Interrogation of tumor metabolism in tissue samplesex vivousing fluorescence lifetime imaging of NAD(P)H

Abstract

Exploring metabolism in human tumors at the cellular level remains a challenge. The reduced form of metabolic cofactor NAD(P)H is one of the major intrinsic fluorescent components in tissues and a valuable indicator of cellular metabolic activity. Fluorescence lifetime imaging (FLIM) enables resolution of both the free and protein-bound fractions of this cofactor, and thus, high sensitivity detection of relative changes in the NAD(P)H-dependent metabolic pathways in real time. However, the clinical use of this technique is still very limited. The applications of metabolic FLIM could be usefully expanded by probing cellular metabolism in tissues ex vivo. For this, however, the development of appropriate tissue preservation protocols is required in order to maintain the optical metabolic characteristics in the ex vivo sample in a state similar to those of the tumor in vivo. Using mouse tumor models of different histological types—colorectal cancer, lung carcinoma and melanoma—we tested eight different methods of tissue handling by comparing NAD(P)H fluorescence decay parameters ex vivo and in vivo as measured with two-photon excited FLIM microscopy. It was found that the samples placed in 10% BSA on ice immediately after excision maintained the same fluorescence lifetimes and free/bound ratios as measured in vivo for at least 3 hours. This protocol was subsequently used for metabolic assessments in fresh postoperative samples from colorectal cancer patients. A high degree of inter- and intra-tumor heterogeneity with a trend to a more oxidative metabolism was detected in T3 colorectal tumors in comparison with normal tumor-distant colon samples. These results suggest that the methodology developed on the basis of FLIM of NAD(P)H in tissues ex vivo show promise for interrogating the metabolic state of patients’ tumors.

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

“…275 Tissues ex vivo have also been imaged to determine whether FLIM can guide surgical resection of tumors. 276 First, Lukina et al compared NAD(P)H FLIM of in vivo and ex vivo samples using mouse models of colorectal cancer, lung carcinoma, and melanoma to determine optimal tissue maintenance protocols to preserve in vivo signals within ex vivo samples. Then, Lukina et al used these protocols to perform NAD(P)H FLIM in postoperative samples obtained from colorectal cancer patients and found significant differences in NAD(P)H lifetimes between normal and malignant specimens.…”

Label-Free Macroscopic Fluorescence Lifetime Imaging of Brain Tumors

“…1×10 6 cells in 10 mL PBS were used for injection into the rat brain. Anaplastic astrocytoma 10-17-2 and glioblastoma 101.8 were obtained by inoculation of homogenized tumor tissue from donor rats (~10 6 tumor cells in 10 mL PBS) into the brain (19). For cells implantation, animals were anesthetized with zoletil (12.5 mg/ kg) and xylazine (1 mg/kg) and immobilized on a stereotaxic unit.…”

On Synthetic Instrument Response Functions of Time-Correlated Single-Photon Counting Based Fluorescence Lifetime Imaging Analysis

“…f (k) ae −(k−1)ts/τ1 + (1 − a)e −(k−1)ts/τ2 (9) where t s is the time bin width. LSD-LE expands the fluorescence single f(t) onto an ordered set of orthonormal Laguerre basis functions b l (k; α) as:…”

“…Compared with fluorescence intensity imaging, FLIM is not only less susceptible to experimental artifacts in excitation/detection setups, optical paths, or fluorophore concentrations, but can also provide abundant cellular information [1][2][3][4]. FLIM offers a unique route for probing and visualizing intracellular physical parameters such as temperature, pH, O 2, and ion concentrations, and it can be promising for cancer diagnosis [5][6][7][8][9]. Furthermore, in combination with Föster resonance energy transfer (FRET) techniques, FLIM-FRET techniques are excellent tools for studying protein-protein interactions, cellular metabolisms, and conformational changes of proteins in living cells [10][11][12].…”