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The article from this special issue was previously published in NMR In Biomedicine , Volume 35, Issue 9, 2022. For completeness we are including the title page of the article below. The full text of the article can be read in Issue 35:9 on Wiley Online Library: https://doi.org/10.1002/nbm.4757.
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Imagen por Resonancia Magnética , Protones , Humanos , Animales , Aminas/química , Técnicas de Cultivo de Célula , Células HEK293 , Imagen por Resonancia Magnética/métodos , Fantasmas de ImagenRESUMEN
Metabolic responses to physiological changes have been detected using chemical exchange saturation transfer (CEST) imaging in clinical settings. Similarly to other MRI techniques, the CEST technique was based originally on phantoms from buffer solutions and was then further developed through animal experiments. However, CEST imaging can capture certain dynamics of metabolism that solution phantoms cannot model. Cell culture phantoms can fill the gap between buffer phantoms and animal models. In this study, we used 1 H NMR and CEST in a B0 field of 9.4 T to investigate HEK293T cells from two-dimensional (2D) cultures, three-dimensional (3D) cultures, and 3D cultures seeded with cell spheroids. Two CEST dips were observed: the magnitude of the amine dip at 2.8 ppm increased during the incubation period, whereas the hydroxyl dip at 1.2 ppm remained approximately the same or modestly increased. We also observed a CEST dip at 2.8 ppm from the 2D culture responding dramatically to doxorubicin treatment. By cross-validating with pH values and the concentrations of amine and hydroxyl protons extracted through 1 H NMR, we observed that they did not correspond to an increase in the amine pool. We believe that the denaturation or degradation of proteins from the fetal bovine serum increased the size of the amine pool. Although 3D culture conditions can be further improved, our study suggests that 3D cultures have the potential to bridge studies of solution phantoms and those on animals.
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Imagen por Resonancia Magnética , Protones , Aminas/química , Animales , Técnicas de Cultivo de Célula , Células HEK293 , Humanos , Imagen por Resonancia Magnética/métodos , Fantasmas de ImagenRESUMEN
In chemical exchange saturation transfer (CEST) imaging, the signal at 2.6 ppm from the water resonance in muscle has been assigned to phosphocreatine (PCr). However, this signal has limited specificity for PCr since the signal is also sensitive to exchange with protein and macromolecular protons when using some conventional quantification methods, and will vary with changes in the water longitudinal relaxation rate. Correcting for these effects while maintaining reasonable acquisition times is challenging. As an alternative approach to overcome these problems, here we evaluate chemical exchange rotation transfer (CERT) imaging of PCr in muscle at 9.4 T. Specifically, the CERT metric, AREXdouble,cpw at 2.6 ppm, was measured in solutions containing the main muscle metabolites, in tissue homogenates with controlled PCr content, and in vivo in rat leg muscles. PCr dominates CERT metrics around 2.6 ppm (although with nontrivial confounding baseline contributions), indicating that CERT is well-suited to PCr specific imaging, and has the added benefit of requiring a relatively small number of acquisitions.
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Músculo Esquelético/química , Resonancia Magnética Nuclear Biomolecular/métodos , Fosfocreatina/análisis , Espectroscopía de Protones por Resonancia Magnética/métodos , Adenosina Trifosfato/análisis , Animales , Creatina/análisis , Glucógeno/análisis , Miembro Posterior , Lactatos/análisis , Músculo Esquelético/diagnóstico por imagen , Ratas , Rotación , Extractos de Tejidos/químicaRESUMEN
PURPOSE: Phospholipids are key constituents of cell membranes and serve vital functions in the regulation of cellular processes; thus, a method for in vivo detection and characterization could be valuable for detecting changes in cell membranes that are consequences of either normal or pathological processes. Here, we describe a new method to map the distribution of partially restricted phospholipids in tissues. METHODS: The phospholipids were measured by signal changes caused by relayed nuclear Overhauser enhancement-mediated CEST between the phospholipid Cho headgroup methyl protons and water at around -1.6 ppm from the water resonance. The biophysical basis of this effect was examined by controlled manipulation of head group, chain length, temperature, degree of saturation, and presence of cholesterol. Additional experiments were performed on animal tumor models to evaluate potential applications of this novel signal while correcting for confounding contributions. RESULTS: Negative relayed nuclear Overhauser dips in Z-spectra were measured from reconstituted Cho phospholipids with cholesterol but not for other Cho-containing metabolites or proteins. Significant contrast was found between tumor and contralateral normal tissue signals in animals when comparing both the measured saturation transfer signal and a more specific imaging metric. CONCLUSION: We demonstrated specific relayed nuclear Overhauser effects in partially restricted phospholipid phantoms and similar effects in solid brain tumors after correcting for confounding signal contributions, suggesting possible translational applications of this novel molecular imaging method, which we name restricted phospholipid transfer.
