Imaging Of Cancer Cells Research Articles

A High-Efficiency Autocatalysis-Oriented Cascade Circuit via Reciprocal Hug-Amplification for Assay-to-Treat Application.

Developing a DNA autocatalysis-oriented cascade circuit (AOCC) via reciprocal navigation of two enzyme-free hug-amplifiers might be desirable for constructing a rapid, efficient, and sensitive assay-to-treat platform. In response to a specific trigger (T), seven functional DNA hairpins were designed to execute three-branched assembly (TBA) and three isotropic hybridization chain reaction (3HCR) events for operating the AOCC. This was because three new inducers were reconstructed in TBA arms to initiate 3HCR (TBA-to-3HCR) and periodic T repeats were resultantly reassembled in the tandem nicks of polymeric nanowires to rapidly activate TBA in the opposite direction (3HCR-to-TBA) without steric hindrance, thereby cooperatively manipulating sustainable AOCC progress for exponential hug-amplification (1:3Nn). Our experimental verifications manifested that the T-dependent AOCC amplifier achieved fast input transduction and efficient fluorescence readout. As predicted, the flexible programming of reactive hairpin species endowed the repeating nicks in productive 3HCR nanowires with great possibilities and accessibilities to graft tailored modular elements, such as G-rich AS1411 aptamers capable of adopting G-quadruplex conformations (G4) that readily facilitated the embedding of zinc(II) protoporphyrin IX (ZnPPIX), a kind of heme oxygenase-1 enzyme inhibitor. Thus, the cascading ZnPPIX/G4 entities acted as fluorescent signal reporters, photosensitizers and anticancer drugs, thereby creating an updated AOCC-based assay-to-treat platform for ultrasensitive biosensing, discernible cell imaging and efficient photodynamic therapy of cancer cells. This would offer a new paradigm to advance the rational integration of dynamic DNA assembly and amplifiable recycling circuits for applicable bioassay and theranostics.

Construction of Multifunctional Hyaluronic Acid Modified Gold Nanoparticles Clocked with Irinotecan and Indocyanine Green: Investigation of Chemotherapy and Cancer Cell Imaging

Construction of Multifunctional Hyaluronic Acid Modified Gold Nanoparticles Clocked with Irinotecan and Indocyanine Green: Investigation of Chemotherapy and Cancer Cell Imaging

Insights into glucose-derived carbon dot synthesis via Maillard reaction: from reaction mechanism to biomedical applications

Carbon dots (CDs) are versatile nanomaterials that are considered ideal for application in bioimaging, drug delivery, sensing, and optoelectronics owing to their excellent photoluminescence, biocompatibility, and chemical stability features. Nitrogen doping enhances the fluorescence of CDs, alters their electronic properties, and improves their functional versatility. N-doped CDs can be synthesized via solvothermal treatment of carbon sources with nitrogen-rich precursors; however, systematic investigations of their synthesis mechanisms have been rarely reported. In this study, we developed a method to synthesize N-doped CDs using the Maillard reaction with glucose and ethanolamine as precursors (namely, G-CDs). Comprehensive characterization of these G-CDs revealed the successful incorporation of nitrogen- and glucose-like functionalities. The optical properties and electronic band structures of G-CDs were analyzed using transient absorption and time-resolved photoluminescence spectroscopy. The prepared G-CDs demonstrated near-infrared photoluminescence, low cytotoxicity, glucose transporter-facilitated cellular uptake, and effective heat generation under an 808-nm laser. Particularly, the cellular uptake of G-CDs was reduced by up to 25% after preincubation with a Glut1 inhibitor. These features are suitable for in vitro biological imaging and photothermal therapy in prostate cancer cells. This paper highlights the potential of G-CDs in clinical applications owing to their multicolor emission, photothermal conversion functionality, and versatile surface structure.

Raman Imaging of Targeted Drug Delivery with DNA-Based Nano-Optical Devices.

