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.

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

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

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.

Multiphoton microscopy assessment of the structure and variability changes of dermal connective tissue in vulvar lichen sclerosus: A pilot study.

In this article, we offer a novel classification of progressive changes in the connective tissue of dermis in vulvar lichen sclerosus (VLS) relying on quantitative assessment of the second harmonic generation (SHG) signal received from formalin fixed and deparaffinized tissue sections. We formulate criteria for distinguishing four degrees of VLS development: Initial-Mild-Moderate-Severe. Five quantitative characteristics (length and thickness type I Collagen fibers, Mean SHG signal intensity, Skewness and Coherence SHG signal) are used to describe the sequential degradation of connective tissue (changes in the structure, orientation, shape and density of collagen fibers) up to the formation of specific homogeneous masses. Each of the degrees has a characteristic set of quantitatively expressed features. We focus on the identification and description of early, initial changes of the dermis as the least specific. The results obtained by us and the proposed classification of the degrees of the disease can be used to objectify the dynamics of tissue changes during treatment.

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.

Late Changes in the Extracellular Matrix of the Bladder after Radiation Therapy for Pelvic Tumors.

Radiation therapy is one of the cardinal approaches in the treatment of malignant tumors of the pelvis. It leads to the development of radiation-induced complications in the normal tissues. Thus, the evaluation of radiation-induced changes in the extracellular matrix of the normal tissue is deemed urgent, since connective tissue stroma degradation plays a crucial role in the development of Grade 3-4 adverse effects (hemorrhage, necrosis, and fistula). Such adverse effects not only drastically reduce the patients' quality of life but can also become life-threatening. The aim of this study is to quantitatively analyze the bladder collagen state in patients who underwent radiation therapy for cervical and endometrial cancer and in patients with chronic bacterial cystitis and compare them to the normal bladder extracellular matrix. MATERIALS AND METHODS: One hundred and five patients with Grade 2-4 of radiation cystitis, 67 patients with bacterial chronic cystitis, and 20 volunteers without bladder pathology were enrolled. Collagen changes were evaluated depending on its hierarchical level: fibrils and fibers level by atomic force microscopy; fibers and bundles level by two-photon microscopy in the second harmonic generation (SHG) mode; general collagen architectonics by cross-polarization optical coherence tomography (CP OCT). RESULTS: The main sign of the radiation-induced damage of collagen fibrils and fibers was the loss of the ordered "basket-weave" packing and a significant increase in the total area of ruptures deeper than 1 µm compared to the intact sample. The numerical analysis of SHG images detected that a decrease in the SHG signal intensity of collagen is correlated with the increase in the grade of radiation cystitis. The OCT signal brightness in cross-polarization images demonstrated a gradual decrease compared to the intact bladder depending on the grade of the adverse event. CONCLUSIONS: The observed correspondence between the extracellular matrix changes at the microscopic level and at the level of the general organ architectonics allows for the consideration of CP OCT as a method of "optical biopsy" in the grading of radiation-induced collagen damage.

Probing Metabolism and Viscosity of Cancer Cells using Fluorescence Lifetime Imaging Microscopy.

Viscosity is an important physical property of a biological membrane, as it is one of the key parameters for the regulation of morphological and physiological state of living cells. Plasma membranes of tumor cells are known to have significant alterations in their composition, structure, and functional characteristics. Along with dysregulated metabolism of glucose and lipids, these specific membrane properties help tumor cells to adapt to the hostile microenvironment and develop resistance to drug therapies. Here, we demonstrate the use of fluorescence lifetime imaging microscopy (FLIM) to sequentially image cellular metabolism and plasma membrane viscosity in live cancer cell culture. Metabolic assessments are performed by detecting fluorescence of endogenous metabolic cofactors, such as reduced nicotinamide adenine dinucleotide NAD(P)H and oxidized flavins. Viscosity is measured using a fluorescent molecular rotor, a synthetic viscosity-sensitive dye, with a strong fluorescence lifetime dependence on the viscosity of the immediate environment. In combination, these techniques enable us to better understand the links between membrane state and metabolic profile of cancer cells and to visualize the changes induced by chemotherapy.

Tracing of intracellular pH in cancer cells in response to Taxol treatment.

