RESUMEN
Proton export is often considered a detoxifying process in animal cells, with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH, thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels, NADPH/NADP+ ratio, and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy, where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth.
Asunto(s)
Carbono/metabolismo , Proliferación Celular , Ácido Láctico/metabolismo , Leucemia Mieloide Aguda/metabolismo , Animales , Transformación Celular Neoplásica/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Ratones Endogámicos C57BL , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/metabolismo , Vía de Pentosa Fosfato , Protones , Células Tumorales CultivadasRESUMEN
Sickle cell disease (SCD) is a monogenic red blood cell (RBC) disorder with high morbidity and mortality. Here, we report, for the first time, the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vascular network of increased numbers of highly tortuous arterioles occupying the majority of the BM cavity, as well as fragmented sinusoidal vessels filled with aggregates of erythroid and myeloid cells. By in vivo imaging, sickle and control RBCs have significantly slow intravascular flow speeds in sickle cell BM but not in control BM. In sickle cell BM, we find increased reactive oxygen species production in expanded erythroblast populations and elevated levels of HIF-1α. The SCD BM exudate exhibits increased levels of proangiogenic growth factors and soluble vascular cell adhesion molecule-1. Transplantation of SCD mouse BM cells into wild-type mice recapitulates the SCD vascular phenotype. Our data provide a model of SCD BM, in which slow RBC flow and vaso-occlusions further diminish local oxygen availability in the physiologic hypoxic BM cavity. These events trigger a milieu that is conducive to aberrant vessel growth. The distorted neovascular network is completely reversed by a 6-week blood transfusion regimen targeting hemoglobin S to <30%, highlighting the plasticity of the vascular niche. A better insight into the BM microenvironments in SCD might provide opportunities to optimize approaches toward efficient and long-term hematopoietic engraftment in the context of curative therapies.
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Anemia de Células Falciformes/complicaciones , Transfusión Sanguínea/métodos , Médula Ósea/patología , Eritrocitos Anormales/patología , Hematopoyesis , Neovascularización Patológica/prevención & control , Esplenomegalia/prevención & control , Animales , Médula Ósea/metabolismo , Eritrocitos Anormales/metabolismo , Femenino , Humanos , Masculino , Ratones , Neovascularización Patológica/etiología , Neovascularización Patológica/patología , Esplenomegalia/etiología , Esplenomegalia/patologíaRESUMEN
Bone marrow endothelial cells (BMECs) form a network of blood vessels that regulate both leukocyte trafficking and haematopoietic stem and progenitor cell (HSPC) maintenance. However, it is not clear how BMECs balance these dual roles, and whether these events occur at the same vascular site. We found that mammalian bone marrow stem cell maintenance and leukocyte trafficking are regulated by distinct blood vessel types with different permeability properties. Less permeable arterial blood vessels maintain haematopoietic stem cells in a low reactive oxygen species (ROS) state, whereas the more permeable sinusoids promote HSPC activation and are the exclusive site for immature and mature leukocyte trafficking to and from the bone marrow. A functional consequence of high permeability of blood vessels is that exposure to blood plasma increases bone marrow HSPC ROS levels, augmenting their migration and differentiation, while compromising their long-term repopulation and survival. These findings may have relevance for clinical haematopoietic stem cell transplantation and mobilization protocols.