Raman spectroscopy (RS) has emerged as a novel optical imaging modality by identifying molecular species through their bond vibrations, offering high specificity and sensitivity in molecule detection. However, its application in intracellular molecular probing has been limited due to challenges in combining vibrational tags with functional probes. DNA nanostructures, known for their high programmability, have been instrumental in fields like biomedicine and nanofabrication. So far, their ability to customize Raman signals remains largely untapped. In this study, a new class of Raman active DNA origami-based hybrid nanodevice (ND) for targeted cancer cell drug delivery and imaging is engineered. The ND is specifically engineered for metastatic prostate cancer treatment, featuring a legumain enzyme-responsive sequence for the controlled release of the chemotherapeutic agent doxorubicin. Integrating RS with precise targeting, the ND enables imaging of aggressive cancer cells and efficient drug delivery with minimal off-target effects. The developed device offers stimuli-responsive behavior, enhanced stability, exceptional tunability, and potent targeting abilities, positioning it as a highly promising strategy for advancing precision cancer imaging and therapy.

Insights into Cancer Cell Imaging Probes Based on Chalcone Scaffolds: Theoretical and Experimental Perspectives.

The research article details the synthesis of chalcone-chromone-based scaffolds via multicomponent reactions. These compounds were characterized using conventional spectroscopic methods, including NMR (1H and 13C), FT-IR, and HR-MS. Among the synthesized scaffolds, AZBNPy stood out, exhibiting exceptional DNA and protein targeting capabilities with superior binding parameters. Molecular docking studies indicated that AZBNPy has potential as a potent anticancer agent and a probe for cancer cell imaging. The findings showed that AZBNPy effectively inhibited cell proliferation and induced cell death by targeting HER-2, PARP-2, and HFR in MCF-7 cells. Additionally, in vitro fluorescence imaging studies confirmed AZBNPy's specificity for cancer cell receptors, displaying strong fluorescence in human breast cancer tissues. The clinical application of AZBNPy as an optical imaging agent holds significant promise in aiding surgeons with the precise identification and removal of cancerous tissues, potentially improving patient outcomes and survival rates.

Gold Nanoparticle Detection with Two-Photon Excitation Fluorescence Lifetime Imaging of NAD(P)H in Cancer Cells: An Analytical Approach to Separate Nanoparticle and NAD(P)H Signals.

Gold nanoparticles (AuNPs) have shown promise for applications in the diagnosis and treatment of different diseases, including cancer. Understanding the effect of AuNPs on metabolic reprogramming in cancer cells at the single cell level is of high importance for improving the efficacy and safety. Fluorescence lifetime imaging microscopy (FLIM) of nicotinamide adenine dinucleotide (phosphate) hydrogen (NAD(P)H) as a main metabolic cofactor and an indicator of metabolic reprogramming in cancer cells enables real-time monitoring of cancer cell metabolism in response to different treatments, including AuNPs. However, NPs such as AuNPs can be a potential source of signals themselves, which provides opportunities to measure the NP internalization, but it is also important to minimize confounding effects on metabolic measurements. In this study, we detected inherent photoluminescence (PL) from the AuNPs in treated prostate cancer cells (PC-3 cell line) as well as in solution at the NAD(P)H emission wavelength. We developed an analysis approach to minimize the confounding effect of the AuNPs' PL on metabolic measurements. On the other hand, we assessed the reliability of the intracellular AuNPs' PL as an estimator of AuNP uptake. To assess if intracellular AuNPs' PL may be dependent on the exposed cell type, we performed NAD(P)H FLIM imaging of AuNP-exposed SKBR-3 breast cancer cells, where we observed a similar AuNP PL but at a much lower level compared to PC-3 cells. We proposed that this difference can be attributed to the different levels of AuNP uptake or varying intracellular microenvironments.

Endoplasmic Reticulum-Targeting Delayed Fluorescent Probe for Dual-mode Nitroreductase Sensing.

Organic thermally activated delayed fluorescence (TADF) materials, known for their long-lived emission properties, are highly sought after for background-free imaging of selective analytes in time-resolved modes. However, their practical application faces significant challenges, including the air sensitivity of triplet states, lack of organelle specificity, and the absence of precise analyte recognition centres. These limitations hinder their effectiveness in detecting key cancer biomarkers such as nitroreductase (NTR). Herein, we present the development of donor (triphenylamine)-acceptor (quinoxaline)-based probes, TPQS and TPNS, which are functionalized with a sulphonamide unit to offer endoplasmic reticulum specificity. TPQS exhibits delayed fluorescence, attributed to a minimal singlet-triplet energy gap, as confirmed by time-resolved fluorescence measurements. Additionally, a nonfluorescent probe, TPNS, is synthesized by introducing a nitro group into the sulphonamide unit of the TPQS backbone, serving as a recognition centre for NTR. Upon reacting with NTR, TPNS displays a "turn-on" luminescence and delayed fluorescence, enabling dual-mode detection of NTR through both confocal fluorescence imaging and time-resolved fluorescence imaging (TRFI) in cancer cells. These findings underscore the potential of delayed fluorescent emitters for the sensitive and specific detection of cancer biomarkers in complex biological environments.

