RESUMO
Pharmacological treatments for advanced hepatocellular carcinoma (HCC) have a partial efficacy. Augmented Na+ content and water retention are observed in human cancers and offer unexplored targets for anticancer therapies. Na+ levels are evaluated upon treatments with the antibiotic cation ionophore Monensin by fluorimetry, ICP-MS, 23Na-MRI, NMR relaxometry, confocal or time-lapse analysis related to energy production, water fluxes and cell death, employing both murine and human HCC cell lines, primary murine hepatocytes, or HCC allografts in NSG mice. Na+ levels of HCC cells and tissue are 8-10 times higher than that of healthy hepatocytes and livers. Monensin further increases Na+ levels in HCC cells and in HCC allografts but not in primary hepatocytes and in normal hepatic and extrahepatic tissue. The Na+ increase is associated with energy depletion, mitochondrial Na+ load and inhibition of O2 consumption. The Na+ increase causes an enhancement of the intracellular water lifetime and death of HCC cells, and a regression and necrosis of allograft tumors, without affecting the proliferating activity of either HCCs or healthy tissues. These observations indicate that HCC cells are, unlike healthy cells, energetically incapable of compensating and surviving a pharmacologically induced Na+ load, highlighting Na+ homeostasis as druggable target for HCC therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Camundongos , Humanos , Animais , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Sódio/metabolismo , Monensin/uso terapêutico , Linhagem Celular , ÁguaRESUMO
This work shows that the longitudinal relaxation differences observed at very low magnetic fields between invasion/migration and proliferation processes on glioma mouse models in vivo are related to differences in the transmembrane water exchange basically linked to the aquaporin expression changes. Three glioma mouse models were used: Glio6 and Glio96 as invasion/migration models and U87 as cell proliferation model. In vivo proton longitudinal relaxation-rate constants (R1) at very low fields were measured by fast field cycling NMR (FFC-NMR). The tumor contribution to the observed proton relaxation rate, R1tum (U87: 12.26 ± 0.64 s−1; Glio6: 3.76 ± 0.88 s−1; Glio96: 6.90 ± 0.64 s−1 at 0.01 MHz), and the intracellular water lifetime, τin (U87: 826 ± 19 ms; Glio6: 516 ± 8 ms; Glio96: 596 ± 15 ms), were found to be good diagnostic hallmarks to distinguish invasion/migration from proliferation (p < 0.01 and 0.001). Overexpression of AQP4 and AQP1 were assessed in invasion/migration models, highlighting the pathophysiological role of these two aquaporins in water exchange that, in turn, determine the lower values in the observed R1 relaxation rate constant in glioma invasion/migration. Overall, our findings demonstrate that τin and R1 (measured at very low fields) are relevant biomarkers, discriminating invasion/migration from proliferation in vivo. These results highlight the use of FFC-NMR and FFC-imaging to assess the efficiency of drugs that could modulate aquaporin functions.
RESUMO
Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fosfatidilinositol 3-Quinase , Animais , Classe Ib de Fosfatidilinositol 3-Quinase , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Inflamação , Camundongos , Peptídeos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismoRESUMO
This study aims to explore whether the water exchange rate constants in tumor cells can act as a hallmark of pathology status and a reporter of therapeutic outcomes. It has been shown, using 4T1 cell cultures and murine allografts, that an early assessment of the therapeutic effect of doxorubicin can be detected through changes in the cellular water efflux rate constant kio. The latter has been estimated by analyzing the magnetization recovery curve in standard NMR T1 measurements when there is a marked difference in the proton relaxation rate constants (R1) between the intra- and the extra-cellular compartments. In cellular studies, T1 measurements were carried out on a relaxometer working at 0.5 T, and the required difference in R1 between the two compartments was achieved via the addition of a paramagnetic agent into the extracellular compartment. For in-vivo experiments, the large difference in the R1 values of the two-compartments was achieved when the T1 measurements were carried out at low magnetic field strengths. This task was accomplished using a Fast Field Cycling (FFC) relaxometer that was properly modified to host a mouse in its probe head. The decrease in kio upon the administration of doxorubicin is the result of the decreased activity of Na+/K+-ATPase, as shown in an independent test on the cellular uptake of Rb ions. The results reported herein suggest that kio can be considered a non-invasive, early and predictive biomarker for the identification of responsive patients immediately from the first doxorubicin treatment.
RESUMO
As conserving surgery is routinely applied for the treatment of early-stage breast cancer, the need for new technology to improve intraoperative margin assessment has become increasingly important. In this study, the potential of fast field-cycling 1H-NMR relaxometry as a new diagnostic tool was evaluated. The technique allows the determination of the tissue proton relaxation rates (R1), as a function of the applied magnetic field, which are affected by the changes in the composition of the mammary gland tissue occurring during the development of neoplasia. The study involved 104 small tissue samples obtained from surgical specimens destined for histopathology. It was found that a good accuracy in margin assessment, i.e., a sensitivity of 92% and a specificity of 85%, can be achieved by using two quantifiers, namely (i) the slope of the line joining the R1 values measured at 0.02 and 1 MHz and (ii) the sum of the R1 values measured at 0.39 and 1 MHz. The method is fast, and it does not rely on the expertise of a pathologist or cytologist. The obtained results suggest that a simplified, low-cost, automated instrument might compete well with the currently available tools in margin assessment.