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Interpretación de Imagen Asistida por Computador , Imagen por Resonancia Magnética , Algoritmos , Animales , Encéfalo , FosfolípidosRESUMEN
Chemical exchange saturation transfer (CEST) can provide metabolite-weighted images in the clinical setting; therefore, understanding the origin of each CEST signal is essential to revealing the changes in diseases at the molecular level, which would provide further insight for diagnoses and treatments. The CEST signal at -1.6 ppm is attributed to the choline methyl group of phosphatidylcholines. The methyl groups have no exchangeable protons, so the corresponding CEST signals must result from the relayed nuclear Overhauser effect (rNOE); however, the detailed mechanism remains unclear. Cholesterol is a major component of biological membranes, and its content is closely related to the dynamics and phases of these lipids. However, cholesterol has a hydroxyl group, which could participate in proton exchange to complete the rNOE process. In this study, we used liposomes containing cholesterol and its analogs (5α-cholestane and progesterone), which presumably have similar capabilities of influencing lipid bilayers, and found that the steroid hydroxyl group is the key to inducing the rNOE at -1.6 ppm. Our results suggest that the origin of the rNOE at -1.6 ppm likely requires an intermolecular NOE between the proton of the choline methyl group and that of the cholesterol hydroxyl group, and a chemical exchange between the cholesterol hydroxyl group and bulk water. However, the phenomenon in which the rNOE at -1.6 ppm appears when the cholesterol concentration is high seems to contradict the in vivo results, suggesting a more complicated mechanism associated with the rNOE at -1.6 ppm in biological membranes.
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Colesterol/química , Imagen por Resonancia Magnética , 1,2-Dipalmitoilfosfatidilcolina/química , Colestanos/química , Liposomas , Ácido Palmítico/químicaRESUMEN
PURPOSE: To test the ability of a novel pulse sequence applied in vivo at 3 Tesla to separate the contributions to the water signal from amide proton transfer (APT) and relayed nuclear Overhauser enhancement (rNOE) from background direct water saturation and semisolid magnetization transfer (MT). The lack of such signal source isolation has confounded conventional chemical exchange saturation transfer (CEST) imaging. METHODS: We quantified APT and rNOE signals using a chemical exchange rotation transfer (CERT) metric, MTRdouble . A range of duty cycles and average irradiation powers were applied, and results were compared with conventional CEST analyses using asymmetry (MTRasym ) and extrapolated magnetization transfer (EMR). RESULTS: Our results indicate that MTRdouble is more specific than MTRasym and, because it requires as few as 3 data points, is more rapid than methods requiring a complete Z-spectrum, such as EMR. In white matter, APT (1.5 ± 0.5%) and rNOE (2.1 ± 0.7%) were quantified by using MTRdouble with a 30% duty cycle and a 0.5-µT average power. In addition, our results suggest that MTRdouble is insensitive to B0 inhomogeneity, further magnifying its speed advantage over CEST metrics that require a separate B0 measurement. However, MTRdouble still has nontrivial sensitivity to B1 inhomogeneities. CONCLUSION: We demonstrated that MTRdouble is an alternative metric to evaluate APT and rNOE, which is fast, robust to B0 inhomogeneity, and easy to process.
