Dual Reporter Bioluminescence Imaging with NanoLuc and Firefly Luciferase.

Bioluminescence imaging is a powerful, broadly utilized method for noninvasive imaging studies in cell-based assays and small animal models of normal physiology and multiple diseases. In combination with molecular engineering of cells and entire organisms using luciferase enzymes, bioluminescence imaging has enabled novel applications including studies of protein-protein interactions, ligand-receptor interactions, cell trafficking, and drug targeting in mouse models. We describe use of a novel luciferase enzyme derived from Oplophorus gracilirostris, NanoLuc, in cell-based assays bioluminescence imaging of tumor-bearing mice.

Functional Isolation of Tumor-Initiating Cells using Microfluidic-Based Migration Identifies Phosphatidylserine Decarboxylase as a Key Regulator.

Isolation of tumor-initiating cells currently relies on markers that do not reflect essential biologic functions of these cells. We proposed to overcome this limitation by isolating tumor-initiating cells based on enhanced migration, a function tightly linked to tumor-initiating potential through epithelial-to-mesenchymal transition (EMT). We developed a high-throughput microfluidic migration platform with automated cell tracking software and facile recovery of cells for downstream functional and genetic analyses.

Fluorescence Lifetime Imaging of a Caspase-3 Apoptosis Reporter.

Caspase-3 is a proteolytic enzyme that functions as a key effector in apoptotic cell death. Determining activity of caspase-3 provides critical information about cancer cell viability and response to treatment. To measure apoptosis in intact cells and living mice, a fluorescence imaging reporter that detects caspase-3 activity by Förster resonance energy transfer (FRET) was used.

Imaging Sensitivity of Quiescent Cancer Cells to Metabolic Perturbations in Bone Marrow Spheroids.

Malignant cells from breast cancer and other common cancers such as prostate and melanoma may persist in bone marrow as quiescent, non-dividing cells that remain viable for years or even decades before resuming proliferation to cause recurrent disease. This phenomenon, referred to clinically as tumor dormancy, poses tremendous challenges to curing patients with breast cancer. Quiescent tumor cells resist chemotherapy drugs that predominantly target proliferating cells, limiting success of neo-adjuvant and adjuvant therapies.

Fluorescence Lifetime Imaging of Apoptosis.

Genetically-encoded fluorescence resonance energy transfer (FRET) reporters are powerful tools to analyze cell signaling and function at single cell resolution in standard two-dimensional cell cultures, but these reporters rarely have been applied to three-dimensional environments. FRET interactions between donor and acceptor molecules typically are determined by changes in relative fluorescence intensities, but wavelength-dependent differences in absorption of light complicate this analysis method in three-dimensional settings. Here we report fluorescence lifetime imaging microscopy (FLIM) with phasor analysis, a method that displays fluorescence lifetimes on a pixel-wise basis in real time, to quantify apoptosis in breast cancer cells stably expressing a genetically encoded FRET reporter.