Strategies for Targeted Imaging of Leukocytes in the Lung

The pulmonary inflammatory response is a tightly regulated multicellular process geared towards protecting the host from invading organisms, yet chronic and exuberant inflammation is a hallmark of many pulmonary diseases including lung cancer, chronic obstructive pulmonary disease (COPD), and asthma. Neutrophils and macrophages are consistently identified as key effector cells in most pulmonary inflammatory diseases. As a result, there has been great effort directed towards understanding the roles of these leukocytes in the context of debilitating lung diseases. In the work presented here, we have focused on the development of molecular imaging based on Positron Emission Tomography (PET) that may facilitate noninvasive evaluation and monitoring of leukocytes in the lung.
I first explored the use of 2'-deoxy-2'-[18F]fluoro-D-glucose (FDG)-PET with compartmental model analysis to probe lung neutrophil metabolic activity. The difficulty with this approach in small animal applications is the requirement for measuring the blood input function during the PET scan, traditionally obtained by invasive means. Therefore, I have: 1) established a noninvasive method for deriving the blood input function directly from gated, dynamic PET images; and 2) determined the feasibility and accuracy of measuring neutrophil metabolic activity with FDG-PET by three-compartment model analysis with an image-derived input function in a mouse model of acute pulmonary inflammation. Based on estimates of the model parameters, the net FDG uptake (Ki) was estimated in lungs following bacteria challenge and was found to be 2.24 times higher compared to controls. Importantly, the mean lung Ki value computed here was not significantly different from previously reported lung values computed from input functions obtained by physical arterial sampling, validating our noninvasive approach.
The second strategy was based on the peptide N-cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF), which was previously reported to be an antagonist to the neutrophil formyl peptide receptor (FPR). We demonstrated that the bioavailability and blood clearance properties of the peptide ligand could be improved by polyethyleneglycol (PEG) conjugation without adversely affecting its binding affinity or neutrophil stimulatory properties. In vivo PET imaging was conducted in mice and the results of these studies demonstrated that this modified peptide has excellent potential for monitoring neutrophilic inflammation in the lungs and may be valuable in evaluating future ant-inflammatory therapies.
In addition to neutrophils, there is also much incentive to develop noninvasive molecular imaging agents that target macrophages. Macrophages, which play an important role promoting inflammatory disorders of the lung, consistently have been shown to exhibit predominantly a M2-like phenotype which can facilitate the growth and spread of lung tumors. The final chapter of this dissertation documents my attempts to target tumor-associated macrophages (TAMs) using liposomes surface-coated with a mannose ligand. This liposome modification has been shown to improve M2 macrophage recognition and internalization via mannose receptor-mediated endocytosis but has not been studied as a macrophage targeting agent for PET imaging applications. Initial studies conducted by us in vitro confirmed that mannosylated (Man3)-liposomes were significantly taken up more by M2 (IL4/13-stimulated) bone marrow-derived macrophages following incubation compared to M1 (LPS/IFN-γ-stimulated) cells. In vivo PET imaging studies were conducted in a mouse model of chemically-induced lung cancer and revealed that Man3-liposomes exhibited a 7-fold higher uptake in tumor tissue compared to normal lung tissue 6 h after intravenous injection. Subsequent ex vivo lung biodistribution and confocal microscopy studies confirmed uptake of Man3-liposomes by TAMs. Finally, co-injection studies revealed that Man3-liposomes exhibited the highest tumor contrast relative to normal lung tissue at the time point studied compared to plain or PEGylated liposomes.