Receptor Mediated Adhesion of Circulating Cells In Immune Cell Interaction and Cancer
Circulating tumor cells (CTCs) disseminating from primary tumor sites travel to distant organs and form secondary tumor sites, known as metastasis. After leaving the primary tumor microenvironment, CTCs enter the circulatory system where they routinely interact with immune cells. This interaction can paradoxically result in both limiting or promoting the metastatic potential of CTCs. The final steps of metastasis involve CTCs invaginating into the underlining tissue and forming secondary tumor sites. One plausible mechanism of CTCs leaving the circulatory system involves a similar process to leukocyte homing - selectin-mediated interaction. The scope of the work presented here can be divided into two parts: (a) exploiting the naturally occurring immune response called NETosis when neutrophils come into contact with cancer cells as a form of cancer therapy, and (b) gaining a better understanding of the mechanism of selectin- ligand interaction that playing a pivotal role in cancer metastasis and leukocyte homing. Immunotherapy is an emerging powerful clinical strategy for cancer therapy. NETosis is an innate immune response elicited by activated neutrophils to fight microbial infections. Activated neutrophils release DNA fibers decorated with anti-microbial proteins called neutrophil extracellular traps (NETs) into the extracellular space to trap and kill surrounding microbes. Here, we show tumor-derived IL-8 released by cancer cells also activates the release of NETs. Until now, there have been no existing technologies that leverage NETs as an anti-tumor drug delivery vehicle. In this study, we describe the re-engineering of neutrophils to express an apoptosis-inducing chimeric protein, supercharged eGFP-TRAIL, on NETs that can ensnare and kill tumor cells while retaining all of their anti-microbial capabilities. We observed significant TRAIL- induced apoptosis in tumor cells captured by TRAIL-decorated NETs. This work demonstrates NETs as a promising technology to deliver protein in response to local cytokine signals. The 3-member (E-, P-, L-) selectin family of cell adhesion molecules facilitates initial leukocyte tethering and subsequent cell rolling during the early stages of the inflammatory response via binding to glycoproteins expressing sialyl LewisX and sialyl LewisA (sLeX/A) to sites of inflammation and trauma. The extracellular microenvironments at these sites often become acidic. We investigated the influence of slightly acidic pH on the binding dynamics of selectins (P-, L-, and E-selectin) to P- selectin glycoprotein ligand-1 (PSGL-1) via computational modeling (molecular dynamics) and experimental rolling assays under shear in vitro. The P-selectin/PSGL-1 binding is strengthened at acidic pH, as evidenced by the formation of a new hydrogen bond (seen computationally) and the observed decrease in the rolling velocities of model cells. In the case of L-selectin/PSGL-1 binding dynamics, the binding strength and frequency increase at acidic pH, as indicated by the greater cell-rolling flux of neutrophils and slower rolling velocities of L-selectin-coated microspheres, respectively. The cell flux is most likely due to an increased population of L-selectin in the high-affinity conformation as pH decreases, whereas the velocities are due to increased L-selectin/PSGL-1 contacts. In contrast to P- and L-selectin, the E- selectin/PSGL-1 binding does not exhibit significant changes at acidic pH levels, as shown both experimentally and computationally. We also investigated the allosteric influence of E-selectin’s structure to ligand binding mechanics. Existing crystal structures of the extracellular lectin/EGF-like domain of E- selectin complexed with sLeX have revealed that E-selectin can exist in two conformation states, a low affinity (bent) conformation, and a high affinity (extended) conformation. The differentiating characteristic of the two conformations is the interdomain angle between the lectin and the EGF-like domain. Using molecular dynamics (MD) simulations we observed that in the absence of tensile force E-selectin undergoes spontaneous switching between the two conformational states at equilibrium. A single amino acid substitution at residue 2 (serine to tyrosine) on the lectin domain favors the extended conformation. Steered molecular dynamics (SMD) simulations of E-selectin and PSGL-1 in conjunction with experimental cell adhesion assays show a longer binding lifetime of E-selectin (S2Y) to PSGL-1 compared to wildtype protein. The findings in this study advance our understanding into how the structural makeup of E-selectin allosterically influences its adhesive dynamics
TRAIL; immunotherapy; netosis; selectin mechanics; Bioengineering
King, Michael R.
Delisa, Matthew; Paszek, Matthew J.
Ph.D., Biomedical Engineering
Doctor of Philosophy
dissertation or thesis