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Mapeo Encefálico , Neoplasias Encefálicas/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Imagen por Resonancia Magnética , Adulto , Algoritmos , Simulación por Computador , Femenino , Humanos , Interpretación de Imagen Asistida por Computador/métodos , Masculino , Persona de Mediana Edad , Modelos Estadísticos , Protones , RotaciónRESUMEN
PURPOSE: Chemical exchange saturation transfer effects at 2 ppm (CEST@2ppm) in brain have previously been interpreted as originating from creatine. However, protein guanidino amine protons may also contribute to CEST@2ppm. This study aims to investigate the molecular origins and specificity of CEST@2ppm in brain. METHODS: Two experiments were performed: (i) samples containing egg white albumin and creatine were dialyzed using a semipermeable membrane to demonstrate that proteins and creatine can be separated by this method; and (ii) tissue homogenates of rat brain with and without dialysis to remove creatine were studied to measure the relative contributions of proteins and creatine to CEST@2ppm. RESULTS: The experiments indicate that dialysis can successfully remove creatine from proteins. Measurements on tissue homogenates show that, with the removal of creatine via dialysis, CEST@2ppm decreases to approximately 34% of its value before dialysis, which indicates that proteins and creatine have comparable contribution to the CEST@2ppm in brain. However, considering the contribution from peptides and amino acids to CEST@2ppm, creatine may have much less contribution to CEST@2ppm. CONCLUSIONS: The contribution of proteins, peptides, and amino acids to CEST@2ppm cannot be neglected. The CEST@2ppm measurements of creatine in rat brain should be interpreted with caution. Magn Reson Med 78:881-887, 2017. © 2017 International Society for Magnetic Resonance in Medicine.
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Encéfalo/diagnóstico por imagen , Procesamiento de Imagen Asistido por Computador/métodos , Imagen por Resonancia Magnética/métodos , Imagen Molecular/métodos , Animales , Química Encefálica , Proteínas/química , RatasRESUMEN
Chemical exchange saturation transfer (CEST) imaging of amine protons exchanging at intermediate rates and whose chemical shift is around 2 ppm may provide a means of mapping creatine. However, the quantification of this effect may be compromised by the influence of overlapping CEST signals from fast-exchanging amines and hydroxyls. We aimed to investigate the exchange rate filtering effect of a variation of CEST, named chemical exchange rotation transfer (CERT), as a means of isolating creatine contributions at around 2 ppm from other overlapping signals. Simulations were performed to study the filtering effects of CERT for the selection of transfer effects from protons of specific exchange rates. Control samples containing the main metabolites in brain, bovine serum albumin (BSA) and egg white albumen (EWA) at their physiological concentrations and pH were used to study the ability of CERT to isolate molecules with amines at 2 ppm that exchange at intermediate rates, and corresponding methods were used for in vivo rat brain imaging. Simulations showed that exchange rate filtering can be combined with conventional filtering based on chemical shift. Studies on samples showed that signal contributions from creatine can be separated from those of other metabolites using this combined filter, but contributions from protein amines may still be significant. This exchange filtering can also be used for in vivo imaging. CERT provides more specific quantification of amines at 2 ppm that exchange at intermediate rates compared with conventional CEST imaging.
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Aminas/química , Imagen por Resonancia Magnética/métodos , Rotación , Animales , Encéfalo/diagnóstico por imagen , Simulación por Computador , Creatina/metabolismo , Neoplasias/diagnóstico , Ratas , Sensibilidad y EspecificidadRESUMEN
Virus protein U (Vpu) from HIV-1, a small membrane protein composed of a transmembrane helical domain and two α-helices in an amphipathic cytoplasmic domain, down modulates several cellular proteins, including CD4, BST-2/CD317/tetherin, NTB-A, and CCR7. The interactions of Vpu with these proteins interfere with the immune system and enhance the release of newly synthesized virus particles. It is essential to characterize the structure and dynamics of Vpu in order to understand the mechanisms of the protein-protein interactions, and potentially to discover antiviral drugs. In this article, we describe investigations of the cytoplasmic domain of Vpu as well as full-length Vpu by NMR spectroscopy. These studies are complementary to earlier analysis of the transmembrane domain of Vpu. The results suggest that the two helices in the cytoplasmic domain form a U-shape. The length of the inter-helical loop in the cytoplasmic domain and the orientation of the third helix vary with the lipid composition, which demonstrate that the C-terminal helix is relatively flexible, providing accessibility for interaction partners.