Tunable Nano-Supramolecules Based on Cucurbiturils for Near-Infrared Phosphorescence Imaging.

Nano-supramolecules based on artificial macrocycles can not only regulate assembly morphology but also boost phosphorescence resonance energy transfer (PRET). Herein, a water-soluble phosphorescence supramolecule was constructed from the hyaluronic acid-modified bromophenylpyridinium (HAPY), cucurbit[n]uril (CB[n], n = 7/8), and energy acceptor phenyl-bridged phenothiazine derivatives, displaying efficient PRET and achieving near-infrared (NIR) phosphorescence by macrocyclic CB[n] and the assembly confinements. As compared with weak phosphorescent nanofibers of HAPY/CB[7], the spherical nanoparticles of HAPY/CB[8] not only gave strong green phosphorescence with extended lifetime to 1.27 ms but also could act as the energy donor and confine cationic phenothiazine in the secondary assemblies, leading to highly efficient PRET efficiency (87.27%) from the phosphors to triplet acceptors, realizing phosphorescence emission at 750 nm and an ultralarge Stokes shift of 440 nm. Ultimately, the nanoassembly achieved by the multiscale confinements boosting PRET was successfully applied in targeted cancer cell imaging, providing new insight for fabricating NIR phosphorescence materials.

Transforming waste into multifunctional nanomaterials: Mg-doped carbon dots from cow dung for Y(III) detection and biomedical applications

Rare earth metal Yttrium (Y3+) plays a vital role in many electronic devices however, discarding these devices eventually contaminates the environmental sources. Herein, we have developed a Mg-doped carbon dots (M-CDs) as a fluorescent probe for selective and sensitive determination of Y3+ in water bodies. The M-CDs having maximum emission at 420 nm upon 310 nm excitation with 20 % quantum yield and which was synthesized from upcycling of agricultural waste i.e., cow dung by simple carbonization followed by hydrothermal method. M-CDs were confirmed using different analytical and characterization techniques as well as stable in different pH and ionic strength solutions. The M-CDs was highly selective towards Y3+ ions over different metal ions with significant blue shift. This fluorescent probe performs a good linear relationship between the different concentrations of Y3+ ion and fluorescence intensity (R2 = 0.99) within the range of 0.2 to 20 μg/mL with a detection limit of 0.019 µg/mL. The study indicates quenching of Y3+ was result of dynamic quenching and the Inner Filter Effect (IFE) effect. Also, the cytotoxicity of M-CDs was checked via CAM assay and significant growth in blood vascularization with healthy growth of chick embryo confirmed the M-CDs were biocompatible. The nontoxic M-CDs was employed for MCF-7 breast cancer cell imaging which gives bright blue fluoresce under UV light excitation. Further, aiming to a circular economy approach the residual carbon remaining after hydrothermal was used as reactivated carbon for the abatement of environmental pollutants. The sustainable, cost-effective and agricultural waste-derived Mg-doped CDs have potential applications in analytical, environmental, forensic as well as biomedical fields.

Substituent groups effects on AIEgens of fluorenone derivatives: Optical properties, bioimaging, and LED devices

Understanding structure-property relationships is crucial for developing materials with a high fluorescence quantum yield (ΦF) and optimizing their performance. In this study, three fluorenone-carbazole-based molecules (FO-Cz1, FO-Cz2, and FO-Cz3) were synthesized, which have similar structures but varying substituent groups (carbazole, 3,6-di‑tert‑butyl‑9H-carbazole, and 3,6-diphenyl-9H-carbazole). The different substituent groups significantly changed the optical properties of the luminogens. The experimental results confirmed that FO-Cz1, FO-Cz2, and FO-Cz3 were AIEgens, and the introduction of a phenyl group gave FO-Cz3 strong αAIE(I/I0) and greater solid fluorescence quantum yields than FO-Cz1 and FO-Cz2. Analysis of photophysical properties, theoretical calculations, and X-ray crystallography revealed that the emission properties of FO-Cz1, FO-Cz2, and FO-Cz3 were influenced by the use of flexible substituent groups. The substituent groups avoided π-π stacking, reduced non-radiative transitions, and enhanced fluorescence in the aggregated state. Substituent groups on the benzene ring gave FO-Cz3 a tightly-packed structure and strong emission. It was used for cancer cell imaging and LED devices, in which FO-Cz3 exhibited high photostability and biocompatibility, making it a superior photosensitizer for bioimaging and applications in OLED devices. This research sheds light on the AIE behaviors of fluorenone complexes and offers valuable insights for the development of organic luminescent materials.

