Using experimental and theoretical approaches, we 1) develop acoustic technologies for cancer treatment, blood coagulation monitoring, and nerve regeneration and 2) study the interactions of blood cells (leukocytes, platelets, red blood cells), tissue resident cells (macrophages, mast cells), and circulating tumor cells with vascular and lymphatic endothelium under pathophysiological conditions such as inflammation, atherosclerosis, thrombosis, and cancer metastasis. We also develop novel methods for rheological characterization of living cells and tissues and use our state-of-the-art computational fluid dynamics models to predict blood flow in vessels with complex geometry. Below you will find a short description of our current research projects.


Figure 1a. Bioflux 200 system installed in the laboratory
Bioflux 200

Figure 1b. Two wells of a Bioflux 48-well plate connected by a microchannel. We grow a confluent layer of endothelial cells in a viewing part of the microchannel
Bioflux 200

Figure 2. VECAM simulation of active deformation of the cell during its entrance into a pore of a square cross section
VECAM

Figure 3. Snapshot of cells migrating in a rectangular micro-channel, according to our numerical simulation. A bigger and less deformable red-colored cell is located closer to the wall than smaller and more deformable green-colored cells.
migration

Figure 4. LPS-activated monocytes anchor breast cancer cells (green) to vascular endothelium
TNF-alpha and histamine

Figure 5. Adhesion of GP Ibalpha-coated microbeads (A,B) and human platelets (C,D) to resting and histamine-stimulated endothelium
Platelet adhesion

Project 1: Quantitative Biomechanical Models of Circulating Cell Interactions

Students involved: Hongzhi Lan, Zhongyi Sheng and Scott Hymel (computational work); Sabrina Lynch and Radhika Josi (experimental work)

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 (dysfunctional) 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. Currently, it is not well understood how endothelial dysfunction and associated leukocyte adhesion develop in the body and how these pathophysiological processes can be prevented or blocked without causing dangerous side effects. In our laboratory, we study the adhesion of leukocytes and other cirulating cells to dysfunctional vascular or lymphatic endothelium 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 simulate both passive and active deformation of adherent cells (Fig. 2) as well as cell adhesion to a compliant substrate. Our computational algorithm can simulate the dynamics of multiple circulating cells with different deformability and size (Fig. 3).

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 (one type of circulating leukocytes) to endothelial cells and accumulation of monocytes/macrophages in the intimal layer of the arterial wall. To identify pro-atherogenic 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 mediators from mast cells and macrophages in endothelial dysfunction and monocyte adhesion.

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. Our recent data showed that LPS-treated monocytes enhanced the arrest of breast cancer cells by anchoring the cancer cells to activated endothelial cells (Fig. 4). We also investigate the effects of dendritic cell interactions with breast cancer cells on activation of lymphatic endothelium, including morphological changes and circulating cell adhesion. The results of our recent experiments indicate that upon exposure to the supernatant of the mixture of dendritic and breast cancer cells, the lymphatic endothelial cell contract. This leads to the formation of gaps in endothelium needed for intravasation.

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. 5) and subsequent growth of a thrombus. We also investigate the process of aggregation of platelets on tumor cells, leading to tumor thrombi that protect circulating tumor cells from recognition by the immune system.

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 systems and computational modeling.



Figure 6. Photograph of the HIFU system installed in the lab
HIFU

Figure 7. Gallery of HepG2 spheroids cultured in 5 mm diameter PDMS wells
spheroid

Figure 8. A mouse with human thyroid cancer xenografts on the left and right sides of the flank. Photograph was taken 3 weeks after treatment. The left xenograft, which was originally smaller than the right xenograft, continues to grow after ethanol treatment. The right xenograft was completed destroyed by ethanol + HIFU treatment, with only a small scar left.
mouse

Project 2: Acoustic Technologies (HIFU, LIFU, acoustic levitation)

Students involved: Hakm Murad, Kevin Luo and Vivien Lu

Focused ultrasound system. Our laboratory is equipped with a fully functional focused ultrasound system (Fig. 6). We built it in 2012, with funding received from the Louisiana Board of Regents/National Science Foundation and Tulane University Senate Committee on Research. It operates at 1.1 MHz or 3.3 MHz frequency, a continuous or pulsed mode, and a broad range of focal acoustic intensities (from 70 to 6000 W/cm2), i.e., it can be used for both low- and high-intensity focused ultrasound (LIFU and HIFU) applications. Our focused ultrasound system is the only system available at Tulane University to conduct preclinical HIFU or LIFU studies. Our current focus is to investigate the tumor destruction (ablation) by HIFU in tissue-mimicking phantoms, excised tissues, multicellular tumor spheroids (Fig. 7), and in vivo (i.e., in mouse xenograft models) (Fig. 8). In this preclinical research, we test HIFU ablation of thyroid, liver, and prostate cancers and collaborate with three clinicians from the Tulane University School of Medicine (Drs. Kandil, Buell, and Lee). We anticipate using this equipment for other applications such as HIFU/LIFU-based adjuvant therapy for cardiovascular disorders and cancer or the LIFU stimulation of nerve regeneration after nerve injury.

Tumor ablation by HIFU and ethanol. Image-guided tumor ablation is a minimally invasive treatment option that can compliment and, in certain cases, replace resection of localized tumors. Ablation can be done via focal deposition of electromagnetic or acoustic energy causing tissue heating and necrosis (thermal ablation) or via local injection of a chemical agent that can directly destruct tissue cells (chemical ablation). The noninvasive nature of HIFU offers an important convenience and benefit to cancer patients over other thermal ablation modalities (e.g., radiofrequency ablation, RFA). In my laboratory, we work on the development of anticancer therapy in which HIFU is synergistically complemented with other therapeutic approaches. Our experimental data showed that one of the therapeutic approaches that can be synergististically combined with HIFU is percutaneous ethanol injection (Fig. 8).