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Membrana Celular/química , Proteínas del Virus de la Inmunodeficiencia Humana/química , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , Proteínas Reguladoras y Accesorias Virales/química , Membrana Celular/metabolismo , Membrana Celular/virología , Dicroismo Circular , Difusión , Dimiristoilfosfatidilcolina/química , VIH-1/metabolismo , VIH-1/fisiología , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Cinética , Proteínas de la Membrana/metabolismo , Micelas , Modelos Moleculares , Éteres Fosfolípidos/química , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteolípidos/química , Proteínas Reguladoras y Accesorias Virales/metabolismoRESUMEN
There is immense cellular and molecular heterogeneity in biological systems. Here, we demonstrate the utility of integrating an inverted light microscope with an ambient ionization source, nanospray electrospray desorption ionization, attached to a high-resolution mass spectrometer to characterize the molecular composition of mouse spinal cords. We detected a broad range of molecules, including peptides and proteins, as well as metabolites such as lipids, sugars, and other small molecules, including S-adenosyl methionine and glutathione, through top-down MS. Top-down analysis revealed variation in the expression of Hb, including the transition from fetal to adult Hb and heterogeneity in Hb subunits consistent with the genetic diversity of the mouse models. Similarly, temporal changes to actin-sequestering proteins ß-thymosins during development were observed. These results demonstrate that interfacing microscopy with ambient ionization provides the means to perform targeted in situ ambient top-down mass spectral analysis to study the pattern of proteins, lipids, and sugars in biologically heterogeneous samples.
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Microscopía/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Tipificación del Cuerpo , Metabolismo de los Hidratos de Carbono , Femenino , Hemoglobinas/genética , Hemoglobinas/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Ratones Transgénicos , Microscopía/instrumentación , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Embarazo , Espectrometría de Masa por Ionización de Electrospray/instrumentación , Médula Espinal/embriología , Espectrometría de Masas en Tándem/instrumentación , Espectrometría de Masas en Tándem/métodos , Timosina/genética , Timosina/metabolismoRESUMEN
Preeclampsia is a life-threatening pregnancy disorder. Current clinical assays cannot predict the onset of preeclampsia until the late 2nd trimester, which often leads to poor maternal and neonatal outcomes. Here we show that Raman spectroscopy combined with machine learning in pregnant patient plasma enables rapid, highly sensitive maternal metabolome screening that predicts preeclampsia as early as the 1st trimester with >82% accuracy. We identified 12, 15 and 17 statistically significant metabolites in the 1st, 2nd and 3rd trimesters, respectively. Metabolic pathway analysis shows multiple pathways corresponding to amino acids, fatty acids, retinol, and sugars are enriched in the preeclamptic cohort relative to a healthy pregnancy. Leveraging Pearson's correlation analysis, we show for the first time with Raman Spectroscopy that metabolites are associated with several clinical factors, including patients' body mass index, gestational age at delivery, history of preeclampsia, and severity of preeclampsia. We also show that protein quantification alone of proinflammatory cytokines and clinically relevant angiogenic markers are inadequate in identifying at-risk patients. Our findings demonstrate that Raman spectroscopy is a powerful tool that may complement current clinical assays in early diagnosis and in the prognosis of the severity of preeclampsia to ultimately enable comprehensive prenatal care for all patients.
