Download Multi-scale Examination of Pancreatic Cancer Cell Biophysics

Multi-scale Examination of Pancreatic Cancer Cell Biophysics
Daniel J. Shea
Department of Chemical and Biomolecular Engineering
Konstantopoulos Lab, Johns Hopkins University
Pancreatic cancer resulted in nearly 42,000 deaths in 2016 making it the third
leading cause of cancer-related death in the United States. The high mortality rate stems
from the fact that treatment options are limited as less than 20% of patients are candidates
for surgical tumor removal because the disease is typically detected after it has
metastasized to other organs. Cancer metastasis is a multistep process where tumor cells
migrate from a primary tumor into the vasculature, where the circulating tumor cell can
then roll on and bind to the vascular epithelial wall before extravasating to a secondary
location. If this cascade is interrupted at any point, metastasis is abrogated. Given its
severity, understanding and treating pancreatic cancer metastasis is critically important in
reducing the high mortality rate associated with the disease.
Selectin-mediated tumor cell tethering and rolling on vascular endothelial cells is
a critical step in the process of cancer metastasis. We recently identified sialofucosylated
mucin16 (MUC16) and podocalyxin (PODXL) as the major functional E- and L-selectin
ligands expressed on the surface of metastatic pancreatic cancer cells. In this work we
explore the critical parameters of selectin-mediated pancreatic tumor cell tethering and
rolling. Using force spectroscopy, we characterized the binding interactions of MUC16
and PODXL to E- and L-selectin at the single-molecule level. To further analyze the
response of these molecular interactions under physiologically relevant regimes, we used
a microfluidic assay in conjunction with a mathematical model to study the biophysics of
selectin-ligand binding as a function of fluid shear stress. Next, we sought to evaluate the
critical parameters of the pancreatic tumor adhesion to hyaluronic acid (HA) mediated by
rolling on E-selectin in physiologically relevant flow. We demonstrate that the ligand-Eselectin interaction initiates adhesion to HA in a twofold manner, both physically slowing
cells and allowing cells to orient close to the surface, increasing the on-rate of adhesion.
We next looked to characterize the presence and absence of giant obscurins in
pancreatic cells and assess their effect on cell migration and cytoskeletal dynamics. Here
we establish that giant obscurin are depleted in PDAC tissue and metastatic pancreatic
cancer cell lines, but are expressed at high levels in normal pancreatic tissue and nontumorigenic human pancreatic ductal epithelial cells (HPDE).
Further, the loss of
obscurins from HPDE cells affects RhoA and PI3K/AKT activity which in turn leads to
major cytoskeletal alterations including increased actin dynamics at cell-cell junctions,
faster microtubule growth and decreased focal adhesion density.
Understanding the biophysics of selectin-ligand bonds and their responses to
physiologically relevant shear stresses as well as identifying biomarkers such as giant
obscurins that increase cell migration and cytoskeletal dynamics is vital for developing
diagnostic assays and/or preventing the metastatic spread of pancreatic tumor cells.
Advisor: Konstantinos Konstantopoulos