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Vasos Sanguíneos/citología , Vasos Sanguíneos/fisiología , Médula Ósea/irrigación sanguínea , Hematopoyesis , Animales , Antígenos Ly/metabolismo , Arterias/citología , Arterias/fisiología , Células de la Médula Ósea/citología , Diferenciación Celular , Movimiento Celular , Autorrenovación de las Células , Supervivencia Celular , Quimiocina CXCL12/metabolismo , Células Endoteliales/fisiología , Femenino , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/citología , Leucocitos/citología , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Nestina/metabolismo , Pericitos/fisiología , Permeabilidad , Plasma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores CXCR4/metabolismoRESUMEN
RATIONALE: Inflammatory stress induced by exposure to bacterial lipopolysaccharide causes hematopoietic stem cell expansion in the bone marrow niche, generating a cellular immune response. As an integral component of the hematopoietic stem cell niche, the bone marrow vasculature regulates the production and release of blood leukocytes, which protect the host against infection but also fuel inflammatory diseases. OBJECTIVE: We aimed to develop imaging tools to explore vascular changes in the bone marrow niche during acute inflammation. METHODS AND RESULTS: Using the TLR (Toll-like receptor) ligand lipopolysaccharide as a prototypical danger signal, we applied multiparametric, multimodality and multiscale imaging to characterize how the bone marrow vasculature adapts when hematopoiesis boosts leukocyte supply. In response to lipopolysaccharide, ex vivo flow cytometry and histology showed vascular changes to the bone marrow niche. Specifically, proliferating endothelial cells gave rise to new vasculature in the bone marrow during hypoxic conditions. We studied these vascular changes with complementary intravital microscopy and positron emission tomography/magnetic resonance imaging. Fluorescence and positron emission tomography integrin αVß3 imaging signal increased during lipopolysaccharide-induced vascular remodeling. Vascular leakiness, quantified by albumin-based in vivo microscopy and magnetic resonance imaging, rose when neutrophils departed and hematopoietic stem and progenitor cells proliferated more vigorously. CONCLUSIONS: Introducing a tool set to image bone marrow either with cellular resolution or noninvasively within the entire skeleton, this work sheds light on angiogenic responses that accompany emergency hematopoiesis. Understanding and monitoring bone marrow vasculature may provide a key to unlock therapeutic targets regulating systemic inflammation.
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Médula Ósea/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Tomografía de Emisión de Positrones/métodos , Nicho de Células Madre , Estrés Fisiológico , Animales , Médula Ósea/patología , Células Progenitoras Endoteliales/citología , Femenino , Inflamación/diagnóstico por imagen , Integrina alfaVbeta3/metabolismo , Lipopolisacáridos/toxicidad , Ratones , Ratones Endogámicos C57BL , Imagen Multimodal/métodosRESUMEN
BACKGROUND: Candida albicans is a dimorphic fungus to which human subjects are exposed early in life, and by adulthood, it is part of the mycobiome of skin and other tissues. Neonatal skin lacks resident memory T (TRM) cells, but in adults the C albicans skin test is a surrogate for immunocompetence. Young adult mice raised under specific pathogen-free conditions are naive to C albicans and have been shown recently to have an immune system resembling that of neonatal human subjects. OBJECTIVE: We studied the evolution of the adaptive cutaneous immune response to Candida species. METHODS: We examined both human skin T cells and the de novo and memory immune responses in a mouse model of C albicans skin infection. RESULTS: In mice the initial IL-17-producing cells after C albicans infection were dermal γδ T cells, but by day 7, αß TH17 effector T cells were predominant. By day 30, the majority of C albicans-reactive IL-17-producing T cells were CD4 TRM cells. Intravital microscopy showed that CD4 effector T cells were recruited to the site of primary infection and were highly motile 10 days after infection. Between 30 and 90 days after infection, these CD4 T cells became increasingly sessile, acquired expression of CD69 and CD103, and localized to the papillary dermis. These established TRM cells produced IL-17 on challenge, whereas motile migratory memory T cells did not. TRM cells rapidly clear an infectious challenge with C albicans more effectively than recirculating T cells, although both populations participate. We found that in normal human skin IL-17-producing CD4+ TRM cells that responded to C albicans in an MHC class II-restricted fashion could be identified readily. CONCLUSIONS: These studies demonstrate that C albicans infection of skin preferentially generates CD4+ IL-17-producing TRM cells, which mediate durable protective immunity.
