Label-free macroscopic fluorescence lifetime imaging of brain tumors

Advanced stage glioma is the most aggressive form of malignant brain tumors with a short survival time. Real-time pathology assisted, or image guided surgical procedures that eliminate tumors promise to improve the clinical outcome and prolong the lives of patients. Our work is focused on the development of a rapid and sensitive assay for intraoperative diagnostics of glioma and identification of optical markers essential for differentiation between tumors and healthy brain tissues. We utilized fluorescence lifetime imaging (FLIM) of endogenous fluorophores related to metabolism of the glioma from freshly excised brains tissues. Macroscopic time-resolved fluorescence images of three intracranial animal glioma models and surgical samples of patients\u27 glioblastoma together with the white matter have been collected. Several established and new algorithms were applied to identify the imaging markers of the tumors. We found that fluorescence lifetime parameters characteristic of the glioma provided background for differentiation between the tumors and intact brain tissues. All three rat tumor models demonstrated substantial differences between the malignant and normal tissue. Similarly, tumors from patients demonstrated statistically significant differences from the peritumoral white matter without infiltration. While the data and the analysis presented in this paper are preliminary and further investigation with a larger number of samples is required, the proposed approach based on the macroscopic FLIM has a high potential for diagnostics of glioma and evaluation of the surgical margins of gliomas

Measurement of Patient-Derived Glioblastoma Cell Response to Temozolomide Using Fluorescence Lifetime Imaging of NAD(P)H

Personalized strategies in glioblastoma treatment are highly necessary. One of the possible approaches is drug screening using patient-derived tumor cells. However, this requires reliable methods for assessment of the response of tumor cells to treatment. Fluorescence lifetime imaging microscopy (FLIM) is a promising instrument to detect early cellular response to chemotherapy using the autofluorescence of metabolic cofactors. Here, we explored FLIM of NAD(P)H to evaluate the sensitivity of patient-derived glioma cells to temozolomide (TMZ) in vitro. Our results demonstrate that the more-responsive cell cultures displayed the longest mean fluorescence lifetime τm after TMZ treatment due to an increase in the protein-bound NAD(P)H fraction α2 associated with a shift to oxidative phosphorylation. The cell cultures that responded poorly to TMZ had generally shorter τm, i.e., were more glycolytic, and showed no or insignificant changes after treatment. The FLIM data correlate well with standard measurements of cellular drug response—cell viability and proliferation index and clinical response in patients. Therefore, FLIM of NAD(P)H provides a highly sensitive, label-free assay of treatment response directly on patient-derived glioblastoma cells and can become an innovative platform for individual drug screening for patients

Genetically encoded far-red fluorescent sensors for caspase-3 activity

Caspase-3 is a key effector caspase that is activated in both extrinsic and intrinsic pathways of apoptosis. Available fluorescent sensors for caspase-3 activity operate in relatively short wavelength regions and are nonoptimal for multiparameter microscopy and whole-body imaging. In the present work, we developed new genetically encoded sensors for caspase-3 activity possessing the most red-shifted spectra to date. These consist of Förster resonance energy transfer (FRET) pairs in which a far-red fluorescent protein (mKate2 or eqFP650) is connected to the infrared fluorescent protein iRFP through a linker containing the DEVD caspase-3 cleavage site. During staurosporine-induced apoptosis of mammalian cells (HeLa and CT26), both mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors showed a robust response (1.6-fold increase of the donor fluorescence intensity). However, eqFP650-DEVD-iRFP displayed aggregation in some cells. For stably transfected CT26 mKate2-DEVD-iRFP cells, fluorescence lifetime imaging (FLIM) enabled us to detect caspase-3 activation due to the increase of mKate2 donor fluorescence lifetime from 1.45 to 2.05 ns. We took advantage of the strongly red-shifted spectrum of mKate2-DEVD-iRFP to perform simultaneous imaging of EGFP-Bax translocation during apoptosis. We conclude that mKate2-DEVD-iRFP is well-suited for multiparameter imaging and also potentially beneficial for in vivo imaging in animal tissues

FLIM of NAD(P)H in Lymphatic Nodes Resolves T-Cell Immune Response to the Tumor

Assessment of T-cell response to the tumor is important for diagnosis of the disease and monitoring of therapeutic efficacy. For this, new non-destructive label-free methods are required. Fluorescence lifetime imaging (FLIM) of metabolic coenzymes is a promising innovative technology for the assessment of the functional status of cells. The purpose of this work was to test whether FLIM can resolve metabolic alterations that accompany T-cell reactivation to the tumors. The study was carried out on C57Bl/6 FoxP3-EGFP mice bearing B16F0 melanoma. Autofluorescence of the immune cells in fresh lymphatic nodes (LNs) was investigated. It was found that fluorescence lifetime parameters of nicotinamide adenine dinucleotide (phosphate) NAD(P)H are sensitive to tumor development. Effector T-cells in the LNs displayed higher contribution of free NADH, the form associated with glycolysis, in all tumors and the presence of protein-bound NADPH, associated with biosynthetic processes, in the tumors of large size. Flow cytometry showed that the changes in the NADH fraction of the effector T-cells correlated with their activation, while changes in NADPH correlated with cell proliferation. In conclusion, FLIM of NAD(P)H in fresh lymphoid tissue is a powerful tool for assessing the immune response to tumor development

