Oxygen Assessment in Tumors In Vivo Using Phosphorescence Lifetime Imaging Microscopy.

The oxygen level in a tumor is a crucial factor for its development and response to therapies. Phosphorescence lifetime imaging (PLIM) with the use of phosphorescent oxygen probes is a highly sensitive, noninvasive optical technique for the assessment of molecular oxygen in living cells and tissues. Here, we present a protocol for microscopic mapping of oxygen distribution in a mouse tumor model in vivo. We demonstrate that PLIM microscopy, in combination with an Ir(III)-based probe, enables visualization of cellular-level heterogeneity of tumor oxygenation.

Effects of Photodynamic Therapy on Tumor Metabolism and Oxygenation Revealed by Fluorescence and Phosphorescence Lifetime Imaging.

This work was aimed at the complex analysis of the metabolic and oxygen statuses of tumors in vivo after photodynamic therapy (PDT). Studies were conducted on mouse tumor model using two types of photosensitizers-chlorin e6-based drug Photoditazine predominantly targeted to the vasculature and genetically encoded photosensitizer KillerRed targeted to the chromatin. Metabolism of tumor cells was assessed by the fluorescence lifetime of the metabolic redox-cofactor NAD(P)H, using fluorescence lifetime imaging. Oxygen content was assessed using phosphorescence lifetime macro-imaging with an oxygen-sensitive probe. For visualization of the perfused microvasculature, an optical coherence tomography-based angiography was used. It was found that PDT induces different alterations in cellular metabolism, depending on the degree of oxygen depletion. Moderate decrease in oxygen in the case of KillerRed was accompanied by an increase in the fraction of free NAD(P)H, an indicator of glycolytic switch, early after the treatment. Severe hypoxia after PDT with Photoditazine resulted from a vascular shutdown yielded in a persistent increase in protein-bound (mitochondrial) fraction of NAD(P)H. These findings improve our understanding of physiological mechanisms of PDT in cellular and vascular modes and can be useful to develop new approaches to monitoring its efficacy.

Correlation of Plasma Membrane Microviscosity and Cell Stiffness Revealed via Fluorescence-Lifetime Imaging and Atomic Force Microscopy.

The biophysical properties of cells described at the level of whole cells or their membranes have many consequences for their biological behavior. However, our understanding of the relationships between mechanical parameters at the level of cell (stiffness, viscoelasticity) and at the level of the plasma membrane (fluidity) remains quite limited, especially in the context of pathologies, such as cancer. Here, we investigated the correlations between cells' stiffness and viscoelastic parameters, mainly determined via the actin cortex, and plasma membrane microviscosity, mainly determined via its lipid profile, in cancer cells, as these are the keys to their migratory capacity. The mechanical properties of cells were assessed using atomic force microscopy (AFM). The microviscosity of membranes was visualized using fluorescence-lifetime imaging microscopy (FLIM) with the viscosity-sensitive probe BODIPY 2. Measurements were performed for five human colorectal cancer cell lines that have different migratory activity (HT29, Caco-2, HCT116, SW 837, and SW 480) and their chemoresistant counterparts. The actin cytoskeleton and the membrane lipid composition were also analyzed to verify the results. The cell stiffness (Young's modulus), measured via AFM, correlated well (Pearson r = 0.93) with membrane microviscosity, measured via FLIM, and both metrics were elevated in more motile cells. The associations between stiffness and microviscosity were preserved upon acquisition of chemoresistance to one of two chemotherapeutic drugs. These data clearly indicate that mechanical parameters, determined by two different cellular structures, are interconnected in cells and play a role in their intrinsic migratory potential.

Macroscopic temporally and spectrally resolved fluorescence imaging enhanced by laser-wavelength multiplexing.

We present a laser scanning system for macroscopic samples that records fully resolved decay curves in individual pixels, resolves the images in 16 wavelength channels, and records simultaneously at several laser wavelengths. By using confocal detection, the system delivers images that are virtually free of lateral scattering and out-of-focus haze. Image formats can be up to 256 × 256 pixels and up to 1024 time channels. We demonstrate the performance of the system both on model experiments with fluorescent micro-beads and on the tumor model in the living mice.

Effects of Paclitaxel on Plasma Membrane Microviscosity and Lipid Composition in Cancer Cells.