Genetically encoded pH-sensors are the promising instrument for intracellular pH (pHi) registration. In tumor tissue the reversed pH gradient is known to be the important hallmark of cancer and regulator of tumor response on chemotherapy. However the effect of chemotherapeutic drugs on the pHi of tumor cells is largely unknown. Here we using genetically encoded ratiometric pH-sensor SypHer2 were able to monitor pHi in vitro in cell monolayer and tumor spheroids and in vivo in tumor xenografts. In tumor cell monolayer different pHi dynamic was revealed in the dying cell and division-arrested surviving cells. The treatment effect of taxol varied in monolayer and tumor spheroids and pHi changes were able to reflect these difference. The tend to pHi decrease in respect to taxol in vivo matched with results obtained for the cell monolayer. Also in both cases the cell cycle-arrest was the main treatment effect in contrast to tumor spheroid, where the cell death was the primary result. These findings elucidate the significance of pHi in the mechanisms of taxol action on cervical cancer cells and will be valuable for development of new approaches for cancer treatment.

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' 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.

Singlet oxygen phosphorescence imaging by superconducting single-photon detector and time-correlated single-photon counting: publisher's note.

This publisher's note contains corrections to Opt. Lett.46, 1217 (2021) OPLEDP0146-959210.1364/OL.415229.

Singlet oxygen phosphorescence imaging by superconducting single-photon detector and time-correlated single-photon counting.

This Letter presents, to the best of our knowledge, a novel optical configuration for direct time-resolved measurements of luminescence from singlet oxygen, both in solutions and from cultured cells on photodynamic therapy. The system is based on the superconducting single-photon detector, coupled to the confocal scanner that is modified for the near-infrared measurements. The recording of a phosphorescence signal from singlet oxygen at 1270 nm has been done using time-correlated single-photon counting. The performance of the system is verified by measuring phosphorescence from singlet oxygen generated by the photosensitizers commonly used in photodynamic therapy: methylene blue and chlorin e6. The described system can be easily upgraded to the configuration when both phosphorescence from singlet oxygen and fluorescence from the cells can be detected in the imaging mode. Thus, co-localization of the signal from singlet oxygen with the areas inside the cells can be done.

Multiphoton tomography in differentiation of morphological and molecular subtypes of breast cancer: A quantitative analysis.

In this study multiphoton tomography, based on second harmonic generation (SHG), and two-photon-excited fluorescence (TPEF) was used to visualize both the extracellular matrix and tumor cells in different morphological and molecular subtypes of human breast cancer. It was shown, that quantified assessment of the SHG based imaging data has great potential to reveal differences of collagen quantity, organization and uniformity in both low- and highly- aggressive invasive breast cancers. The values of quantity and uniformity of the collagen fibers distribution were significantly higher in low-aggressive breast cancer compared to the highly-aggressive subtypes, while the value representing collagen organization was lower in the former type. Additionally, it was shown, that TPEF detection of elastin fibers and amyloid protein may be used as a biomarker of detection the low-aggressive breast cancer subtype. Thus, TPEF/SHG imaging offers the possibility of becoming a useful tool for the rapid diagnosis of various subtypes of breast cancer during biopsy as well as for the intraoperative determinination of tumor-positive resection margins.

FUCCI-Red: a single-color cell cycle indicator for fluorescence lifetime imaging.

The phase of the cell cycle determines numerous aspects of cancer cell behaviour including invasiveness, ability to migrate and responsiveness to cytotoxic drugs. To non-invasively monitor progression of cell cycle in vivo, a family of genetically encoded fluorescent indicators, FUCCI (fluorescent ubiquitination-based cell cycle indicator), has been developed. Existing versions of FUCCI are based on fluorescent proteins of two or more different colors fused to cell-cycle-dependent degradation motifs. Thus, FUCCI-expressing cells emit light of different colors in different phases providing a robust way to monitor cell cycle progression by fluorescence microscopy and flow cytometry but limiting the possibility to simultaneously visualize other markers. To overcome this limitation, we developed a single-color variant of FUCCI, called FUCCI-Red, which utilizes two red fluorescent proteins with distinct fluorescence lifetimes, mCherry and mKate2. Similarly to FUCCI, these proteins carry cell cycle-dependent degradation motifs to resolve G1 and S/G2/M phases. We showed utility of FUCCI-Red by visualizing cell cycle progression of cancer cells in 2D and 3D cultures and monitoring development of tumors in vivo by confocal and fluorescence lifetime imaging microscopy (FLIM). Single-channel registration and red-shifted spectra make FUCCI-Red sensor a promising instrument for multiparameter in vivo imaging applications, which was demonstrated by simultaneous detection of cellular metabolic state using endogenous fluorescence in the blue range.