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BACKGROUND: While magnetic resonance imaging (MRI) is the imaging modality of choice for the evaluation of patients with brain tumors, it may still be challenging to differentiate glioblastoma multiforme (GBM) from solitary brain metastasis (SBM) due to their similar imaging features. This study aimed to evaluate the features extracted of dual-tree complex wavelet transform (DTCWT) from routine MRI protocol for preoperative differentiation of glioblastoma (GBM) and solitary brain metastasis (SBM). METHODS: A total of 51 patients were recruited, including 27 GBM and 24 SBM patients. Their contrast-enhanced T1-weighted images (CET1WIs), T2 fluid-attenuated inversion recovery (T2FLAIR) images, diffusion-weighted images (DWIs), and apparent diffusion coefficient (ADC) images were employed in this study. The statistical features of the pre-transformed images and the decomposed images of the wavelet transform and DTCWT were utilized to distinguish between GBM and SBM. RESULTS: The support vector machine (SVM) showed that DTCWT images have a better accuracy (82.35%), sensitivity (77.78%), specificity (87.50%), and the area under the curve of the receiver operating characteristic curve (AUC) (89.20%) than the pre-transformed and conventional wavelet transform images. By incorporating DTCWT and pre-transformed images, the accuracy (86.27%), sensitivity (81.48%), specificity (91.67%), and AUC (93.06%) were further improved. CONCLUSIONS: Our studies suggest that the features extracted from the DTCWT images can potentially improve the differentiation between GBM and SBM.
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The chemical exchange saturation transfer (CEST) signal at -1.6 ppm is attributed to the choline methyl on phosphatidylcholines and results from the relayed nuclear Overhauser effect (rNOE), that is, rNOE(-1.6). The formation of rNOE(-1.6) involving the cholesterol hydroxyl is shown in liposome models. We aimed to confirm the correlation between cholesterol content and rNOE(-1.6) in cell cultures, tissues, and animals. C57BL/6 mice (N = 9) bearing the C6 glioma tumor were imaged in a 7 T MRI scanner, and their rNOE(-1.6) images were cross-validated through cholesterol staining with filipin. Cholesterol quantification was obtained using an 18.8-T NMR spectrometer from the lipid extracts of the brain tissues from another group of mice (N = 3). The cholesterol content in the cultured cells was manipulated using methyl-ß-cyclodextrin and a complex of cholesterol and methyl-ß-cyclodextrin. The rNOE(-1.6) of the cell homogenates and their cholesterol levels were measured using a 9.4-T NMR spectrometer. The rNOE(-1.6) signal is hypointense in the C6 tumors of mice, which matches the filipin staining results, suggesting that their tumor region is cholesterol deficient. The tissue extracts also indicate less cholesterol and phosphatidylcholine contents in tumors than in normal brain tissues. The amplitude of rNOE(-1.6) is positively correlated with the cholesterol concentration in the cholesterol-manipulated cell cultures. Our results indicate that the cholesterol dependence of rNOE(-1.6) occurs in cell cultures and solid tumors of C6 glioma. Furthermore, when the concentration of phosphatidylcholine is carefully considered, rNOE(-1.6) can be developed as a cholesterol-weighted imaging technique.
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Rapid and accurate response to targeted therapies is critical to differentiate tumors that are resistant to treatment early in the regimen. In this work, we demonstrate a rapid, noninvasive, and label-free approach to evaluate treatment response to molecular inhibitors in breast cancer (BC) cells with Raman spectroscopy (RS). Metabolic reprogramming in BC was probed with RS and multivariate analysis was applied to classify the cells into responsive or nonresponsive groups as a function of drug dosage, drug type, and cell type. Metabolites identified with RS were then validated with mass spectrometry (MS). We treated triple-negative BC cells with Trametinib, an inhibitor of the extracellular-signal-regulated kinase (ERK) pathway. Changes measured with both RS and MS corresponding to membrane phospholipids, amino acids, lipids and fatty acids indicated that these BC cells were responsive to treatment. Comparatively, minimal metabolic changes were observed post-treatment with Alpelisib, an inhibitor of the mammalian target of rapamycin (mTOR) pathway, indicating treatment resistance. These findings were corroborated with cell viability assay and immunoblotting. We also showed estrogen receptor-positive MCF-7 cells were nonresponsive to Trametinib with minimal metabolic and viability changes. Our findings support that oncometabolites identified with RS will ultimately enable rapid drug screening in patients ensuring patients receive the most effective treatment at the earliest time point.