The cell membrane is an important regulator for the cytotoxicity of chemotherapeutic agents. However, the biochemical and biophysical effects that occur in the membrane under the action of chemotherapy drugs are not fully described. In the present study, changes in the microviscosity of membranes of living HeLa-Kyoto tumor cells were studied during chemotherapy with paclitaxel, a widely used antimicrotubule agent. To visualize the microviscosity of the membranes, fluorescence lifetime imaging microscopy (FLIM) with a BODIPY 2 fluorescent molecular rotor was used. The lipid profile of the membranes was assessed using time-of-flight secondary ion mass spectrometry ToF-SIMS. A significant, steady-state decrease in the microviscosity of membranes, both in cell monolayers and in tumor spheroids, was revealed after the treatment. Mass spectrometry showed an increase in the unsaturated fatty acid content in treated cell membranes, which may explain, at least partially, their low microviscosity. These results indicate the involvement of membrane microviscosity in the response of tumor cells to paclitaxel treatment.

Cell-in-Cell Structures in Gastrointestinal Tumors: Biological Relevance and Clinical Applications.

This review summarizes information about cell-in-cell (CIC) structures with a focus on gastrointestinal tumors. The phenomenon when one cell lives in another one has attracted an attention of researchers over the past decades. We briefly discuss types of CIC structures and mechanisms of its formation, as well as the biological basis and consequences of the cell-engulfing process. Numerous clinico-histopathological studies demonstrate the significance of these structures as prognostic factors, mainly correlated with negative prognosis. The presence of CIC structures has been identified in all gastrointestinal tumors. However, the majority of studies concern pancreatic cancer. In this field, in addition to the assessment of the prognostic markers, the attempts to manipulate the ability of cells to form CISs have been done in order to stimulate the death of the inner cell. Number of CIC structures also correlates with genetic features for some gastrointestinal tu-mors. The role of CIC structures in the responses of tumors to therapies, both chemotherapy and immunotherapy, seems to be the most poorly studied. However, there is some evidence of involvement of CIC structures in treatment failure. Here, we summarized the current literature on CIC structures in cancer with a focus on gastrointestinal tumors and specified future perspectives for investigation.

Biocompatible Phosphorescent O2 Sensors Based on Ir(III) Complexes for In Vivo Hypoxia Imaging.

In this work, we obtained three new phosphorescent iridium complexes (Ir1-Ir3) of general stoichiometry [Ir(N^C)2(N^N)]Cl decorated with oligo(ethylene glycol) fragments to make them water-soluble and biocompatible, as well as to protect them from aggregation with biomolecules such as albumin. The major photophysical characteristics of these phosphorescent complexes are determined by the nature of two cyclometallating ligands (N^C) based on 2-pyridine-benzothiophene, since quantum chemical calculations revealed that the electronic transitions responsible for the excitation and emission are localized mainly at these fragments. However, the use of various diimine ligands (N^N) proved to affect the quantum yield of phosphorescence and allowed for changing the complexes' sensitivity to oxygen, due to the variations in the steric accessibility of the chromophore center for O2 molecules. It was also found that the N^N ligands made it possible to tune the biocompatibility of the resulting compounds. The wavelengths of the Ir1-Ir3 emission maxima fell in the range of 630-650 nm, the quantum yields reached 17% (Ir1) in a deaerated solution, and sensitivity to molecular oxygen, estimated as the ratio of emission lifetime in deaerated and aerated water solutions, displayed the highest value, 8.2, for Ir1. The obtained complexes featured low toxicity, good water solubility and the absence of a significant effect of biological environment components on the parameters of their emission. Of the studied compounds, Ir1 and Ir2 were chosen for in vitro and in vivo biological experiments to estimate oxygen concentration in cell lines and tumors. These sensors have demonstrated their effectiveness for mapping the distribution of oxygen and for monitoring hypoxia in the biological objects studied.

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.

Monitoring the Intracellular pH and Metabolic State of Cancer Cells in Response to Chemotherapy Using a Combination of Phosphorescence Lifetime Imaging Microscopy and Fluorescence Lifetime Imaging Microscopy.

The extracellular matrix (ECM), in which collagen is the most abundant protein, impacts many aspects of tumor physiology, including cellular metabolism and intracellular pH (pHi), as well as the efficacy of chemotherapy. Meanwhile, the role of collagen in differential cell responses to treatment within heterogeneous tumor environments remains poorly investigated. In the present study, we simultaneously monitored the changes in pHi and metabolism in living colorectal cancer cells in vitro upon treatment with a chemotherapeutic combination, FOLFOX (5-fluorouracil, oxaliplatin and leucovorin). The pHi was followed using the new pH-sensitive probe BC-Ga-Ir, working in the mode of phosphorescence lifetime imaging (PLIM), and metabolism was assessed from the autofluorescence of the metabolic cofactor NAD(P)H using fluorescence lifetime imaging (FLIM) with a two-photon laser scanning microscope. To model the ECM, 3D collagen-based hydrogels were used, and comparisons with conventional monolayer cells were made. It was found that FOLFOX treatment caused an early temporal intracellular acidification (reduction in pHi), followed by a shift to more alkaline values, and changed cellular metabolism to a more oxidative state. The presence of unstructured collagen markedly reduced the cytotoxic effects of FOLFOX, and delayed and diminished the pHi and metabolic responses. These results support the observation that collagen is a factor in the heterogeneous response of cancer cells to chemotherapy and a powerful regulator of their metabolic behavior.