Visualization of intracellular hydrogen peroxide with HyPer, a genetically encoded fluorescent probe

The fluorescent sensor HyPer allows monitoring of intracellular H O levels with a high degree of sensitivity and specificity. Here, we provide a detailed protocol of ratiometric imaging of HO produced by cells during phagocytosis, including instructions for experiments on different commercial confocal systems, namely, Leica SP2, Leica SP5, and Carl Zeiss LSM, as well as wide-field Leica 6000 microscope. The general experimental scheme is easily adaptable for imaging HO production by various cell types under a variety of conditions

Properties of Fluorescent Far-Red Anti-TNF Nanobodies

Upregulation of the expression of tumor necrosis factor (TNF-α, TNF) has a significant role in the development of autoimmune diseases. The fluorescent antibodies binding TNF may be used for personalized therapy of TNF-dependent diseases as a tool to predict the response to anti-TNF treatment. We generated recombinant fluorescent proteins consisting of the anti-TNF module based on the variable heavy chain (VHH) of camelid antibodies fused with the far-red fluorescent protein Katushka (Kat). Two types of anti-TNF VHH were developed: one (BTN-Kat) that was bound both human or mouse TNF, but did not neutralize their activity, and a second (ITN-Kat) that was binding and neutralizing human TNF. BTN-Kat does not interfere with TNF biological functions and can be used for whole-body imaging. ITN-Kat can be evaluated in humanized mice or in cells isolated from humanized mice. It is able to block human TNF (hTNF) activities both in vitro and in vivo and may be considered as a prototype of a theranostic agent for autoimmune diseases

Simultaneous Probing of Metabolism and Oxygenation of Tumors In Vivo Using FLIM of NAD(P)H and PLIM of a New Polymeric Ir(III) Oxygen Sensor

Tumor cells are well adapted to grow in conditions of variable oxygen supply and hypoxia by switching between different metabolic pathways. However, the regulatory effect of oxygen on metabolism and its contribution to the metabolic heterogeneity of tumors have not been fully explored. In this study, we develop a methodology for the simultaneous analysis of cellular metabolic status, using the fluorescence lifetime imaging microscopy (FLIM) of metabolic cofactor NAD(P)H, and oxygen level, using the phosphorescence lifetime imaging (PLIM) of a new polymeric Ir(III)-based sensor (PIr3) in tumors in vivo. The sensor, derived from a polynorbornene and cyclometalated iridium(III) complex, exhibits the oxygen-dependent quenching of phosphorescence with a 40% longer lifetime in degassed compared to aerated solutions. In vitro, hypoxia resulted in a correlative increase in PIr3 phosphorescence lifetime and free (glycolytic) NAD(P)H fraction in cells. In vivo, mouse tumors demonstrated a high degree of cellular-level heterogeneity of both metabolic and oxygen states, and a lower dependence of metabolism on oxygen than cells in vitro. The small tumors were hypoxic, while the advanced tumors contained areas of normoxia and hypoxia, which was consistent with the pimonidazole assay and angiographic imaging. Dual FLIM/PLIM metabolic/oxygen imaging will be valuable in preclinical investigations into the effects of hypoxia on metabolic aspects of tumor progression and treatment response

Red Light-Emitting Water-Soluble Luminescent Iridium-Containing Polynorbornenes: Synthesis, Characterization and Oxygen Sensing Properties in Biological Tissues In Vivo

New water-soluble polynorbornenes P1–P4 containing oligoether, amino acid groups and luminophoric complexes of iridium(III) were synthesized by ring-opening metathesis polymerization. The polymeric products in organic solvents and in water demonstrate intense photoluminescence in the red spectral region. The polymers P1 and P3 with 1-phenylisoquinoline cyclometalating ligands in iridium fragments reveal 4–6 fold higher emission quantum yields in solutions than those of P2 and P4 that contain iridium complexes with 1-(thien-2-yl)isoquinoline cyclometalating ligands. The emission parameters of P1–P4 in degassed solutions essentially differ from those in the aerated solutions showing oxygen-dependent quenching of phosphorescence. Biological testing of P1 and P3 demonstrates that the polymers do not penetrate into live cultured cancer cells and normal skin fibroblasts and do not possess cytotoxicity within the concentrations and time ranges reasonable for biological studies. In vivo, the polymers display longer phosphorescence lifetimes in mouse tumors than in muscle, as measured using phosphorescence lifetime imaging (PLIM), which correlates with tumor hypoxia. Therefore, preliminary evaluation of the synthesized polymers shows their suitability for noninvasive in vivo assessments of oxygen levels in biological tissues