Thiol-linked hyaluronic acid-mediated encapsulation of RCR-stabilized gold nanoclusters for hyaluronidase sensing and cellular imaging

Encapsulating peptide-stabilized gold nanoclusters (AuNCs) with thiolated hyaluronic acid (HA-SH) and selectively adding cysteine to the peptide sequence increased their photoluminescence. We found that peptide compositions with cysteine in the middle emitted the most. RCR-stabilized AuNCs can be purified using size-exclusion chromatography to characterize their optical characteristics, chemical composition, and possible structure. Our findings show that RCR-stabilized AuNCs have a unique chemical structure, microsecond photoluminescence lifetime, good quantum yield, and near-infrared emission peak. Due to Au-S bonding and electrostatic interactions, RCR-stabilized AuNCs were encapsulated with HA-SH to create nanocomposites. HA-SH-AuNCs had a longer emission peak, greater particle size, and better photostability than RCR-stabilized AuNCs. HAase break down HA in HA-SH-AuNCs, changing their structure and size. Thus, centrifugation makes it easier to separate HA-SH-AuNCs from HAase-digested ones. Similar to earlier sensors, HA-SH-AuNCs have great sensitivity and selectivity for HAase, with a linear range of 0.5–6.0 U/mL and a detection limit of 0.39 U/mL. They were useful for urine HAase determination, with spike recovery of 103 % to 107 %. HA-SH-AuNCs further served as a platform for targeted imaging of CD44 receptor-expressing cancer cells, demonstrating bioimaging and clinical diagnostic potential.

Grafting a chromophore on AMD070 analogues for CXCR4 bioimaging: Chemical synthesis and in vitro assessment of the inhibition properties of the CXCR4 receptor

Thank to their particular pharmacokinetics, the use of small organic molecules can be a very promising alternative to macromolecular targeting biomolecules (i.e. antibodies, peptides…) for specific imaging of tumours. Herein, the potential of two AMD070-like inhibitors as CXCR4-targeting units for specific imaging of cancer cells, and the influence of chromophore-grafting on their recognition properties was investigated.

Phospholipid micelles-encapsulated perovskite nanocrystals via dual solvent exchange for human hela cervical cancer cells imaging

Polyethylene glycol-conjugated phospholipid micelles encapsulated CsPbBr3 nanocrystals were prepared through dual solvent exchange method. This strategy was applied for the first time to micellar encapsulation of CsPbBr3 perovskites, circumventing the purification step. The as-prepared sample showed high solubility and maintained high photoluminescence performance even after five days in water. More importantly, the encapsulation of phospholipid micelles toward CsPbBr3 nanocrystals greatly improved the imaging efficiency of human hela cervical cancer cells (Hela cells).

Cumulative learning based segmentation aided cell mixtures classification in digital holographic microscopy

The tumor microenvironment consists of several components like tumor cells, normal healthy cells, extracellular matrix, secreted factors, and other non-cellular components. The interaction among these components is crucial for tumor progression and metastasis. Identifying the presence of tumor cells among the normal healthy cells during the initial stages of cancer will aid in its early detection and treatment. One of the main features that differentiates a cancer cell from a normal healthy cell distinctively is its morphology. In this study, the effectiveness of machine learning algorithms is evaluated in classifying cells co-cultured in a Petri dish. To achieve this, digital holographic microscopy is used to capture the quantitative phase images of human dermal fibroblast and A375 human melanoma cancer cells co-cultured at 1:1, 1:2, and 2:1 ratios. Cell segmentation was performed using a cumulative learning approach. Subsequently, morphological and phase-based features were extracted to train the machine learning algorithms for binary classification of cells. Phase-based features in addition to the morphological features were found to provide improved performance of the classifier. The classifier demonstrated an average F1-score of 0.9 during testing.