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Candida albicans/fisiología , Candidiasis/inmunología , Piel/inmunología , Subgrupos de Linfocitos T/fisiología , Células Th17/fisiología , Inmunidad Adaptativa , Adulto , Animales , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Inmunocompetencia , Memoria Inmunológica , Recién Nacido , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Piel/microbiologíaRESUMEN
Mitochondrial fatty acid oxidation (FAO) is essential for hematopoietic stem cell (HSC) self-renewal; however, the mechanism by which mitochondrial metabolism controls HSC fate remains unknown. Here, we show that within the hematopoietic lineage, HSCs have the largest mitochondrial NADPH pools, which are required for proper HSC cell fate and homeostasis. Bioinformatic analysis of the HSC transcriptome, biochemical assays, and genetic inactivation of FAO all indicate that FAO-generated NADPH fuels cholesterol synthesis in HSCs. Interference with FAO disturbs the segregation of mitochondrial NADPH toward corresponding daughter cells upon single HSC division. Importantly, we have found that the FAO-NADPH-cholesterol axis drives extracellular vesicle (EV) biogenesis and release in HSCs, while inhibition of EV signaling impairs HSC self-renewal. These data reveal the existence of a mitochondrial NADPH-cholesterol axis for EV biogenesis that is required for hematopoietic homeostasis and highlight the non-stochastic nature of HSC fate determination.
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Vesículas Extracelulares , Células Madre Hematopoyéticas , NADP/metabolismo , Células Madre Hematopoyéticas/metabolismo , Diferenciación Celular/fisiología , Autorrenovación de las CélulasRESUMEN
Lipid droplets are complex organelles that exhibit highly dynamic behavior in early Drosophila embryo development. Imaging lipid droplet motion provides a robust platform for the investigation of shuttling by kinesin and dynein motors, but methods for imaging are either destructive or deficient in resolution and penetration to study large populations of droplets in an individual embryo. Here we report real-time imaging and quantification of droplet motion in live embryos using a recently developed technique termed "femtosecond-stimulated Raman loss" microscopy. We captured long-duration time-lapse images of the developing embryo, tracked single droplet motion within large populations of droplets, and measured the velocity and turning frequency of each particle at different apical-to-basal depths and stages of development. To determine whether the quantities for speed and turning rate measured for individual droplets are sufficient to predict the population distributions of droplet density, we simulated droplet motion using a velocity-jump model. This model yielded droplet density distributions that agreed well with experimental observations without any model optimization or unknown parameter estimation, demonstrating the sufficiency of a velocity-jump process for droplet trafficking dynamics in blastoderm embryos.
Asunto(s)
Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Espacio Intracelular/metabolismo , Metabolismo de los Lípidos , Microscopía/métodos , Movimiento , Espectrometría Raman , Animales , Desarrollo Embrionario , Modelos Biológicos , Factores de TiempoRESUMEN
A major challenge in creating and optimizing therapeutics in the fight against cancer is visualizing and understanding the microscale spatiotemporal treatment response dynamics that occur in patients. This is especially true for photodynamic therapy (PDT), where therapeutic optimization relies on understanding the interplay between factors such as photosensitizer localization and uptake, in addition to light dose and delivery rate. In vitro 3D culture systems that recapitulate many of the biological features of human disease are powerful platforms for carrying out detailed studies on PDT response and resistance. Current techniques for visualizing these models, however, often lack accuracy due to the perturbative nature of the sample preparation, with light attenuation complicating the study of intact models. Optical coherence tomography (OCT) is an ideal method for the long-term, non-perturbative study of in vitro models and their response to PDT. Monitoring the response of 3D models to PDT by time-lapse OCT methods promises to provide new perspectives and open the way to cancer treatment methodologies that can be translated towards the clinic.
RESUMEN
Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ's microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.
