Project 1. Quantitative Biomechanical Models of Circulating Cell Interactions with Applications in Inflammation, Atherosclerosis, Thrombosis, Sickle Cell Disease, and Cancer Metastasis

Funding: Skolkovo Foundation, Louisiana Board of Regents, Newcomb College Institute

Students involved: Hongzhi Lan (leukocyte migration modeling), Carol Chen (experimental work: leukocyte and tumor cell adhesion assays), Gisele Calderon (experiments on breast cancer cell interactions with monocytic and endothelial cells), You Lu (modeling thrombus formation and rupture), Teddy Brown and Sabrina Lynch (experimental work: platelet adhesion), Zerick Dunbar (experimental work: sickle cell adhesion)

Inflammation. Blood-borne leukocytes (white blood cells) are the first line of defense against invading pathogens. They are recruited from peripheral blood into infected tissues during inflammation through a complex series of events involving leukocyte capture by activated endothelial cells, leukocyte rolling on and firm adhesion to endothelium, and leukocyte transendothelial migration (diapedesis). These events, collectively known as leukocyte extravasation or leukocyte adhesion cascade, are mediated by the interplay of inflammatory mediators and cell adhesion molecules of the selectin and integrin families. In our laboratory, we study the leukocyte adhesion cascade by using in vitro systems (a parallel-plate flow chamber and Bioflux 200 microfluidic shear flow system that permits up to 24 cell adhesion assays in parallel, Fig. 1) and our custom three-dimensional computational algoritm of deformable cell adhesion, known as VECAM (ViscoElastic Cell Adhesion Model). VECAM can predict both passive and active deformation of adherent cells (Fig. 2) as well as cell adhesion to a compliant substrate. Understanding the leukocyte adhesion cascade is critical to the development of therapies for inflammatory disorders.

Atherosclerosis, a leading cause of myocardial infarction and stroke, is now recognized as a chronic inflammation in the walls of arteries. This condition develops as a result of oxidative damage to vascular endothelium, leading to increased adhesion of monocytes to endothelial cells and accumulation of monocytes/macrophages in the intimal layer of the arterial wall. To identify proatherogenic factors (chemicals, flow disturbances) and thus to develop the therapy targeted to atherosclerosis, we study adhesive interactions of human monocytes with human vascular endothelial cells activated by a combination of factors such as oxidized low-density lipoprotein (OxLDL), TNF-alpha, histamine, LPS, and serotonin. We are interested to understand the interplay between mediators released from tissue resident macrophages and mast cells in the development of cardiovascular disease. Our preliminary study indicate a synergy between TNF-alpha (released from macrophages) and histamine (released from mast cells) in monocyte-endothelium adhesion (Fig. 3). In this effort, we collaborate with Prof. D. Neil Granger (LSU Health Sciences Center at Shreveport).

Thrombosis. The adhesion and accumulation of platelets (thrombocytes) on collagen at a site of vascular injury result in a blood clot that seals the injured vessel. However, interactions of platelets with intact but dysfunctional endothelium cause the formation of a thrombus that occludes the blood flow. Arterial thrombosis developed at atherosclerotic plaques in large vessels supplying oxygen to the heart and brain is especially dangerous. This is because it may lead to the rupture of the plaque from underlying tissue and blockage of arteries downstream, thus causing myocardial infarction or stroke. Our interests are in understanding how inflammatory mediators (e.g., histamine and TNF-alpha) influence platelet adhesion to vascular endothelium (Fig. 4) and subsequent growth of a thrombus.

Sickle cell disease. Red blood cells that carry oxygen to distant tissues in the body becomes more rigid and sticky and change their shape in patients with sickle cell disease (SCD) due to aggregation of the abnormal hemoglobin molecules (hemoglobin-S) inside the cells. Interactions of sickle red blood cells with vascular endothelial cells and leukocytes are the main reason why SCD patients have severe vaso-occlusive pain episodes and low life expectancy. We study these interactions using our microfluidic flow systems and computational modeling.