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.

Effect of Collagen Matrix on Doxorubicin Distribution and Cancer Cells' Response to Treatment in 3D Tumor Model.

The extracellular matrix (ECM) plays an important role in regulation of many aspects of tumor growth and response to therapies. However, the specifics of the interaction of chemotherapeutic agents with cancer cells in the presence of collagen, the major component of ECM, is still poorly investigated. In this study, we explored distribution of doxorubicin (DOX) and its effects on cancer cells' metabolism in the presence of collagen with different structures in 3D models. For this, a combination of second harmonic generation imaging of collagen and multiphoton fluorescence microscopy of DOX, and metabolic cofactor NAD(P)H was used. It was found that collagen slowed down the diffusion of DOX and thus decreased the cellular drug uptake. Besides nuclei, DOX also targeted mitochondria leading to inhibition of oxidative phosphorylation, which was more pronounced in the cells growing in the absence of collagen. As a result, the cells in collagen displayed better viability upon treatment with DOX. Taken together, our data illustrate that tumor collagen contributes to heterogeneous and sub-optimal response to DOX and highlight the challenges in improving drug delivery and efficacy.

Corrigendum: Highly invasive fluorescent/bioluminescent patient-derived orthotopic model of glioblastoma in mice.

[This corrects the article DOI: 10.3389/fonc.2022.897839.].

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.

Highly Invasive Fluorescent/Bioluminescent Patient-Derived Orthotopic Model of Glioblastoma in Mice.

Development of the novel diagnostic and therapeutic approaches in neuro-oncology requires tumor models that closely reproduce the biological features of patients' tumors. Patient-derived xenografts (PDXs) are recognized as a valuable and the most "close-to-patient" tool for preclinical studies. However, their establishment is complicated by the factors related to both the surgical material and technique of the orthotopic implantation. The aim of this work was to develop a patient-derived glioblastoma multiform (GBM) model that stably co-expresses luciferase and a far-red fluorescent protein for monitoring of tumor progression in the brain and, using this model, to validate new diagnostic methods-macroscopic fluorescence lifetime imaging (macro-FLIM) and cross-polarization optical coherence tomography (CP OCT). The established model was similar to the original patient's GBM in terms of histological and immunohistochemical features and possessed reproducible growth in nude mice, which could be observed by both fluorescence and bioluminescence imaging. Our results demonstrated the high potential of macro-FLIM and CP OCT for intraoperative differentiation of GBM from the white matter. Thus, the dual-labeled PDX model of GBM proved to be an excellent approach for observation of tumor development by optical methods.

Development of resistance to 5-fluorouracil affects membrane viscosity and lipid composition of cancer cells.

The investigations reported here were designed to determine whether the bulk plasma membrane is involved in mechanisms of acquired resistance of colorectal cancer cells to 5-fluorouracil (5-FU). Fluorescence lifetime imaging microscopy (FLIM) of live cultured cells stained with viscosity-sensitive probe BODIPY 2 was exploited to non-invasively assess viscosity in the course of treatment and adaptation to the drug. In parallel, lipid composition of membranes was examined with the time-of-flight secondary ion mass spectrometry (ToF-SIMS). Our results showed that a single treatment with 5-FU induced only temporal changes of viscosity in 5-FU sensitive cells immediately after adding the drug. Acquisition of chemoresistance was accompanied by persistent increase of viscosity, which was preserved upon treatment without any changes. Lipidomic analysis revealed that the resistant cells had a lower level of monounsaturated fatty acids and increased sphingomyelin or decreased phosphatidylcholine in their membranes, which partly explain increase of the viscosity. Thus, we propose that a high membrane viscosity mediates the acquisition of resistance to 5-FU.

Insight into redox regulation of apoptosis in cancer cells with multiparametric live-cell microscopy.