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Magnetic resonance imaging (MRI) is extensively used in clinical and basic biomedical research. However, MRI detection of pH changes still poses a technical challenge. Chemical exchange saturation transfer (CEST) imaging is a possible solution to this problem. Using saturation transfer, alterations in the exchange rates between the solute and water protons because of small pH changes can be detected with greater sensitivity. In this study, we examined a fatigued skeletal muscle model in electrically stimulated mice. The measured CEST signal ratio was between 1.96 ppm and 2.6 ppm in the z-spectrum, and this was associated with pH values based on the ratio between the creatine (Cr) and the phosphocreatine (PCr). The CEST results demonstrated a significant contrast change at the electrical stimulation site. Moreover, the pH value was observed to decrease from 7.23 to 7.15 within 20 h after electrical stimulation. This pH decrease was verified by 31P magnetic resonance spectroscopy and behavioral tests, which showed a consistent variation over time.
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Creatinina/metabolismo , Imagen por Resonancia Magnética/métodos , Músculo Esquelético/metabolismo , Fosfocreatina/metabolismo , Algoritmos , Animales , Conducta Animal , Calibración , Creatina/análisis , Estimulación Eléctrica , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos C57BL , Imagen Molecular/métodos , Fantasmas de Imagen , Radioisótopos de Fósforo , Protones , Reproducibilidad de los ResultadosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Precise monitoring of specific biomarkers in biological fluids with accurate biodiagnostic sensors is critical for early diagnosis of diseases and subsequent treatment planning. In this work, we demonstrated an innovative biodiagnostic sensor, portable reusable accurate diagnostics with nanostar antennas (PRADA), for multiplexed biomarker detection in small volumes (~50 µl) enabled in a microfluidic platform. Here, PRADA simultaneously detected two biomarkers of myocardial infarction, cardiac troponin I (cTnI), which is well accepted for cardiac disorders, and neuropeptide Y (NPY), which controls cardiac sympathetic drive. In PRADA immunoassay, magnetic beads captured the biomarkers in human serum samples, and gold nanostars (GNSs) "antennas" labeled with peptide biorecognition elements and Raman tags detected the biomarkers via surface-enhanced Raman spectroscopy (SERS). The peptide-conjugated GNS-SERS barcodes were leveraged to achieve high sensitivity, with a limit of detection (LOD) of 0.0055 ng/ml of cTnI, and a LOD of 0.12 ng/ml of NPY comparable with commercially available test kits. The innovation of PRADA was also in the regeneration and reuse of the same sensor chip for ~14 cycles. We validated PRADA by testing cTnI in 11 de-identified cardiac patient samples of various demographics within a 95% confidence interval and high precision profile. We envision low-cost PRADA will have tremendous translational impact and be amenable to resource-limited settings for accurate treatment planning in patients.
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The overexpression of immunomarker programmed cell death protein 1 (PD-1) and engagement of PD-1 to its ligand, PD-L1, are involved in the functional impairment of cluster of differentiation 8+ (CD8+) T cells, contributing to cancer progression. However, heterogeneities in PD-L1 expression and variabilities in biopsy-based assays render current approaches inaccurate in predicting PD-L1 status. Therefore, PD-L1 screening alone is not predictive of patient response to treatment, which motivates us to simultaneously detect multiple immunomarkers engaged in immune modulation. Here, we have developed multimodal probes, immunoactive gold nanostars (IGNs), that accurately detect PD-L1+ tumor cells and CD8+ T cells simultaneously in vivo, surpassing the limitations of current immunoimaging techniques. IGNs integrate the whole-body imaging of positron emission tomography with high sensitivity and multiplexing of Raman spectroscopy, enabling the dynamic tracking of both immunomarkers. IGNs also monitor response to immunotherapies in mice treated with combinatorial PD-L1 and CD137 agonists and distinguish responders from those nonresponsive to treatment. Our results showed a multifunctional nanoscale probe with capabilities that cannot be achieved with either modality alone, allowing multiplexed immunologic tumor profiling critical for predicting early response to immunotherapies.