Zebrafish: A Versatile Animal Model in Biomedical Research

Zebrafish (Danio rerio) has emerged as a powerful animal model in biomedical research, offering a unique combination of genetic tractability, rapid development, and conserved gene function with humans. This review highlights the significance of zebrafish in understanding various human diseases, including neurodegenerative disorders, metabolic disorders, cancer, and more. The zebrafish genome, with over 70% similarity to the human genome, allows for the modelling of human diseases with high fidelity. The ease of genetic manipulation, high fecundity, and low maintenance costs make zebrafish an attractive model for large-scale genetic screens and drug discovery. Zebrafish models of neurodegenerative diseases, such as Parkinson's disease, Alzheimer's disease, and Huntington's disease, have been developed, providing insights into disease mechanisms and potential therapeutic targets. Similarly, zebrafish models of metabolic disorders, including diabetes, dyslipidaemia, and atherosclerosis, have been established, enabling the study of disease pathogenesis and prevention strategies.The zebrafish has also been used to model various types of cancer, including leukaemia, melanoma, and pancreatic cancer, facilitating the discovery of novel oncogenes and tumor suppressors. The transparent nature of zebrafish embryos and larvae allows for in vivo imaging of cancer cells, enabling the study of cancer cell behavior and response to therapy. Furthermore, zebrafish has been used to investigate brain-to-organ communication, cell transplantation, and cell-cell interactions, providing insights into the complex interactions between different tissues and organs. The development of novel tools, such as CRISPR/Cas9 genome editing and single-cell RNA sequencing, has further expanded the capabilities of zebrafish research.

Spatially Selective Self-Amplified Imaging of Chemotherapy-Induced Cancer Senescence via Reversal of Impaired Ferritinophagy.

Real-time monitoring of chemotherapy-induced senescence (CIS) in cancer remains a challenging task that would lead to new insights into the adaptive mechanisms of cancer therapy and provide guidance for cancer management. Here, we designed a tailor-made nanoprobe capable of imaging CIS in a sequential activation and self-amplified manner by reversing senescence-related impaired ferritinophagy. It contains an amphipathic polymer as a spatially responsive vehicle, a Fe2+-activable dye as the reporter, and an autophagy inducer as the signal amplifier. Owing to metabolic changes, the nanoprobe preferentially enriches in senescent cancer cells, leading to in situ activation and fluorescence switching of the reporter by labile Fe2+. Meanwhile, the inducer restores ferritinophagy and promotes autophagic degradation of accumulated ferritin, facilitating conversion of ferritin-bound iron into Fe2+ for amplified imaging in senescent cancer cells yet keeping inert in nonsenescent cells. Of note, the accumulation and activation of the nanoprobe and sustained ferritin degradation occur at the same subcellular location, thus minimizing the diffusion process-induced nonspecific responses. The feasibility of this strategy is successfully demonstrated in both living cells and animal models. This work offers a new way for therapeutic evaluation and a basic understanding of the roles of senescence in cancer treatment.

The Acid-Stimulated Self-Assembled DNA Nanonetwork for Sensitive Detection and Living Cancer Cell Imaging of MicroRNA-221.

Herein, a novel functional DNA structure, acid-stimulated self-assembly DNA nanonetwork (ASDN), was proposed for miRNA-221 sensitive detection and high-resolution living cancer cell imaging. Significantly, the self-assembly of ASDN only occurred in the acidic extracellular environment of cancer cells, which could be endocytosed by cancer cells to eliminate the interference of noncancer cells and deliver the ASDN into cancer cells. Subsequently, endogenous miRNA-221 could trigger the catalytic hairpin assembly within ASDN, resulting in the separation of the fluorophore Cy5 and the quencher BHQ2 to recover the substantial Cy5 fluorescence signals, thus achieving signal amplification for sensitive detection of miRNA-221 with a detection limit of 5.5 pM, as well as facilitating high-resolution and low-background imaging of miRNA-221 in cancer cells. In consequence, this strategy provides an innovative DNA nanonetwork to distinguish cancer cells from other cells for sensitive detection of biomarkers, offering a meaningful reference for the application of DNA nanostructure self-assembly technology in relevant fundamental research and disease diagnosis.

