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Lipofuscin autofluorescence reports the responses of immune-checkpoint-blockade (ICB) therapy in mice and human 3D tumor slices (TSC). A Twophoton fluorescence microscopy (λ ex = 1060 nm) of drug-induced lipofuscin fluorescence (red color) and second harmonic generation imaging of collagen networks (green color) in 3D-TSCs. Scale bar: 50 μm. B Average TPF intensities of lipofuscin fluorescence in 3D-TSCs treated with cisplatin, αPD-1, and αPD-L1 for four days. Lifetime traces of lipofuscin fluorescence in the control group, chemotherapy (cisplatin) group, and ICB therapy groups (αPD-1 and αPD-L1). C Lifetime traces of
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Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like auto-fluorescence acutely accumula...
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... explore its potential in drug sensitivity tests, we analyzed the correlation of red autofluorescence to cell apoptosis in a time-and dose-dependent manner in cisplatin-treated MDA-MB-231 cells. With the Annexin V/PI assay ( Supplementary Fig. S5A), the IC 50 dosage would be approximately 30 μM at 72 h posttreatment ( Supplementary Figs. S5B-D), which indicates MDA-MB-231 cells have high tolerance to the cisplatin. ...
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... signatures. To explore its potential in drug sensitivity tests, we analyzed the correlation of red autofluorescence to cell apoptosis in a time-and dose-dependent manner in cisplatin-treated MDA-MB-231 cells. With the Annexin V/PI assay ( Supplementary Fig. S5A), the IC 50 dosage would be approximately 30 μM at 72 h posttreatment ( Supplementary Figs. S5B-D), which indicates MDA-MB-231 cells have high tolerance to the cisplatin. Due to the overgrowth-induced cell death (Supplementary Fig. S5A), the control and sublethal groups have a constant background death rate. Examined by TPF microscopy, the control groups (0 μM, 0 h) have few red fluorescent cells in the field of view. As drug ...
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... which indicates MDA-MB-231 cells have high tolerance to the cisplatin. Due to the overgrowth-induced cell death (Supplementary Fig. S5A), the control and sublethal groups have a constant background death rate. Examined by TPF microscopy, the control groups (0 μM, 0 h) have few red fluorescent cells in the field of view. ...
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... further test how early the apoptosis fate could be determined, we washed the drug away at the 12th and 24th h post-treatment and further cultured them in a fresh medium for 24 h. The cells still showed elevated apoptosis from 5.9% (12 h w/drug) and 12.0% (24 h w/drug) to 13.1% (12 h w/+ 24 h w/o drug) and 27.4% (24 h w/+ 24 h w/o drug), respectively ( Supplementary Fig. S5E). They still followed the trend of a growing death rate after the drug withdrawal from incubation. ...
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... lipofuscin-like reporting approach to evaluate anti-cancer drug efficacy in a three-dimensional tumor slice culture (3D-TSC) model. 3D-TSC could even preserve the tumor microenvironment and immune cells, which is critical for evaluating immunecheckpoint-blockade (ICB) therapy. By imaging drug-induced lipofuscin-like fluorophore (red channel in Fig. 5A) and collagen (green color in Fig. 5A) in cells, we investigated the 3D-TSC treatment dynamics over 4 days. After treatment with 25 μM cisplatin, 2.5 μg/mL αPD-1 (anti-PD1), and 2.5 μg/mL αPD-L1 (anti-PD-L1), we also observed enhanced lipofuscin-like fluorescence intensity in the tumor slices, and the lifetime increased to a much ...
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... to evaluate anti-cancer drug efficacy in a three-dimensional tumor slice culture (3D-TSC) model. 3D-TSC could even preserve the tumor microenvironment and immune cells, which is critical for evaluating immunecheckpoint-blockade (ICB) therapy. By imaging drug-induced lipofuscin-like fluorophore (red channel in Fig. 5A) and collagen (green color in Fig. 5A) in cells, we investigated the 3D-TSC treatment dynamics over 4 days. After treatment with 25 μM cisplatin, 2.5 μg/mL αPD-1 (anti-PD1), and 2.5 μg/mL αPD-L1 (anti-PD-L1), we also observed enhanced lipofuscin-like fluorescence intensity in the tumor slices, and the lifetime increased to a much longer 950 ps under αPD-1 and αPD-1 ...
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... dynamics over 4 days. After treatment with 25 μM cisplatin, 2.5 μg/mL αPD-1 (anti-PD1), and 2.5 μg/mL αPD-L1 (anti-PD-L1), we also observed enhanced lipofuscin-like fluorescence intensity in the tumor slices, and the lifetime increased to a much longer 950 ps under αPD-1 and αPD-1 treatment compared with that of the control group (573.4 ps) (Fig. 5B). However, for cisplatin treatment, the intensity and lifetime of lipofuscinlike fluorescence were slightly lower than those of the ICB therapy groups (Fig. 5B and C). This phenomenon implies that more tumor cells were eradicated by ICB therapy than by cisplatin treatment. The stressinduced red fluorescence intensity and lifetime change ...
