Non-Invasive Imaging of Tumors by Monitoring Autotaxin Activity Using an Enzyme-Activated Near-Infrared Fluorogenic Substrate

Damian Madan,Colin G Ferguson,Charles A Testa,Won Yong Lee,Glenn D Prestwich,Matthew Bogyo

doi:10.1371/journal.pone.0079065

Abstract

Autotaxin (ATX), an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA) from lysophosphatidylcholine (LPC). Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2) that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR) region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.

Highlights

Autotaxin (ATX) and its lipid product lysophosphatidic acid (LPA) are key players in cell proliferation and motility and are central to both normal physiology and the physiology of several diseases
LPA binds at least six G protein-coupled receptors (GPCRs) to trigger multiple downstream signaling cascades, such as activation of Rho and Ras small GTPases
Overexpression of ATX contributes to the pathophysiology of invasive cancers [4,5], and ATX levels are elevated in various cancers such as melanoma, breast cancer, renal cancer, non-small-cell lung cancer, neuroblastoma, hepatocellular carcinoma, glioblastoma multiforme, Hodgkin’s lymphoma, and thyroid cancer [6]

Summary

Introduction

Autotaxin (ATX) and its lipid product lysophosphatidic acid (LPA) are key players in cell proliferation and motility and are central to both normal physiology and the physiology of several diseases. To address the unmet need to non-invasively measure ATX activity in vivo we developed a near-infrared imaging probe, AR-2 (Figure 2). Fluorescence intensity correlated with ATX levels, and decreased signal was observed when animals were pretreated with an ATX inhibitor.

Results

Conclusion