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Biomarcadores de Tumor/análisis , Oro/química , Inmunoterapia , Melanoma/diagnóstico por imagen , Melanoma/terapia , Nanopartículas del Metal/química , Imagen Óptica , Animales , Antígeno B7-H1/agonistas , Antígeno B7-H1/análisis , Antígeno B7-H1/genética , Biomarcadores de Tumor/agonistas , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Ratones , Tamaño de la Partícula , Propiedades de Superficie , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/análisis , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/genéticaRESUMEN
In this work, we demonstrate the targeted diagnosis of immunomarker programmed death ligand 1 (PD-L1) and simultaneous detection of epidermal growth factor receptor (EGFR) in breast cancer tumors in vivo using gold nanostars (AuNS) with multiplexed surface enhanced Raman spectroscopy (SERS). Real-time longitudinal tracking with SERS demonstrated maximum accumulation of AuNS occurred 6 h post intravenous (IV) delivery, enabling detection of both biomarkers simultaneously. Raman signal correlating to both PD-L1 and EGFR decreased by â¼30% in control tumors where receptors were pre-blocked prior to AuNS delivery, indicating both the sensitivity and specificity of SERS in distinguishing tumors with different levels of PD-L1 and EGFR expression. Our in vivo study was combined with the first demonstration of ex vivo SERS spatial maps of whole tumor lesions that provided both a qualitative and quantitative assessment of biomarker status with near cellular-level resolution. High resolution SERS maps also provided an overview of AuNS distribution in tumors which correlated well with the vascular density. Mass spectrometry showed AuNS accumulation in tumor and liver, and clearance via spleen, and electron microscopy revealed AuNS were endocytosed in tumors, Kupffer cells in the liver, and macrophages in the spleen. This study demonstrates that SERS-based diagnosis mediated by AuNS provides an accurate measure of multiple biomarkers both in vivo and ex vivo, which will ultimately enable a clinically-translatable platform for patient-tailored immunotherapies and combination treatments.
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Neoplasias de la Mama/diagnóstico , Oro , Nanopartículas del Metal , Espectrometría Raman , Antígeno B7-H1/análisis , Receptores ErbB/análisis , Humanos , Sensibilidad y EspecificidadRESUMEN
In this study, we demonstrate the theranostic capability of actively targeted, site-specific multibranched gold nanoantennas (MGNs) in triple-negative breast cancer (TNBC) cells in vitro. By utilizing multiplexed surface-enhanced Raman scattering (SERS) imaging, enabled by the narrow peak widths of Raman signatures, we simultaneously targeted immune checkpoint receptor programmed death ligand 1 (PDL1) and the epidermal growth factor receptor (EGFR) overexpressed in TNBC cells. A 1:1 mixture of MGNs functionalized with anti-PDL1 antibodies and Raman tag 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) and MGNs functionalized with anti-EGFR antibodies and Raman tag para-mercaptobenzoic acid (pMBA) were incubated with the cells. SERS imaging revealed a cellular traffic map of MGN localization by surface binding and receptor-mediated endocytosis, enabling targeted diagnosis of both biomarkers. Furthermore, cells incubated with anti-EGFR-pMBA-MGNs and illuminated with an 808 nm laser for 15 min at 4.7 W/cm2 exhibited photothermal cell death only within the laser spot (indicated by live/dead cell fluorescence assay). Therefore, this study not only provides an optical imaging platform that can track immunomarkers with spatiotemporal control but also demonstrates an externally controlled light-triggered therapeutic approach enabling receptor-specific treatment with biocompatible theranostic nanoprobes.