RESUMO
Nowadays, magnetic resonance imaging (MRI) is one of the key, noninvasive modalities to detect and stage cancer which benefits from contrast agents (CA) to differentiate healthy from tumor tissue. An innovative class of MRI CAs is represented by Gd-loaded gold nanoparticles. The size, shape and chemical functionalization of Gd-loaded gold nanoparticles appear to affect the observed relaxation enhancement of water protons in their suspensions. The herein reported results shed more light on the determinants of the relaxation enhancement brought by Gd-loaded concave cube gold nanoparticles (CCGNPs). It has been found that, in the case of nanoparticles endowed with concave surfaces, the relaxivity is remarkably higher compared to the corresponding spherical (i.e., convex) gold nanoparticles (SPhGNPs). The main determinant for the observed relaxation enhancement is represented by the occurrence of a large contribution from second sphere water molecules which can be exploited in the design of high-efficiency MRI CA.
RESUMO
Tumour-associated macrophages (TAM) are forced by cancer cells to adopt an anti-inflammatory phenotype and secrete factors to promote tumour invasion thus being responsible for poor patient outcome. The aim of this study is to develop a clinically applicable, non-invasive method to obtain a quantitative TAM detection in tumour tissue. The method is based on longitudinal proton relaxation rate (R1) measurements at low field (0.01-1 MHz) to assess the localization of ferumoxytol (clinical approved iron oxide particles) in TAM present in melanoma tumours, where R1 = 1/T1. R1 at low magnetic fields appears highly dependent on the intra or extra cellular localization of the nanoparticles thus allowing an unambiguous TAM quantification. R1 profiles were acquired on a Fast Field-Cycling relaxometer equipped with a 40 mm wide bore magnet and an 11 mm solenoid detection coil placed around the anatomical region of interest. The R1 values measured 3 h and 24 h after the injection were significantly different. At 24 h R1 exhibited a behavior similar to "in vitro" ferumoxytol-labelled J774A.1 macrophages whereas at 3 h, when the ferumoxytol distribution was extracellular, R1 exhibited higher values similar to that of free ferumoxytol in solution. This finding clearly indicated the intracellular localization of ferumoxytol at 24 h, as confirmed by histological analysis (Pearls and CD68 assays). This information could be hardly achievable from measurements at a single magnetic field and opens new horizons for cell tracking applications using FFC-MRI.
Assuntos
Meios de Contraste , Melanoma , Animais , Compostos Férricos , Óxido Ferroso-Férrico , Humanos , Imageamento por Ressonância Magnética , Camundongos , Macrófagos Associados a TumorRESUMO
Nanotheranostic reagents that integrate magnetic resonance imaging (MRI) and photothermal therapy (PTT) offer a promising strategy for the treatment of human disease. However, classic gadolinium (Gd)-based T1-MRI contrast agents are limited by their low relaxivity. To address this, we produced Gd-loaded Tobacco mosaic virus (TMV) particles coated with the mussel-inspired biopolymer polydopamine (PDA). Such biocompatible nanotheranostic reagents can be used to facilitate PTT, guided by multimodal magnetic resonance/photoacoustic imaging. The r1-relaxivity of the Gd-TMV-PDA particles at 60 MHz was â¼80 mM-1 s-1, compared to 13.63 mM-1 s-1 for the uncoated Gd-TMV particles. The Gd-TMV-PDA particles also promoted strong near-infrared absorption with high photothermal conversion efficiency (28.9%) and demonstrated excellent photoacoustic contrast. Multimodal imaging and PTT resulted in the effective killing of PC-3 prostate cancer cells. Gd-TMV-PDA nanoparticles therefore offer a promising theranostic approach that can now be tested in vivo in cancer models.
Assuntos
Meios de Contraste/química , Indóis/química , Polímeros/química , Vírus do Mosaico do Tabaco/química , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Meios de Contraste/síntese química , Gadolínio/química , Humanos , Raios Infravermelhos , Imageamento por Ressonância Magnética , Microscopia Confocal , Nanopartículas/química , Nanopartículas/toxicidade , Neoplasias/diagnóstico , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , Técnicas Fotoacústicas , FototerapiaRESUMO
1 H Fast Field Cycling NMR (FFC-NMR) relaxometry is proposed as a powerful method to investigate tumour stroma in vivo upon the administration of a Gd-based contrast agent. To perform this study, an FFC-NMR equipment endowed with a wide bore magnet was used for the acquisition of Nuclear Magnetic Resonance Dispersion profiles on healthy muscle and tumour tissue in living mice. At magnetic field strengths < of ca. 1 MHz, the differences in the relaxation rates of the intra and extracellular compartment become of the same order of magnitude of the exchange rate across the cellular membranes. Under this condition, the water exchange rate between the two compartments yields to a biexponential magnetization recovery that can be analysed by fitting the experimental data with the two-Site eXchange (2SX) model. Using this model, it was possible to obtain, for the two compartments, both relaxation properties and water kinetic constants for water exchange across cell membranes. The method allowed us to determine the effect of the "matrix" on the water proton relaxation times and, in turn, to get some insights of the composition of this compartment, till now, largely unknown.
