirmed by a lack of tumor spheroid formation in vitro and complete tumor regression in vivo. These results show that mechanochemical disruption inhibits uncontrolled proliferation and reduces tumorigenicity and aggressiveness of HCC cells through ROS overproduction and associated activation of TNF- and Fas-mediated cell death signaling. Our study identifies a novel curative therapeutic approach that can prevent the development of aggressive HCC phenotypes.

Mechanochemical Disruption Suppresses Metastatic Phenotype and Pushes Prostate Cancer Cells toward Apoptosis.

Chemical-based medicine that targets specific oncogenes or proteins often leads to cancer recurrence due to tumor heterogeneity and development of chemoresistance. This challenge can be overcome by mechanochemical disruption of cancer cells via focused ultrasound (FUS) and sensitizing chemical agents such as ethanol. We demonstrate that this disruptive therapy decreases the viability, proliferation rate, tumorigenicity, endothelial adhesion, and migratory ability of prostate cancer cells in vitro. It sensitized the cells to TNFR1-- and Fas--mediated apoptosis and reduced the expression of metastatic markers CD44 and CD29. Using a prostate cancer xenograft model, we observed that the mechanochemical disruption led to complete tumor regression in vivo. This switch to a nonaggressive cell phenotype was caused by ROS and Hsp70 overproduction and subsequent impairment of NFκB signaling. FUS induces mechanical perturbations of diverse cancer cell populations, and its combination with agents that amplify and guide remedial cellular responses can stop lethal cancer progression. IMPLICATIONS: Mechanochemical disruption therapy in which FUS is combined with ethanol can be curative for locally aggressive and castration-resistant prostate cancer.

Design of a system to measure light scattering from individual cells excited by an acoustic wave.

A system to measure light scattering from individual cells excited by an acoustic wave was designed, and tests were performed on live Jurkat cells. Cells passing in a laminar stream within a water bath were excited by a focused ultrasound pulse, while the scattered light from a laser beam was monitored at various scattering angles. The cells were modeled as viscoelastic liquid drops, which return to equilibrium via shape oscillations after an acoustically-induced deformation. The Fast Fourier Transform of the scattered light signal was used to extract information about the highly-damped resonant frequencies of the cells, and the detected frequencies are consistent with theoretical predictions.

Focused Ultrasound-Triggered Release of Tyrosine Kinase Inhibitor From Thermosensitive Liposomes for Treatment of Renal Cell Carcinoma.

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 FUS 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%, not significant) or TKI/TSL (71.0% ± 3.65%, p < 0.001) at 96 h 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 FUS in clinical practice make the proposed combination of targeted chemotherapy, nanotechnology, and FUS a promising platform for enhanced drug delivery and cancer treatment.

Oxidized low-density lipoprotein contributes to atherogenesis via co-activation of macrophages and mast cells.

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 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.