RESUMEN
Extracellular vesicles (EV) are a family of cell-originating, membrane-enveloped nanoparticles with diverse biological function, diagnostic potential, and therapeutic applications. While EV can be abundant in circulation, their small size (â¼4 order of magnitude smaller than cells) has necessitated bulk analyses, making many more nuanced biological explorations, cell of origin questions, or heterogeneity investigations impossible. Here we describe a single EV analysis (SEA) technique which is simple, sensitive, multiplexable, and practical. We profiled glioblastoma EV and discovered surprising variations in putative pan-EV as well as tumor cell markers on EV. These analyses shed light on the heterogeneous biomarker profiles of EV. The SEA technology has the potential to address fundamental questions in vesicle biology and clinical applications.
Asunto(s)
Vesículas Extracelulares/patología , Glioblastoma/patología , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Diseño de Equipo , Vesículas Extracelulares/química , Glioblastoma/química , Glioblastoma/diagnóstico por imagen , Humanos , Dispositivos Laboratorio en un Chip , Imagen Óptica/instrumentación , Imagen Óptica/métodosRESUMEN
Eribulin mesylate was developed as a potent microtubule-targeting cytotoxic agent to treat taxane-resistant cancers, but recent clinical trials have shown that it eventually fails in many patient subpopulations for unclear reasons. To investigate its resistance mechanisms, we developed a fluorescent analog of eribulin with pharmacokinetic (PK) properties and cytotoxic activity across a human cell line panel that are sufficiently similar to the parent drug to study its cellular PK and tissue distribution. Using intravital imaging and automated tracking of cellular dynamics, we found that resistance to eribulin and the fluorescent analog depended directly on the multidrug resistance protein 1 (MDR1). Intravital imaging allowed for real-time analysis of in vivo PK in tumors that were engineered to be spatially heterogeneous for taxane resistance, whereby an MDR1-mApple fusion protein distinguished resistant cells fluorescently. In vivo, MDR1-mediated drug efflux and the three-dimensional tumor vascular architecture were discovered to be critical determinants of drug accumulation in tumor cells. We furthermore show that standard intravenous administration of a third-generation MDR1 inhibitor, HM30181, failed to rescue drug accumulation; however, the same MDR1 inhibitor encapsulated within a nanoparticle delivery system reversed the multidrug-resistant phenotype and potentiated the eribulin effect in vitro and in vivo in mice. Our work demonstrates that in vivo assessment of cellular PK of an anticancer drug is a powerful strategy for elucidating mechanisms of drug resistance in heterogeneous tumors and evaluating strategies to overcome this resistance.
Asunto(s)
Antineoplásicos/farmacocinética , Benzopiranos/administración & dosificación , Resistencia a Antineoplásicos/efectos de los fármacos , Furanos/farmacocinética , Isoquinolinas/administración & dosificación , Cetonas/farmacocinética , Neoplasias/tratamiento farmacológico , Tetrazoles/administración & dosificación , Moduladores de Tubulina/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Benzopiranos/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Química Farmacéutica , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Femenino , Humanos , Isoquinolinas/química , Ratones Desnudos , Microscopía Confocal , Microscopía Fluorescente , Nanopartículas , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Recombinantes de Fusión/metabolismo , Tetrazoles/química , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Pharmacokinetic analysis at the organ level provides insight into how drugs distribute throughout the body, but cannot explain how drugs work at the cellular level. Here we demonstrate in vivo single-cell pharmacokinetic imaging of PARP-1 inhibitors and model drug behaviour under varying conditions. We visualize intracellular kinetics of the PARP-1 inhibitor distribution in real time, showing that PARP-1 inhibitors reach their cellular target compartment, the nucleus, within minutes in vivo both in cancer and normal cells in various cancer models. We also use these data to validate predictive finite element modelling. Our theoretical and experimental data indicate that tumour cells are exposed to sufficiently high PARP-1 inhibitor concentrations in vivo and suggest that drug inefficiency is likely related to proteomic heterogeneity or insensitivity of cancer cells to DNA-repair inhibition. This suggests that single-cell pharmacokinetic imaging and derived modelling improve our understanding of drug action at single-cell resolution in vivo.