Cancer Metastasis. Tumor cells switch to a motile, metastatic phenotype when they break away from their primary tumor. This leads to their migration into blood or lymphatic vessels (intravasation) and then to their interactions with the endothelium and migration across the endothelium into tissue at distant sites (extravasation), where tumor cells form metastases. We conduct both experimental and computational studies to elucidate the factors that influence the interactions of circulating tumor cells with leukocytes and vascular and lymphatic endothelium.

Project 2. Cancer Ablation by High-Intensity Focused Ultrasound (HIFU)

Funding: Louisiana Board of Regents and Tulane University Provost's Office

Students involved: Carol Chen and Nguyen Hoang (HIFU experiments), Sithira Ratnayaka, Taylor Hillburn and Uchenna Onwuegbusi (tumor spheroid culture)

Motivation. Image-guided tumor ablation is a non-surgical treatment option for cancer. In this approach, the destruction of cancerous masses is achieved through direct, local application of a chemical agent or through localized heating of tumor tissue via the absorption of the electromagnetic or acoustic wave energy. Although the existing tumor ablation modalities treat well small tumors, they are largely ineffective for metastases and single tumor lesions greater than 5 cm in size. The overall goal of this project is to develop a novel method for controlled ablation of large and metastatic tumor masses in which high intensity focused ultrasound (HIFU) will be complemented with the techniques that reduce the cavitation threshold locally in the tumor. Our current focus is on the combination of HIFU and percutaneous ethanol injection (PEI). The proposed activities may ultimately provide a non-surgical treatment option for patients with late-stage cancer.

Research progress. Our laboratory has a fully functional HIFU system (Fig. 5). We conduct 1) tissue phantom experiments, 2) in vitro studies with multicellular tumor spheroids, 3) ex vivo studies with tumor tissue specimens, and 4) computational modeling the effect of acoustic cavition on tumor ablation. In collaboration with Dr. Myers' laboratory in the Center for Devices and Radiological Health at U.S. Food & Drug Administration, we have performed preliminary experiments for this project. In this study, hydrogel-based tissue-mimicking material (TMM) phantoms and excised bovine liver samples (Fig. 6) were treated with ethanol (Fig. 7) and insonated with a focused ultrasound transducer. The results of this study indicate that the treatment of tissue phantom and bovine liver with ethanol reduces the cavitation threshold in these materials. As a result of the increased cavitation bubble activity, a sudden rise in temperature in ethanol-treated samples occurred at a lower acoustic power than that in untreated ones (Fig. 8). We established collaboration with clinicians at Tulane University School of Medicine (Drs. Buell, Kandil, and Lee) to begin translational studies of PEI-HIFU ablation of liver, thyroid, kidney, and prostate cancers.

Project 3. Computational Models of Intracranial Aneurysm Growth and Rupture

Students/postdocs involved: Weixiong Wang

In this project, we use computational fluid dynamics to study blood flow in the human vertebrobasilar system (ones of the major arteries in the brain) under normal conditions and in the presence of one or several aneurysms (including the case of a giant aneurysm that forms as a result of coalescense of two aneurysms, Fig. 9). The geometry of our computational models is reconstructed from the images of the vessels extracted from deceased patients in the laboratory of our collaborator (Dr. Arthur Ulm) in the Department of Neurosurgery at the LSU Health Sciences Center. Using our model, we determine "weak spots" in the vessel walls at which new aneurysms can develop. We also study through numerical simulations whether surgical treatment of intracranial aneurysms (e.g., filling an aneurysm with wires or Onyx solution) may lead to further complications.