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... fluorescence intensity in the tumor slices, and the lifetime increased to a much longer 950 ps under αPD-1 and αPD-1 treatment compared with that of the control group (573.4 ps) (Fig. 5B). However, for cisplatin treatment, the intensity and lifetime of lipofuscinlike fluorescence were slightly lower than those of the ICB therapy groups (Fig. 5B and C). This phenomenon implies that more tumor cells were eradicated by ICB therapy than by cisplatin treatment. The stressinduced red fluorescence intensity and lifetime change are wellcorrelated with conventional cell viability assay ( Supplementary Figs. S16A and B). As a double-check, we also employed green fluorescence protein (GFP) ...
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... also applied this technique to patient-derived tumor slices of colon cancer and nasopharyngeal cancer samples. There was a significant increase in lipofuscin-like fluorescence intensity in colon cancer after treatment with αPD-L1 for 7 days ( Fig. 5D and E). But nasopharyngeal cancer tumor slice (NPC) showed different therapy responses between the two patients. For patient NPC-1, the lipofuscin-like fluorescence only slightly increased while αPD-L1 treated NPC-2 exhibited a significant increase (Fig. 5D-F). This practical method is translatable to clinical ...
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... fluorescence intensity in colon cancer after treatment with αPD-L1 for 7 days ( Fig. 5D and E). But nasopharyngeal cancer tumor slice (NPC) showed different therapy responses between the two patients. For patient NPC-1, the lipofuscin-like fluorescence only slightly increased while αPD-L1 treated NPC-2 exhibited a significant increase (Fig. 5D-F). This practical method is translatable to clinical ...
Citations
... 46 For this, shikonin has demonstrated promise as a necroptosis-inducing agent, 47 the effects of which have recently been probed using autofluorescence FLIM of the red channel attributed to lipofuscin. 30 Hence, CT26 fragments were either left untreated or treated with H 2 O 2 , heat shock, staurosporine, or shikonin 2 h after staining with either PI or Caspase 3/7 Red, an exogenous label for apoptosis. Spatial maps of the NMS and corresponding PI images comparing treated and untreated fragments are shown in Figs. ...
Significance: To enable non-destructive longitudinal assessment of drug agents in intact tumor tissue without the use of disruptive probes, we have designed a label-free method to quantify the health of individual tumor cells in excised tumor tissue using multiphoton fluorescence lifetime imaging microscopy (MP-FLIM).
Aim: Using murine tumor fragments which preserve the native tumor microenvironment, we seek to demonstrate signals generated by the intrinsically fluorescent metabolic co-factors nicotinamide adenine dinucleotide phosphate [NAD(P)H] and flavin adenine dinucleotide (FAD) correlate with irreversible cascades leading to cell death.
Approach: We use MP-FLIM of NAD(P)H and FAD on tissues and confirm viability using standard apoptosis and live/dead (Caspase 3/7 and propidium iodide, respectively) assays.
Results: Through a statistical approach, reproducible shifts in FLIM data, determined through phasor analysis, are shown to correlate with loss of cell viability. With this, we demonstrate that cell death achieved through either apoptosis/necrosis or necroptosis can be discriminated. In addition, specific responses to common chemotherapeutic treatment inducing cell death were detected.
Conclusions: These data demonstrate that MP-FLIM can detect and quantify cell viability without the use of potentially toxic dyes, thus enabling longitudinal multi-day studies assessing the effects of therapeutic agents on tumor fragments.
... Proliferation migration was reflected by fluorescence intensity and the number of cells transferred to the torso. Some drugs not only reduce the growth of the primary tumor [97] but also effectively control the proliferation and metastasis process [66]. ...
Developing anticancer drugs is a complex and time-consuming process. The inability of current laboratory models to reflect important aspects of the tumor in vivo limits anticancer medication research. Zebrafish is a rapid, semi-automated in vivo screening platform that enables the use of non-invasive imaging methods to monitor morphology, survival, developmental status, response to drugs, locomotion, or other behaviors. Zebrafish models are widely used in drug discovery and development for anticancer drugs, especially in conjunction with live imaging techniques. Herein, we concentrated on the use of zebrafish live imaging in anticancer therapeutic research, including drug screening, efficacy assessment, toxicity assessment, and mechanism studies. Zebrafish live imaging techniques have been used in numerous studies, but this is the first time that these techniques have been comprehensively summarized and compared side by side. Finally, we discuss the hypothesis of Zebrafish Composite Model, which may provide future directions for zebrafish imaging in the field of cancer research.