Cellular redox status and the level of reactive oxygen species (ROS) are important regulators of apoptotic potential, playing a crucial role in the growth of cancer cell and their resistance to apoptosis. However, the relationships between the redox status and ROS production during apoptosis remain poorly explored. In this study, we present an investigation on the correlations between the production of ROS, the redox ratio FAD/NAD(P)H, the proportions of the reduced nicotinamide cofactors NADH and NADPH, and caspase-3 activity in cancer cells at the level of individual cells. Two-photon excitation fluorescence lifetime imaging microscopy (FLIM) was applied to monitor simultaneously apoptosis using the genetically encoded sensor of caspase-3, mKate2-DEVD-iRFP, and the autofluorescence of redox cofactors in colorectal cancer cells upon stimulation of apoptosis with staurosporine, cisplatin or hydrogen peroxide. We found that, irrespective of the apoptotic stimulus used, ROS accumulation correlated well with both the elevated pool of mitochondrial, enzyme-bound NADH and caspase-3 activation. Meanwhile, a shift in the contribution of bound NADH could develop independently of the apoptosis, and this was observed in the case of cisplatin. An increase in the proportion of bound NADPH was detected only in staurosporine-treated cells, this likely being associated with a high level of ROS production and their resulting detoxification. The results of the study favor the discovery of new therapeutic strategies based on manipulation of the cellular redox balance, which could help improve the anti-tumor activity of drugs and overcome apoptotic resistance.

Label-free sensing of cells with fluorescence lifetime imaging: The quest for metabolic heterogeneity.

Molecular, morphological, and physiological heterogeneity is the inherent property of cells which governs differences in their response to external influence. Tumor cell metabolic heterogeneity is of a special interest due to its clinical relevance to tumor progression and therapeutic outcomes. Rapid, sensitive, and noninvasive assessment of metabolic heterogeneity of cells is a great demand for biomedical sciences. Fluorescence lifetime imaging (FLIM), which is an all-optical technique, is an emerging tool for sensing and quantifying cellular metabolism by measuring fluorescence decay parameters of endogenous fluorophores, such as NAD(P)H. To achieve accurate discrimination between metabolically diverse cellular subpopulations, appropriate approaches to FLIM data collection and analysis are needed. In this paper, the unique capability of FLIM to attain the overarching goal of discriminating metabolic heterogeneity is demonstrated. This has been achieved using an approach to data analysis based on the nonparametric analysis, which revealed a much better sensitivity to the presence of metabolically distinct subpopulations compared to more traditional approaches of FLIM measurements and analysis. The approach was further validated for imaging cultured cancer cells treated with chemotherapy. These results pave the way for accurate detection and quantification of cellular metabolic heterogeneity using FLIM, which will be valuable for assessing therapeutic vulnerabilities and predicting clinical outcomes.

Fluorescence lifetime-based pH mapping of tumors in vivo using genetically encoded sensor SypHerRed.

Changes in intracellular pH (pHi) reflect metabolic states of cancer cells during tumor growth and dissemination. Therefore, monitoring of pHi is essential for understanding the metabolic mechanisms that support cancer progression. Genetically encoded fluorescent pH sensors have become irreplaceable tools for real-time tracking pH in particular subcellular compartments of living cells. However, ratiometric readout of most of the pH probes is poorly suitable to measure pH in thick samples ex vivo or tissues in vivo including solid tumors. Fluorescence lifetime imaging (FLIM) is a promising alternative to the conventional fluorescent microscopy. Here, we present a quantitative approach to map pHi in cancer cells and tumors in vivo, relying on fluorescence lifetime of a genetically encoded pH sensor SypHerRed. We demonstrate the utility of SypHerRed in visualizing pHi in cancer cell culture and in mouse tumor xenografts using fluorescence lifetime imaging microscopy and macroscopy. For the first time to our knowledge, the absolute pHi value is obtained for tumors in vivo by an optical technique. In addition, we demonstrate the possibility of simultaneous detection of pHi and endogenous fluorescence of metabolic cofactor NADH, which provides a complementary insight into metabolic aspects of cancer. Fluorescence lifetime-based readout and red-shifted spectra make pH sensor SypHerRed a promising instrument for multiparameter in vivo imaging applications.

The Role of Plasma Membrane Viscosity in the Response and Resistance of Cancer Cells to Oxaliplatin.

Maintenance of the biophysical properties of membranes is essential for cell survival upon external perturbations. However, the links between a fluid membrane state and the drug resistance of cancer cells remain elusive. Here, we investigated the role of membrane viscosity and lipid composition in the responses of cancer cells to oxaliplatin and the development of chemoresistance. Plasma membrane viscosity was monitored in live colorectal cancer cells and tumor xenografts using two-photon excited fluorescence lifetime imaging microscopy (FLIM) using the fluorescent molecular rotor BODIPY 2. The lipid profile was analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS). It was found that the plasma membrane viscosity increased upon oxaliplatin treatment, both in vitro and in vivo, and that this correlated with lower phosphatidylcholine and higher cholesterol content. The emergence of resistance to oxaliplatin was accompanied by homeostatic adaptation of the membrane lipidome, and the recovery of lower viscosity. These results suggest that maintaining a constant plasma membrane viscosity via remodeling of the lipid profile is crucial for drug resistance in cancer.