Intellectual Merit: Cell adhesion, mediated via highly specific and tightly controlled receptor-ligand (R-L) interactions, plays a pivotal role in diverse biological events. The kinetics of R-L binding imparts unique properties that allow cells to interact with one another amidst the challenge of physiological stresses, such as fluid shear in the vasculature. In the physiological setting, receptors and their respective ligands are anchored to the surfaces of apposing cells; thus, R-L binding is a two-dimensional (2D) process. Although sophisticated biophysical techniques have recently been developed to measure the unstressed 2D affinity of receptor-ligand pairs, they fail to disclose its dependence on applied force1,2. This dependence must be elucidated, however, as forces associated with fluid flow in the vasculature modulate the kinetics of R-L bonds mediating cell-cell adhesion. An integrated experimental and mathematical approach will be developed to determine the 2D kinetic constants of R-L interactions as a function of hydrodynamic shear. This approach exploits the concept of encounter time for the physicochemical reaction of R-L binding. In experiment, lithographic and microfluidic methods will be used to create zones of length L functionalized with proteins at controlled surface density and orientation. A flowing cell with velocity U and a protein-coated zone L will interact with the protein for an encounter time (ô=L/U), allowing the 2D receptor-ligand kinetic parameters to be determined from mathematical modeling. The model will be fully informed by the highly complex response of R-L bonds to applied shear force, with all parameters measured from accompanying experiments. In proof of concept experiments and analysis have identified key asymptotes that apply to the results of the proposed work.
The 2D kinetic and micromechanical properties of critical R-L interactions pertinent to pancreatic cancer metastasis will be studied herein. The proposed work is fundamental, addressing basic science issues, with potential results that can be leveraged in engineering devices and therapeutic interventions. The focus of this study is at the level of R-L biophysical characterization in terms of transport kinetics. Specifically, a mechanistic interpretation will be provided for two discrete adhesion steps (i.e., rolling and arrest) mediated by distinct R-L pairs: selectin binding to mucin 16 (MUC16) and podocalyxin-like protein (PCLP), and fibrin (ogen) binding to the standard form of CD44 (CD44s), respectively. This will be achieved by investigating the respective kinetic (e.g., 2D on- and off- rates) and micromechanical (e.g., tensile strength) properties of the aforementioned R-L pairs using single-molecule force-spectroscopy, microfluidics and micropatterning along with mathematical modeling. Moreover, a quantitative understanding of how selectin-ligand tethering facilitates CD44s-fibrin(ogen) mediated firm adhesion at elevated levels of shear stress will be developed. This study will determine the rate-limiting parameters in this cascade of events, such as the lengths of selectin- or fibrin(ogen)-coated zones and their site densities, necessary to support downstream firm adhesion by CD44s-fibrin(ogen) binding at elevated shear stress levels. Finally, this project will determine how these parameters are modulated by selective individual knockdown of MUC16 and PCLP. Experiments will be guided by computational modeling of cell rolling/adhesion.
Broader Impacts: Scientific/ Technological: This work will advance the knowledge in the field of cell biophysics. Specifically, this research will have broad impact in the basic scientific understanding of R-L processes that permeate biology. Knowledge of the kinetics of R-L-mediated cell adhesion in physiologically relevant settings will provide design parameters needed to engineer sensors, to target biological entities based on recognition, to design molecules to interrupt adverse or pathological adhesion events, such as those in cancer, and, generically, to interrogate biological events. These fundamental measurements will be performed in the specific context of pancreatic cancer. While this study focuses on a specific example, the experimental and mathematical framework developed in this project will be broadly applicable to other (patho)physiological processes occurring in the vasculature. Mentoring of Female and Under-represented Students: Students from outreach initiatives will be welcome to work on small research projects associated with this research. (PI's and co-PI's personal contacts, via laison with Baltimore Polytechnic high-school, Penn's ACS Project SEED, REU programs). Student Participation: Undergraduate/high school students regularly perform research in the PI's/co-PI's labs; often women or from underrepresented groups. Pre- and Post-doctoral mentoring: Pre- and post-doctoral career development is a priority in the PIs' laboratories.