Assuntos
Meios de Contraste/análise , Matriz Extracelular/metabolismo , Gadolínio/análise , Imageamento por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Animais , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Meios de Contraste/administração & dosagem , Feminino , Cinética , Camundongos Endogâmicos BALB C , Imagem Multimodal/métodos , Água/químicaRESUMO
It was established through inâ vivo T1 measurements at low magnetic fields that tumour cells display proton T1 values that are markedly longer than those shown by healthy tissue. Moreover, it has been found that the elongation of T1 parallels the aggressiveness of the investigated tumour. The T1 lengthening is associated with an enhanced water exchange rate across the transcytolemmal membrane through an overexpression/upregulation of GLUT1 and Na+ /K+ ATPase transporters. It follows that the intracellular water lifetime represents a hallmark of tumour cells that can be easily monitored by measuring T1 at different magnetic field strengths ranging from 0.2 to 200â mT.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Água Corporal/metabolismo , Neoplasias Mamárias Experimentais/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Animais , Transportador de Glucose Tipo 1/metabolismo , Xenoenxertos , Imageamento por Ressonância Magnética/métodos , Neoplasias Mamárias Experimentais/enzimologia , Neoplasias Mamárias Experimentais/patologia , Camundongos , Prótons , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
A novel fluorescein/Gd-DOTAGA containing nanoprobe for the visualization of tumors by optical and Magnetic Resonance Imaging (MRI) is reported herein. It is based on the functionalization of the surface of small mesoporous silica nanoparticles (MSNs) (â¼30 nm) with the arginine-glycine-aspartic (RGD) moieties, which are known to target αvß3 integrin receptors overexpressed in several tumor cells. The obtained nanoprobe (Gd-MSNs-RGD) displays good stability, tolerability and high relaxivity (37.6 mM(-1) s(-1) at 21.5 MHz). After a preliminary evaluation of their cytotoxicity and targeting capability toward U87MG cells by in vitro fluorescence and MR imaging, the nanoprobes were tested in vivo by T1-weighted MR imaging of xenografted murine tumor models. The obtained results demonstrated that the Gd-MSNs-RGD nanoprobes are good reporters both in vitro and in vivo for the MR-visualization of tumor cells overexpressing αvß3 integrin receptors.
Assuntos
Integrina alfaVbeta3/genética , Imageamento por Ressonância Magnética/métodos , Técnicas de Diagnóstico Molecular/métodos , Neoplasias/diagnóstico , Imagem Óptica/métodos , Dióxido de Silício/química , Animais , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/química , Gadolínio/química , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Neoplasias/genética , Porosidade , Regulação para Cima/genéticaRESUMO
Fluorine MRI ((19) F MRI) is receiving an increasing attention as a viable alternative to proton-based MRI ((1) H MRI) for dedicated application in molecular imaging. The (19) F nucleus has a high gyromagnetic ratio, a 100% natural abundance and is furthermore hardly present in human tissues allowing for hot spot MR imaging. The applicability of (19) F MRI as a molecular and cellular imaging technique has been exploited, ranging from cell tracking to detection and imaging of tumors in preclinical studies. In addition to applications, developing new contrast materials with improved relaxation properties has also been a core research topic in the field, since the inherently low longitudinal relaxation rates of perfluorocarbon compounds result in relatively low imaging efficiency. Borrowed from (1) H MRI, the incorporation of lanthanides, specifically Gd(III) complexes, as signal modulating ingredients in the nanoparticle formulation has emerged as a promising approach to improvement of the fluorine signal. Three different perfluorocarbon emulsions were investigated at five different magnetic field strengths. Perfluoro-15-crown-5-ether was used as the core material and Gd(III)DOTA-DSPE, Gd(III)DOTA-C6-DSPE and Gd(III)DTPA-BSA as the relaxation altering components. While Gd(III)DOTA-DSPE and Gd(III)DOTA-C6-DSPE were favorable constructs for (1) H NMR, Gd(III)DTPA-BSA showed the strongest increase in (19F) R(1). These results show the potential of the use of paramagnetic lipids to increase (19F) R(1) at clinical field strengths (1.5-3 T). At higher field strengths (6.3-14 T), gadolinium does not lead to an increase in (19F) R(1) compared with emulsions without gadolinium, but leads to an significant increase in (19F) R(2). Our data therefore suggest that the most favorable situation for fluorine measurements is at high magnetic fields without the inclusion of gadolinium constructs.