Early detection of thermal image based T1 breast cancer using enhanced multiwavelet denoised convolution neural network with region based analysis

Medical Thermography image is used for the detection of breast cancer at an earlier stage. Thermography image shows the temperature change in the body due to cells. The metabolic rate of cancer cells is high compared to normal cells. A high metabolic rate increases the blood flow in cancer cells. High blood leads to changes in body temperature. The change in body temperature is used for cancer cell detection at an earlier stage. However, T1-stage cancer cells are smaller and have small temperature differences undetected with thermography. In this paper, a T1-stage cancer cell is heated by an external source; then, thermal images are acquired for earlier detection of small-size cancer cells. External heat source amplifies T1 stage cancer cell temperature. Amplified cancer cell images are analyzed using the proposed Multiwavelet-Deep Denoised Convolutional Neural Network (MWTDnCNN) algorithm for T1 cancer cell detection. Amplified T1 stage cancer cell has higher thermal conductivity (k) and heat capacity (Cp), which helps to detect T1 cancer cell tissue due to the enhanced pixel feature. The proposed MWTDnCNN algorithm has a T1-stage cancer cell detection accuracy of about 98% compared with traditional algorithms. The proposed MWTDnCNN algorithm detects T1-stage cancer of size 1.29 mm.

The Inhibitory Effects of Anti-GPC3 Antibody on Wnt/β-Catenin Signaling Pathway as a Biological Therapy in Liver Cancer.

To investigate the effects of anti-GPC3 antibody on the Wnt/catenin pathway in liver cancer biology, thus providing a new target for the biological treatment of the disease. A total of 12 BALB/C experimental nude mice were selected as experimental objects. The mice were all male, weighed 15-20g and aged 4-5weeks. First, mouse liver cancer models were constructed. Then, the HepG2 liver cancer cells in logarithmic growth period were inoculated into the caudal veins of mouse models. On the 3rd day after inoculation, the tumor formations of nude mice were observed. Second, the anti-GPC3 antibody was designed and constructed, and the activity of anti-GPC3 antibody was detected. The mouse models were divided into the experimental group (EG) and the control group (CG), with 6 mice in each group. In the EG, mice were injected with 3mg/kg of anti-GPC3 antibody in the caudal veins once a day for three consecutive days, while in the CG mice were injected with the same amount of saline as the control. Mice in both groups received 3 injections in total. After the last administration, all mice were euthanized using the decapitation method following anesthesia with ethyl ether. The liver cancer cells of nude mice were extracted and cultured in DMEM medium. The effects of anti-GPC3 antibody on Wnt/β-catenin in liver cancer nude mice and the effects of anti-GPC3 antibody on epithelial-mesenchymal transition (EMT) in mouse liver cancer cells were observed. The bodyweight, liver weight and index of mice in the EG increased significantly (P < 0.05). The serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) levels of mice in the EG were reduced than those in the CG (P < 0.05). Compared with the CG, the superoxide dismutase (SOD) concentration increased, while the malonaldehyde (MDA) concentration decreased in the liver tissues of mice in the EG (P < 0.05). There was binding activity between GPC3 recombinant protein and the antibody; the affinity constant was KD = 1.4 × 10-6 M, compared with the commercial anti-GPC3 antibody, the affinity constant was lower. The interference effects of siRNA on anti-GPC3 antibody were detected by Western Blot. After the injection of anti-GPC3 antibody, the expression of β-catenin siRNA in liver cancer cells decreased significantly. The optical microscope images of mouse liver cancer cells in groups were compared. Through down-regulating the Wnt/β-catenin by anti-GPC3 antibody, the morphology of epithelial cells was maintained, the cells were arranged orderly, the cubic structure was kept stable, and the occurrence of EMT was reduced. However, in the CG, the structure of mouse liver cancer cells was disordered, the obvious EMT occurred (P < 0.05). Through down-regulating the expression of Wnt/β-catenin by anti-GPC3 antibody, the invasiveness, and metastasis of liver cancer cells could be effectively inhibited. Compared with the CG, the number of cells passing through the chamber was significantly reduced (P < 0.05). The anti-GPC3 antibody had inhibitory effect on Wnt/β-catenin signaling pathway. The occurrence of EMT and the invasiveness of liver cancer cells were inhibited effectively. Therefore, the anti-GPC3 antibody could be used as a reference for clinical molecular targeted therapy of liver cancer.

Simultaneous assessment of NAD(P)H and flavins with multispectral fluorescence lifetime imaging microscopy at a single excitation wavelength of 750nm.

Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. The use of a multispectral detector in combination with an excitation at a single wavelength of 750nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.