... (d) 9 -color composite images presented by multiplex immunofluorescence provide a powerful tool for studying cell interactions and the tumor microenvironment [92] , with scale bar of 100 μm 特邀综述 第 51 卷 第 9 期/2024 年 5 月/中国激光 organoid levels based on autofluorescence from the reduced form of NAD(P)H [94] , with scale bar of 400 μm; (b) twophoton fluorescence microscopy for detecting collagen, lipofuscin, and flavin autofluorescence in tissue models can predict tumor responses to chemotherapy and immunotherapy [95] , with scale bar of 24 μm; (c) obtaining lipofuscin autofluorescence lifetime parameter through twophoton fluorescence imaging and fluorescence lifetime imaging (FLIM) techniques can distinguish between apoptosis and necrosis in individual cell level [96] , with scale bar of 50 μm 特邀综述 第 51 卷 第 9 期/2024 年 5 月/中国激光 ...
... Trinh et al. have demonstrated that the NAD(P)H fluorescence lifetime (NAD(P)H bound/free ratio) increases as cancer cells become less proliferative as a result of drug treatment [114], thus indicating their drug sensitivity. Similarly, Yan et al. have reported a FLIM based on lipofuscin-like autofluorescence, which shows acute accumulation during the cell death process and can distinguish necrosis from apoptosis [94]. ...
Simple Summary
Studies aimed at prediction of chemotherapeutic efficacy using patient-derived ex vivo cultures (referred to here as “functional testing”) have been increasing. The present review provides information on the various types of ex vivo cultures and endpoint assays that employ a range of surrogate biomarkers of drug response. As ex vivo cultures for functional testing, two-dimensional cultures, spheroids, organoids, explants (including histoculture), microfluid-based culture, and micro-organospheres are introduced. The endpoint assays described include ATP-based bulk assay, dynamic BH3 profiling, optical metabolic imaging, fluorescence lifetime imaging microscopy, fluorescent dye-based assay, mass accumulation rate assay, live cell imaging-based assay, and immunostaining for drug-specific response biomarkers. The advantages and disadvantages of these culture systems and endpoint assays are discussed.
Abstract
Prediction of therapeutic outcomes is important for cancer patients in order to reduce side effects and improve the efficacy of anti-cancer drugs. Currently, the most widely accepted method for predicting the efficacy of anti-cancer drugs is gene panel testing based on next-generation sequencing. However, gene panel testing has several limitations. For example, only 10% of cancer patients are estimated to have druggable mutations, even if whole-exome sequencing is applied. Additionally, even if optimal drugs are selected, a significant proportion of patients derive no benefit from the indicated drug treatment. Furthermore, most of the anti-cancer drugs selected by gene panel testing are molecularly targeted drugs, and the efficacies of cytotoxic drugs remain difficult to predict. Apart from gene panel testing, attempts to predict chemotherapeutic efficacy using ex vivo cultures from cancer patients have been increasing. Several groups have retrospectively demonstrated correlations between ex vivo drug sensitivity and clinical outcome. For ex vivo culture, surgically resected tumor tissue is the most abundant source. However, patients with recurrent or metastatic tumors do not usually undergo surgery, and chemotherapy may be the only option for those with inoperable tumors. Therefore, predictive methods using small amounts of cancer tissue from diagnostic materials such as endoscopic, fine-needle aspirates, needle cores and liquid biopsies are needed. To achieve this, various types of ex vivo culture and endpoint assays using effective surrogate biomarkers of drug sensitivity have recently been developed. Here, we review the variety of ex vivo cultures and endpoint assays currently available.
... LF can be excited with broad UV-VIS laser lines in confocal fluorescence microscopy, and it fluoresces with a spectrum covering 570-605 nm (Jung et al. 2009;Croce et al. 2016). LF can also be excited using 2P-microscopy at 1060 nm, with detection ranging from 604-679 nm (Yan et al. 2023). Combining 2P excitation with FLIM, LF autofluorescence has been shown to discriminate between necrosis and apoptosis with single-cell resolution (Yan et al. 2023). ...
... LF can also be excited using 2P-microscopy at 1060 nm, with detection ranging from 604-679 nm (Yan et al. 2023). Combining 2P excitation with FLIM, LF autofluorescence has been shown to discriminate between necrosis and apoptosis with single-cell resolution (Yan et al. 2023). Several results have suggested heterogeneity in LF spectral properties (Marmorstein et al. 2002;Warburton et al. 2007;Yakovleva et al. 2020). ...