RESUMEN
Advances in intravital microscopy (IVM) have enabled the studies of cellular organization and dynamics in the native microenvironment of intact organisms with minimal perturbation. The abilities to track specific cell populations and monitor their interactions have opened up new horizons for visualizing cell biology in vivo, yet the success of standard fluorescence cell labeling approaches for IVM comes with a "dark side" in that unlabeled cells are invisible, leaving labeled cells or structures to appear isolated in space, devoid of their surroundings and lacking proper biological context. Here we describe a novel method for "filling in the void" by harnessing the ubiquity of extracellular (interstitial) fluid and its ease of fluorescence labelling by commonly used vascular and lymphatic tracers. We show that during routine labeling of the vasculature and lymphatics for IVM, commonly used fluorescent tracers readily perfuse the interstitial spaces of the bone marrow (BM) and the lymph node (LN), outlining the unlabeled cells and forming negative contrast images that complement standard (positive) cell labeling approaches. The method is simple yet powerful, offering a comprehensive view of the cellular landscape such as cell density and spatial distribution, as well as dynamic processes such as cell motility and transmigration across the vascular endothelium. The extracellular localization of the dye and the interstitial flow provide favorable conditions for prolonged Intravital time lapse imaging with minimal toxicity and photobleaching.
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Medios de Contraste/química , Microscopía Intravital , Animales , Automatización , Médula Ósea/diagnóstico por imagen , Femenino , Colorantes Fluorescentes/química , Ganglios Linfáticos/diagnóstico por imagen , Masculino , Ratones Endogámicos C57BL , Microscopía Fluorescente , Flujo Sanguíneo Regional , Factores de TiempoRESUMEN
Acute myeloid leukemia (AML) is a high remission, high relapse fatal blood cancer. Although mTORC1 is a master regulator of cell proliferation and survival, its inhibitors have not performed well as AML treatments. To uncover the dynamics of mTORC1 activity in vivo, fluorescent probes are developed to track single cell proliferation, apoptosis and mTORC1 activity of AML cells in the bone marrow of live animals and to quantify these activities in the context of microanatomical localization and intra-tumoral heterogeneity. When chemotherapy drugs commonly used clinically are given to mice with AML, apoptosis is rapid, diffuse and not preferentially restricted to anatomic sites. Dynamic measurement of mTORC1 activity indicated a decline in mTORC1 activity with AML progression. However, at the time of maximal chemotherapy response, mTORC1 signaling is high and positively correlated with a leukemia stemness transcriptional profile. Cell barcoding reveals the induction of mTORC1 activity rather than selection of mTORC1 high cells and timed inhibition of mTORC1 improved the killing of AML cells. These data define the real-time dynamics of AML and the mTORC1 pathway in association with AML growth, response to and relapse after chemotherapy. They provide guidance for timed intervention with pathway-specific inhibitors.
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Leucemia Mieloide Aguda/tratamiento farmacológico , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación hacia Abajo , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Modelos Biológicos , Células 3T3 NIH , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Transcriptoma/genética , Resultado del TratamientoRESUMEN
In spite of the outstanding properties of single-walled carbon nanotubes (SWNTs), the coexistence of metallic and semiconducting SWNTs as a result of synthesis has hindered their electronic and photonic applications. We demonstrate a pump-probe microscopy method for fast, contact-free mapping of metallicity in individual SWNTs. We employ the phase of transient absorption as a contrast to discriminate metallic and semiconducting SWNTs. Furthermore, we have clarified the phase dependence on the pump or probe wavelengths and the energy structure of SWNTs. Our imaging method holds the potential of serving as a high-speed metallicity-mapping tool to assist the development of SWNT-based nanoelectronics.
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Metales/química , Microscopía/instrumentación , Nanotecnología/métodos , Nanotubos de Carbono/química , Fenómenos Ópticos , Absorción , SemiconductoresRESUMEN
Proper chemical imaging tools are critical to the pharmaceutical industry due to growing regulatory demand for intermediate and end-product content uniformity testing. Herein we demonstrate stimulated Raman scattering (SRS) imaging of active pharmaceutical ingredient (API) and four excipients within tablets. Tablets from six manufactures were imaged with a speed of 53 s per frame of 512 × 512 pixels (i.e., 200 µs per pixel) and a lateral spatial resolution as high as 0.62 µm. The SRS chemical imaging was compared to confocal Raman mapping and coherent anti-Stokes Raman scattering (CARS) chemical imaging in terms of speed and chemical selectivity. The acquisition speed of SRS imaging is ca. 10(4) times faster than confocal Raman mapping and SRS technique showed superior to CARS chemical selectivity for studied samples. Our data demonstrate the potential of SRS microscopy in high-speed screening of pharmaceutical solid dosage forms.
