Research

The human body represents an exquisite feat of bioengineering.  Physical and biochemical stimuli actively regulate cell function.  Our research is directed at understanding how physical cues (i.e., confinement or fluid shear stress) regulate cell responses pertinent to cancer metastasis and inflammation using physiologically relevant in vitro and in vivo models.  This is accomplished through the synthesis of engineering and microtechnology principles with quantitative modeling and concepts from biophysics, biochemistry and molecular cell biology.

Some of the key research contributions by the Konstantopoulos lab are (1) the discovery of novel selectin ligands that mediate tumor cell adhesion in the vasculature, (2) the biophysical characterization of these adhesive interactions at the single-molecule level, and (3) the elucidation of novel signaling mechanisms during migration through physically confined microenvironments. Specifically, by integrating expertise in microfluidics, imaging, cell & molecular biology and mathematical modeling, we discovered a new mechanism of tumor cell migration in confined spaces that is driven by water permeation.

Cell Migration

migration-in-3-um-channel-9hoursCell motility is influenced by the complex interplay between signaling mechanisms and the physical cues of the microenvironment.  Much of what we know about the mechanisms of cell migration stems from in vitro studies using two-dimensional (2D), unconfined surfaces.  Cell locomotion in 2D is driven by cycles of actin protrusion, integrin-mediated adhesion and myosin-dependent contraction.  However,  2D assays of cell migration fail to recapitulate the physiological tissue environment.  In vivo, cells migrate  within 3D extracellular matrices and through pre-existing tissue tracks (longitudinal channels).  These channels form between the connective tissue and the basement membrane of epithelium, nerve or muscle.  We use photolithography to fabricate longitudinal channels of prescribed dimensions similar to those encountered in vivo.  Using our device, we can directly compare and contrast the mechanisms of 2D (unconfined) to confined migration.  We recently discovered a new mechanism of tumor cell migration in confined spaces that is driven by water permeation.  We are currently deciphering the intracellular signaling mechanisms used by normal and tumor cells during migration through confined microenvironments.

 

Cell Adhesion

Colon carcinoma cell adhesion to immobilized platelets in shear flow.The Konstantopoulos lab has made significant contributions to understanding how fluid shear affects the binding kinetics and receptor specificity of cell-cell adhesion events pertinent to cancer metastasis and inflammation/infection, from both an experimental and theoretical viewpoint.  Most notably, our studies have revealed a paradigm in which the coordinated action of a “rapid” selectin-dependent pathway followed by a “slow” integrin-mediated process is required for maximal binding of tumor cells to either blood platelets, polymorphonuclear (PMN) leukocytes or endothelial cells under physiological flow conditions.  We also extended the applicability of this two-step paradigm to the adhesion of Staphylococcus aureus to platelets pertinent to the process of bacterial infection, with the exception that distinct molecular constituents mediate the “rapid” tethering step.  These studies have paved the way for investigating the kinetic and mechanical properties of key receptor-ligand bonds at the single-molecule level using cutting-edge biophysical technologies, as well as isolating novel molecular structures from the surfaces of metastatic tumor cells and leukocytes that could represent potential therapeutic targets.

Nanoscale Mechanics

nanoscale mechanics

Schematic of a molecular force probe.

Receptor/ligand-mediated cellular interactions play a pivotal role in a number of diverse patho-physiological processes such as cancer metastasis, inflammation and thrombosis.  Characterizing the biophysical and biochemical nature of key receptor-ligand interactions will therefore provide a rational basis for designing potent, specific receptor antagonists to combat the aforementioned pathological processes.  Using single-molecule force spectroscopy, we probe in situ the kinetic and micromechanical properties of individual receptor-ligand bonds.  Moreover, the two-dimensional (2D) binding kinetic constants of receptor-ligand pairs are determined using a microfluidic device in conjunction with mathematical modeling.

 

 

Receptor Biochemistry

Blot rolling assay

Blot rolling assay

Selectins (E-, P- and L-selectin) facilitate metastasis and tumor cell arrest in the microvasculature by mediating specific interactions between selectin-expressing host cells and selectin ligands on tumor cells.  Selectins are known to recognize sialofucosylated oligosaccharides, such as sialyl Lewis x (sLex) and its isomer sLea.  sLex is absent from normal pancreas tissue, but its expression progressively increases with higher-grade pancreatic intraepithelial neoplasia lesions and pancreatic adenocarcinoma, and correlates with hepatic metastasis and poorer prognosis.  By identifying the major functional selectin ligands on pancreatic cancer cells, characterizing their biochemical properties, and delineating their pathophysiological function, we will provide the basis to engineer novel therapeutic agents that will selectively block tumor-host cell binding events, and thus interfere with metastatic spread without impairing other important selectin-mediated physiological processes.  Our studies may also identify targets for developing diagnostic assays and/or effective cancer cell drug-delivery therapies.  Using bioengineering, biochemical, molecular and cell biology techniques (e.g. blot rolling assays, 2D gels, tandem mass spectrometry, antibody production, immunoprecipitation studies, RNAi), we identify, isolate and characterize the major functional selectin ligands on metastatic pancreatic cancer cells.

Quantitative Modeling

cell_rolling_side_1

Simulation of cell rolling.

Simulation of Cell-Substrate and Cell-Cell Interactions in Shear Flow

We have developed the first 3-D computational model based on immersed boundary method to predict receptor-mediated rolling and adhesion of deformable cells in shear flow coupled to a Monte Carlo method simulating the stochastic receptor-ligand interactions.  We have extended this line of research to cell-cell interactions in bulk suspensions.

 

 

Intracellular Signaling

Proposed cascade of signaling event in human T/C-28a2 chondrocytic cells subjected to high shear stress.

Proposed cascade of signaling event in human T/C-28a2 chondrocytic cells subjected to high shear stress.

Elucidation of Intracellular Signaling Pathways
Cells of the human body utilize feedback control mechanisms which are known to act on the level of DNA, RNA and protein, in order to maintain and optimize cell metabolism and function.  These control mechanisms are commonly referred to as signaling pathways.  When cellular homeostasis is disrupted, various signaling pathways are activated by distinct or common stimuli in an attempt to return cellular conditions to a basal state.  Disease states typically arise when signaling pathways malfunction, and therefore provide excellent targets for therapeutics.  However, due to the non-linear nature of signaling pathways coupled with the limits of current discovery techniques, a majority of active signaling pathways remain unknown.  In addition, those pathways that have been investigated remain only partially elucidated, thus making therapeutic discovery very challenging.  The process of drug discovery and design may be significantly simplified if potentially therapeutic signaling pathways are identified and manipulated using artificial or natural inducers or inhibitors.  To this end, our overall goal is to delineate dominant signal transduction pathways directly involved in the onset of a diseased cellular state. In particular, we aim to identify regulatory molecules (proteins, microRNAs etc) that may serve as potential therapeutic targets using computational functional genomics coupled with sophisticated molecular biology techniques.