Over the past decade, the utilization of advanced fluorescence microscopy technologies has presented numerous opportunities to study or re-investigate autofluorescent molecules and harmonic generation signals as molecular biomarkers and biosensors for in vivo cell and tissue studies. The label-free approaches benefit from the endogenous fluorescent molecules within the cell and take advantage of their spectroscopy properties to address biological questions. Harmonic generation can be used as a tool to identify the occurrence of fibrillar or lipid deposits in tissues, by using second and third-harmonic generation microscopy. Combining autofluorescence with novel techniques and tools such as fluorescence lifetime imaging microscopy (FLIM) and hyperspectral imaging (HSI) with model-free analysis of phasor plots has revolutionized the understanding of molecular processes such as cellular metabolism. These tools provide quantitative information that is often hidden under classical intensity-based microscopy. In this short review, we aim to illustrate how some of these technologies and techniques may enable investigation without the need to add a foreign fluorescence molecule that can modify or affect the results. We address some of the most important autofluorescence molecules and their spectroscopic properties to illustrate the potential of these combined tools. We discuss using them as biomarkers and biosensors and, under the lens of this new technology, identify some of the challenges and potentials for future advances in the field.
... But these light sources also excite autofluorescence from NAD(P)H and FAD, which will interfere with the lipofuscin quantification. Here, with near-infrared two-photon excitation at 1040 nm, we could selectively excite the red-end autofluorescence of lipofuscin and avoid the co-excitation problems [57,58]. That's how we successfully identify the specific lipofuscin-like red autofluorescence in macrophages. ...
Rationale: Prediabetes can be reversed through lifestyle intervention, but its main pathologic hallmark, insulin resistance (IR), cannot be detected as conveniently as blood glucose testing. In consequence, the diagnosis of prediabetes is often delayed until patients have hyperglycemia. Therefore, developing a less invasive diagnostic method for rapid IR evaluation will contribute to the prognosis of prediabetes. Adipose tissue is an endocrine organ that plays a crucial role in the development and progression of prediabetes. Label-free visualizing the prediabetic microenvironment of adipose tissues provides a less invasive alternative for the characterization of IR and inflammatory pathology. Methods: Here, we successfully identified the differentiable features of prediabetic adipose tissues by employing the metabolic imaging of three endogenous fluorophores NAD(P)H, FAD, and lipofuscin-like pigments. Results: We discovered that 1040-nm excited lipofuscin-like autofluorescence could mark the location of macrophages. This unique feature helps separate the metabolic fluorescence signals of macrophages from those of adipocytes. In prediabetes fat tissues with IR, we found only adipocytes exhibited a low redox ratio of metabolic fluorescence and high free NAD(P)H fraction a1. This differential signature disappears for mice who quit the high-fat diet or high-fat-high-sucrose diet and recover from IR. When mice have diabetic hyperglycemia and inflamed fat tissues, both adipocytes and macrophages possess this kind of metabolic change. As confirmed with RNA-seq analysis and histopathology evidence, the change in adipocyte's metabolic fluorescence could be an indicator or risk factor of prediabetic IR. Conclusion: Our study provides an innovative approach to diagnosing prediabetes, which sheds light on the strategy for diabetes prevention.
Low physical performance is closely associated with skeletal muscle dysfunction and metabolic disorders such as
obesity, diabetes and cardiovascular disease. The aim of the present study was to characterize the differences in liver
mitochondrial function in an aged rat model with congenital low and high running capacity (LCR/HCR) of generation
44. In addition to monitoring basal and succinate-mediated H2O2-induced fluorescence, NAD(P)H and lipofuscin
fluorescence lifetime imaging (FLIM) was applied in frozen rat liver tissue. Reduced VO2max, increased muscle wasting
and increased body mass were observed in LCR compared to HCR rats. Succinate load suggested a deterioration of
intact liver mitochondria in LCR rats: ROS production was greater, accompanied by a limited NADH increase at the
same mitochondrial membrane potential (DYm). Complex I- and Complex II-driven ADP-coupled ATP production was
the same. The NAD(P)H lifetime of cytosol at LCR rats significantly shifted toward free NAD(P)H lifetime values
(Gmax= 0,60 ± 0,03) compared to normal young rats (Gmax= 0,54 ± 0,04) indicating a sensitive transition from oxidative
phosphorylation to glycolysis. Interestingly, the shift was more pronounced in case of HCR rats (Gmax= 0,69 ± 0,03). In
conclusion, age shifts metabolism from oxidative phosphorylation to glycolysis in liver. The beneficial epigenetic
difference coupled to high running capacity helps to slow the deterioration of physical and mitochondrial fitness, as
reflected in the irreversible accumulation of the oxidative stress marker lipofuscin. Accordingly, NAD(P)H and
lipofuscin FLIM imaging may provide a sensitive, predictive approach to study early effects of metabolic syndrome and
ageing.
Keywords: Ageing, NAD(P)H FLIM, Lipofuscin, Epigenetics, Metabolic syndrome, Mitochondria, Liver