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Espectrometría Raman/métodos , Comprimidos/química , Amlodipino/análisis , Microscopía Confocal/métodos , Preparaciones Farmacéuticas/química , VibraciónRESUMEN
Understanding of cellular interactions with a nanostructure requires tracking directly the nanostructure. Current investigation is challenged by the lack of a strong, intrinsic signal from the nanostructure. We demonstrate intensive four-wave mixing and third-harmonic generation signals from dimension-controllable silicon nanowires as small as 5 nm in diameter. The nonlinear optical signals observed from the nanowires are highly photostable with an intensity level of 10 times larger than that observed from silver nanoparticles of comparable sizes. This intrinsic optical signal enabled intravital imaging of nanowires circulating in the peripheral blood of a mouse and mapping of nanowires accumulated in the liver and spleen, opening up further opportunities to investigate in vivo cellular response to nanomaterials as a function of size, aspect ratio, and surface chemistry.
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Hígado/metabolismo , Nanocables/análisis , Silicio/análisis , Bazo/metabolismo , Animales , Fluorescencia , Hígado/química , Ratones , Nanomedicina , Silicio/metabolismo , Bazo/químicaRESUMEN
Multimodal nonlinear optical microscopy is a valuable tool to study complex biological samples. We present an easy-to-operate approach to perform coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), second harmonic generation (SHG), and third-harmonic generation (THG) imaging using a single laser source composed of an 80 MHz femtosecond (fs) laser, an optical parametric oscillator (OPO), and a PPLN crystal for frequency doubling. The platform allows vibrationally resonant CARS imaging of CH-rich myelin sheath in fresh spinal tissues and lipid bodies in live cells. Multimodal nonlinear optical imaging and microspectroscopy analysis of fresh liver tissues are demonstrated.
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Microscopía/métodos , Microespectrofotometría/métodos , Dinámicas no Lineales , Animales , Membrana Celular/química , Conducta Alimentaria , Rayos Láser , Lípidos/química , Hígado/fisiología , Nanopartículas/química , Espectrometría Raman , Tejido Subcutáneo/fisiología , Titanio/química , VibraciónRESUMEN
Skates and rays are commercially important fish in South Korea, and among them, Beringraja pulchra has the highest economic value. However, the similar morphological traits among skates and rays are often exploited for seafood fraud. Here, we designed both Beringraja pulchra-specific and skate-universal primer sets, capable of detecting short sequences in the cytochrome oxidase subunit I gene, and developed highly sensitive and reliable quantitative real-time PCR (qPCR) assays to differentiate between Beringraja pulchra and other skate and ray species. AΔCq method based on differences in the amplification efficiency was developed, validated, and then used to confirm the presence of Beringraja pulchra in twenty-six commercial skate products. The averageΔCq value obtained for other skate species (18.94⯱â¯3.46) was significantly higher than that of Beringraja pulchra (1.18⯱â¯0.15). For on-site applications, we developed an ultra-fast qPCR assay, allowing for completion of the entire analytical procedure within 30â¯min.