LIFU for nerve regeneration. One interesting extension of our focused ultrasound research is the use of LIFU for nerve regeneration. In rat models of sciatic nerve injury, low-intensity ultrasound was shown to increase nerve fiber density, stimulate rapid nerve regeneration in biodegradable Schwann cell-lined conduits implanted at a site of the injury, and improve the outcomes of functional tests such as sciatic functional index. According to in vitro data, low-intensity ultrasound first causes neurite retraction and cell body shrinkage, which in turn favors new neurite formation and nerve fiber regeneration. The exact mechanism by which ultrasound activates neurons is unknown, and no clinical studies or in vitro experiments with human neurons were conducted to test this therapeutic effect of ultrasound. We use our focused ultrasound system to find the conditions necessary for optimal growth of nerves in vitro and in vivo. In this effort, our laboratory collaborates with the laboratory of Prof. Moore (Biomedical Engineering, Tulane University).

Acoustic levitation. We are in process of building an accoustic levitation system that will be used as a diagonstic tool for various biomedical applications including blood coagulation. Other details cannot be shared because this project is "know-how".


Figure 9. Ensemble-averaged MSD vs. lag time and measured viscosity for 5% dextran-water solution. The data of three independent experiments agree with each other after deterministic motion cancellation (DMC). There is a close agreement between the high-shear-rate apparent viscosity of 5% dextran-water solution, measured with Advanced Rheometer 2000 (TA Instruments), and the value of viscosity measured with the MPTM with DMC.
MPTM1


Figure 10. Giant intracranial aneurysm in the vertebrobasilar system of a diseased patient (LEFT) and the geometry of the corresponding computational model (RIGHT).
levitation

Project 3: Advanced Methods for Rheological Characterization of Biological Materials
Students/postdocs involved: Kevin Luo

In this project, my interest is to develop state-of-the-art methods for accurate rheological measurement of circulating cells (e.g., leukocytes, platelets, tumor cells), blood and other biological fluids, and reactive materials which properties can change with time due to polymerization dynamics and other active processes. Specifically, we are developing the following rheological methods: 1) acoustic tweezing rheometry (details are not given because of "know-how"), 2) multiple-particle-tracking microrheology for live cell measurements, 3) slotted rotor/slotted plate techniques for accurate characterization of blood rheology. We also develop computational models for the flow of blood and other complex fluids, characterized by non-Newtonian viscoelastic properties, in channels of different geometry (e.g., corresponding to the geometry of rheometric systems, capillary sprouts, and aneurysm sacs).

Multiple-particle-tracking microrheology (MPTM) is a noncontact method well suited for measurement of local rheological properties of biological materials including circulating cells. Here, spherical particles of small size are suspended (or they can be naturally present) in a quiescent sample. 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 of the sample. 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. The Stokes-Einstein theory works only if we neglect hydrodynamic interactions between particles. Another issue is that it is impossible 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. Our laboratory works on the development of the improved version of MPTM that implements the “deterministic motion cancellation” algorithm in analysis of the experimental datamicr. We have conducted microrheological experiments with different fluids (water, dextran-in-water and agarose-in-water solutions) and showed that the deterministic motion cancellation led to the values of the shear viscosity of these solutions that agreed much better with the published data than the values measured by conventional MPTM (Fig. 9).

Slotted rotor / slotted plate techniques. To reduce wall slip effects on rheological measurement, we work on a novel design of a rotational rheometer in which the rotor has open slots. We also develop a slotted plate device for sensitive measure of blood yield stress and viscosity. In collaboration with Dr. De Kee (Prof. of Chemical & Biomedical Engineering at Tulane University), we have tested these ideas via computational fluid dynamics (CFD) simulations. Our computational work indicates that 1) a rheometer equipped with a slotted rotor can measure the fluid properties with enhanced accuracy and less sensitivity to the wall slip velocity than a rheometer with a non-slotted rotor; 2) a double concentric cylinder rheometer with slotted rotor (DCCR/SR) is able to accurately measure rheological properties of a wider spectrum of test fluids than a vane rheometer because of significant reduction of the end and secondary flow effects; and 3) wall slip effects and pressure drag force can be substantially reduced by adopting a thin plate with sharp front and rear edges, high slot area ratio, and large number of slots, thus increasing sensitivity of the slotted plate device to yield stress measurement. Using these data, we plan to develop prototypes of slotted plate and slotted rotor rheometers for accurate measurement of rheological properties of human blood. To complete these tasks, we have established collaboration with TA Instruments, a leading manufacturer of rheometric systems.

CFD modeling of blood flow. We have developod computational models of blood flow that predict the distribution of fluid shear stresses in capillaries with sprouts. In these models, we use sprout geometries typical for microvasculature and considered non-permeable and permeable vessel wall conditions. We also have computational models that simulate blood flow in the human vertebra-basilar 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 formed as a result of coalescence of two aneurysms, Fig. 10). The geometry of these models (Fig. 10) was reconstructed from the images of the vessels extracted from deceased patients. Using the models, we have determined "weak spots" in the vessel walls at which new aneurysms can develop. We also studied whether surgical treatment of intracranial aneurysms (e.g., filling an aneurysm with wires or Onyx solution) may lead to further complications. Additionally, we showed that 1) yield stress fluids can be used for aneurysm embolization provided the yield stress value is 20 Pa or higher, 2) there exists a strong flow recirculation in the empty aneurysm which is reduced by coil treatment but can be minimized by yield stress fluid treatment, and 3) the size of the inflow jet impingement zone on the aneurysm wall is reduced most considerably when the aneurysm is filled with a yield stress fluid material.