Students/postdocs involved: Susan Teng (microrheology), Weixiong Wang (microrheology, DCCR/SR)

Multiple-particle-tracking microrheology (MPTM) is a noncontact rheological method based on the Stokes-Einstein theory of Brownian motion. In this method, spherical particles of small size are suspended (or they can be naturally present) in a quiescent test fluid. The trajectories of these particles are recorded by a high-speed camera (attached to a microscope). Assuming the particles undergo random motion, the Stokes-Einstein relationship is applied to the mean-square-displacement (MSD) vs. lag time data to determine the rheological properties (shear viscosity, relaxation times, etc.) of the test fluid (Fig. 10). A big advantage of this method is it does not introduce significant disturbances to fluid structure during measurement. This is extremely important for biological systems because they can be easily activated when interacting with solid surfaces of conventional rheometric systems (including also a micropipette aspiration system). If the test fluid has endogenous particles (leukocytes, for examples, has granules that can be tracked), MPTM does not lead to any changes in the fluid properties. As a result, this method is well suited for rheological measurements of leukocytes and other living cells and is the only one that can be used to directly measure cellular biomechanics in vivo. However, the biggest issue with MPTM is the absence of the theory that adequately describes particles' trajectories. This leads to significant errors in rheological measurements with MPTM, especially when studying living cells. First, the Stokes-Einstein theory works only if we neglect hydrodynamic interactions between particles. Second, it is very difficult if possible to completely eliminate the deterministic motion of a test fluid during measurement because of thermal convection of the fluid, fluctuations and inclination of the experimental platform, and active transport of particles as, for example, in the case of cytoskeleton remodeling. In our laboratory, we conduct MPTM measurements of different biological fluids (hydrogels, cytoskeletal protein solutions, leukocytes) and develop advanced models that account for anomalous diffusion associated with determinstic motion of suspended particles. We already showed that our modification of the MPTM leads to the values of shear viscosity of water and dextran solutions that agree much better with with the published data than the values measured by conventional MPTM (Figs. 11 and 12). Our overall goal in this subproject is to develop the microrheological method that is able to accurately measure rheology of living cells including changes in rheological properties with cytoskeleton remodeling in activated cells.

Acoustc tweezing rheometry. Another noncontact rheological method we develop in our laboratory utilizes an acoustic levitation technique (Fig. 13). This approach is similar to optical tweezers but here we use acoustic radiation pressure generated by a standing ultrasound field to balance the gravitational force. By modulating the acoustic wave amplitude at low frequency, we are able to induce shape oscillations of a spheroidal sample trapped in the air gap between the levitation transducer and reflector. We have previously developed the theory that relates the fluid rheology and its shape oscillation. This theory as well as direct numerical simulation are used in this subproject to extract rheological properties from the shape deformation of levitating samples. Our plan is to adapt this noncontact method for measurement of blood clot rheology and then use it for rheological measurements of other biolgical materials. Our collaborator (Dr. R. Glynn Holt, Department of Mechanical Engineering at Boston University) and we have already developed several protocols for the generation of spheroidal blood clot samples from coagulating bovine blood and showed that these samples can be forced into shape oscillations during acoustic levitation. Knowledge of blood clot/thrombus rheology is critically important for the development of appropriate strategies for dissolution of attached or dislodged thrombi that can block small arteries downstream leading to heart attach or stroke.

Double concentric cylinder rheometer with slotted rotor (DCCR/SR). In this effort, we colalborate with Dr. De Kee's laboratory (Department of Chemical & Biomolecular Engineering, Tulane University) on modification of the double concentric cylinder rheometer to reduce its systematic errors due to wall slip, secondary flow, and end effects. Specifically, we propose a novel design of this rheometer in which the rheometer rotor is perforated with slots. We showed through computational fluid dynamics simulations that our design is able to accurately measure rheological properties of suspensions including blood. Now, our tasks are 1) a parameter sensitivity analysis and design optimization; 2) fabrication and testing of a prototype of the optimal rheometer design, and 3) application of the prototype for rheological measurement of biological fluids. To complete these tasks, we have established collaboration with TA Instruments, a leading manufacturer of rheometric systems.