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Productos Pesqueros/análisis , Análisis de los Alimentos/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Rajidae/genética , Animales , Cartilla de ADN , Complejo IV de Transporte de Electrones/genética , Proteínas de Peces/genética , Reproducibilidad de los Resultados , República de Corea , Especificidad de la EspecieRESUMEN
The bone marrow is a unique microenvironment where blood cells are produced and released into the circulation. At the top of the blood cell lineage are the hematopoietic stem cells (HSC), which are thought to reside in close association with the bone marrow vascular endothelial cells (Morrison and Scadden, Nature 505:327-334, 2014). Recent efforts at characterizing the HSC niche have prompted us to make close examinations of two distinct types of blood vessel in the bone marrow, the arteriolar vessels originating from arteries and sinusoidal vessels connected to veins. We found the two vessel types to exhibit different vascular permeabilites, hemodynamics, cell trafficking behaviors, and oxygen content (Itkin et al., Nature 532:323-328, 2016; Spencer et al., Nature 508:269-273, 2014). Here, we describe a method to quantitatively measure the permeability and hemodynamics of arterioles and sinusoids in murine calvarial bone marrow using intravital microscopy.
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Arteriolas/citología , Médula Ósea/crecimiento & desarrollo , Capilares/citología , Permeabilidad Capilar , Células Madre Hematopoyéticas/citología , Hemodinámica , Microscopía Intravital/métodos , Animales , Arteriolas/metabolismo , Médula Ósea/metabolismo , Capilares/metabolismo , Movimiento Celular , Células Madre Hematopoyéticas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Three-dimensional in vitro tumor models are highly useful tools for studying tumor growth and treatment response of malignancies such as ovarian cancer. Existing viability and treatment assessment assays, however, face shortcomings when applied to these large, complex, and heterogeneous culture systems. Optical coherence tomography (OCT) is a noninvasive, label-free, optical imaging technique that can visualize live cells and tissues over time with subcellular resolution and millimeters of optical penetration depth. Here, we show that OCT is capable of carrying out high-content, longitudinal assays of 3D culture treatment response. We demonstrate the usage and capability of OCT for the dynamic monitoring of individual and combination therapeutic regimens in vitro, including both chemotherapy drugs and photodynamic therapy (PDT) for ovarian cancer. OCT was validated against the standard LIVE/DEAD Viability/Cytotoxicity Assay in small tumor spheroid cultures, showing excellent correlation with existing standards. Importantly, OCT was shown to be capable of evaluating 3D spheroid treatment response even when traditional viability assays failed. OCT 3D viability imaging revealed synergy between PDT and the standard-of-care chemotherapeutic carboplatin that evolved over time. We believe the efficacy and accuracy of OCT in vitro drug screening will greatly contribute to the field of cancer treatment and therapy evaluation.
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Antineoplásicos/farmacología , Rastreo Celular/métodos , Neoplasias Ováricas/tratamiento farmacológico , Fármacos Fotosensibilizantes/farmacología , Esferoides Celulares/efectos de los fármacos , Tomografía de Coherencia Óptica/métodos , Carboplatino/farmacología , Técnicas de Cultivo de Célula , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Combinación de Medicamentos , Sinergismo Farmacológico , Femenino , Humanos , Imagenología Tridimensional/métodos , Modelos Biológicos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fotoquimioterapia , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Esferoides Celulares/ultraestructura , Tiazinas/farmacología , Células Tumorales CultivadasRESUMEN
Gelatin, a purified protein derived mostly from pig skin and bovine tissue, is used widely in both food and pharmaceutical industries. Here, to determine the species of origin of capsule gelatin, we developed a sensitive and reliable test using the polymerase chain reaction (PCR) method, which included 1) species-specific or universal primer sets, designed to detect short 16S ribosomal RNA (rRNA) gene sequences from cow, pig, and fish (tilapia) as well as genes encoding the large subunit of plant ribulose-1,5-bisphosphate carboxylase oxygenase and 2) species-specific PCR coupled with whole-genome amplification. This method was used to verify manufacturing label claims of 28 gelatin capsule samples sold as dietary supplements. The results from 27 samples were consistent with gelatin-related information on the manufacturer label, while one sample that mentioned tilapia gelatin was found to contain only bovine DNA. This rapid method can therefore be used to verify the authenticity of gelatin capsules.