Expression of EMT-Related Genes in Hybrid E/M Colorectal Cancer Cells Determines Fibroblast Activation and Collagen Remodeling.

Collagen, the main non-cellular component of the extracellular matrix (ECM), is profoundly reorganized during tumorigenesis and has a strong impact on tumor behavior. The main source of collagen in tumors is cancer-associated fibroblasts. Cancer cells can also participate in the synthesis of ECM; however, the contribution of both types of cells to collagen rearrangements during the tumor progression is far from being clear. Here, we investigated the processes of collagen biosynthesis and remodeling in parallel with the transcriptome changes during cancer cells and fibroblasts interactions. Combining immunofluorescence, RNA sequencing, and second harmonic generation microscopy, we have explored the relationships between the ratio of epithelial (E) and mesenchymal (M) components of hybrid E/M cancer cells, their ability to activate fibroblasts, and the contributions of both cell types to collagen remodeling. To this end, we studied (i) co-cultures of colorectal cancer cells and normal fibroblasts in a collagen matrix, (ii) patient-derived cancer-associated fibroblasts, and (iii) mouse xenograft models. We found that the activation of normal fibroblasts that form dense collagen networks consisting of large, highly oriented fibers depends on the difference in E/M ratio in the cancer cells. The more-epithelial cells activate the fibroblasts more strongly, which correlates with a dense and highly ordered collagen structure in tumors in vivo. The more-mesenchymal cells activate the fibroblasts to a lesser degree; on the other hand, this cell line has a higher innate collagen remodeling capacity. Normal fibroblasts activated by cancer cells contribute to the organization of the extracellular matrix in a way that is favorable for migratory potency. At the same time, in co-culture with epithelial cancer cells, the contribution of fibroblasts to the reorganization of ECM is more pronounced. Therefore, one can expect that targeting the ability of epithelial cancer cells to activate normal fibroblasts may provide a new anticancer therapeutic strategy.

Monitoring membrane viscosity in differentiating stem cells using BODIPY-based molecular rotors and FLIM.

Membrane fluidity plays an important role in many cell functions such as cell adhesion, and migration. In stem cell lines membrane fluidity may play a role in differentiation. Here we report the use of viscosity-sensitive fluorophores based on a BODIPY core, termed "molecular rotors", in combination with Fluorescence Lifetime Imaging Microscopy, for monitoring of plasma membrane viscosity changes in mesenchymal stem cells (MSCs) during osteogenic and chondrogenic differentiation. In order to correlate the viscosity values with membrane lipid composition, the detailed analysis of the corresponding membrane lipid composition of differentiated cells was performed by time-of-flight secondary ion mass spectrometry. Our results directly demonstrate for the first time that differentiation of MSCs results in distinct membrane viscosities, that reflect the change in lipidome of the cells following differentiation.

Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose.

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

In vivo metabolic and SHG imaging for monitoring of tumor response to chemotherapy.

Although chemotherapy remains one of the main types of treatment for cancer, treatment failure is a frequent occurrence, emphasizing the need for new approaches to the early assessment of tumor response. The aim of this study was to search for indicators based on optical imaging of cellular metabolism and of collagen in tumors in vivo that enable evaluation of their response to chemotherapy. The study was performed on a mouse colorectal cancer model with the use of cisplatin, paclitaxel, and irinotecan. The metabolic activity of the tumor cells was assessed using fluorescence lifetime imaging of the metabolic cofactor reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H. Second harmonic generation (SHG) imaging was used to analyze the extent and properties of collagen within the tumors. We detected an early decrease in the free/bound NAD(P)H ratio in all treated tumors, indicating a shift toward a more oxidative metabolism. Monitoring of collagen showed an early increase in the amount of collagen followed by an increase in the extent of its orientation in tumors treated with cisplatin and paclitaxel, and decrease in collagen content in the case of irinotecan. Our study suggests that changes in cellular metabolism and fibrotic stroma organization precede morphological alterations and tumor size reduction, and that this indicates that NAD(P)H and collagen can be considered as intrinsic indicators of the response to treatment. This is the first time that these parameters have been investigated in tumors in vivo in the course of chemotherapy with drugs having different mechanisms of action. © 2018 International Society for Advancement of Cytometry.