Luminescence Lifetime Imaging Microscopy For Analysis Of Oxygen In The Cancer Cell

Abstract

Molecular oxygen (O2), the key component of aerobic metabolism, plays important roles in normal and cancer cells and tissues. A real‐time and quantitative O2 imaging in 3D cell models would allow to discriminate complex effects of hypoxia on cell function in complex (heterogeneous) cell populations at subcellular resolution. Here, we used luminescence lifetime imaging (FLIM, PLIM) to develop and evaluate phosphorescent O2 probes with 3D spheroid and multi‐cellular aggregate models. We tested rat embryonic neurosphere culture with cationic nanoparticle O2 probes (MM2, NanO2) and developed O2 imaging protocol, multiplexed with analysis of distribution of specific protein markers such as HIF‐2α. To address the problem of efficient in‐depth staining, we designed two novel intracellular O2 probes: small molecule Pt‐Glc and nanoparticle PA2. Both probes displayed high analytical performance (brightness, photostability, O2 calibration) and were evaluated with rat pheochromocytoma PC12 and human colon cancer HCT116 cell aggregates. Resting cells cultured in 3D aggregates of >50 μm size displayed significant deoxygenation even under ambient atmospheric O2 levels. Still, they showed high viability and experienced different levels of oxygenation, upon treatment with mitochondrial uncouplers, inhibitors or pharmacological drugs. This data confirms the importance of probing of cellular O2 for experiments with 3D cell models. When combined with other live cell imaging dyes, such as markers of necrosis and apoptosis, the method allows for multi‐parameter quantitative assessment of cell physiology within relevant 3D micro‐environment.Supported by Science Foundation Ireland (SFI) grant 13/SIRG/2144.