RESUMEN
Organ-specific colonization suggests that specific cell-cell recognition is essential. Yet, very little is known about this particular interaction. Moreover, tumor cell lodgement requires binding under shear stress, but not static, conditions. Here, we successfully isolate the metastatic populations of cancer stem/tumor-initiating cells (M-CSCs). We show that the M-CSCs tether more and roll slower than the non-metastatic (NM)-CSCs, thus resulting in the preferential binding to the peritoneal mesothelium under ascitic fluid shear stress. Mechanistically, this interaction is mediated by P-selectin expressed by the peritoneal mesothelium. Insulin-like growth factor receptor-1 carrying an uncommon non-sulfated sialyl-Lewisx (sLex) epitope serves as a distinct P-selectin binding determinant. Several glycosyltransferases, particularly α1,3-fucosyltransferase with rate-limiting activity for sLex synthesis, are highly expressed in M-CSCs. Tumor xenografts and clinical samples corroborate the relevance of these findings. These data advance our understanding on the molecular regulation of peritoneal metastasis and support the therapeutic potential of targeting the sLex-P-selectin cascade.
Asunto(s)
Líquido Ascítico , Carcinoma/secundario , Adhesión Celular , Hidrodinámica , Células Madre Neoplásicas/metabolismo , Oligosacáridos/metabolismo , Neoplasias Ováricas/patología , Selectina-P/metabolismo , Neoplasias Peritoneales/secundario , Animales , Carcinoma/metabolismo , Línea Celular Tumoral , Epitelio/metabolismo , Femenino , Fucosiltransferasas/metabolismo , Células HEK293 , Humanos , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias Ováricas/metabolismo , Neoplasias Peritoneales/metabolismo , Peritoneo/metabolismo , Receptor IGF Tipo 1/metabolismo , Antígeno Sialil Lewis X , Estrés MecánicoRESUMEN
In this study, we develop a method to detect multiple DNAs of foodborne pathogens by encapsulating emulsion droplets for loop-mediated isothermal amplification (LAMP). In contrast to the traditional bulk-phase LAMP, which involves a labor-intensive mixing process, with our method, different primers are automatically mixed with DNA samples and LAMP buffers after picoinjection. By directly observing and analyzing the fluorescence intensity of the resultant droplets, one can detect DNA from different pathogens, with a detection limit 500 times lower than that obtained by bulk-phase LAMP. We further demonstrate the ability to quantify bacteria concentration by detecting bacterial DNA in practical samples, showing great potential in monitoring water resources and their contamination by pathogenic bacteria.
Asunto(s)
Bacterias/aislamiento & purificación , ADN Bacteriano/análisis , Contaminación de Alimentos/análisis , Técnicas Analíticas Microfluídicas/métodos , Bacterias/genética , Enfermedades Transmitidas por los Alimentos/prevención & control , Dispositivos Laboratorio en un Chip , Límite de Detección , Técnicas de Amplificación de Ácido Nucleico/métodos , Sensibilidad y Especificidad , Aguas Residuales/análisisRESUMEN
Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.
Asunto(s)
Técnicas Analíticas Microfluídicas/métodos , Neoplasias Ováricas/patología , Cavidad Peritoneal/patología , Neoplasias Peritoneales/diagnóstico , Esferoides Celulares/patología , Línea Celular Tumoral , Progresión de la Enfermedad , Epitelio/patología , Femenino , Humanos , Modelos Biológicos , Metástasis de la Neoplasia/diagnóstico , Neoplasias Peritoneales/secundario , Microambiente TumoralRESUMEN
We present a wash-free high-sensitivity immunoassay of C-reactive proteins with droplet microfluidics. Microbeads are encapsulated within droplets for the immunoassay, and the droplets are scanned by a fluorescence detection platform to quantify the amount of proteins captured on the microbeads. The limit of detection determined by our platform is 0.01 µg mL-1, which is ten times more sensitive than conventional high-sensitivity C-reactive protein assays. With the decrease in diffusion distance within droplets, the immunoassay requires only half of the time required for similar conventional approaches. This approach for carrying out immunoassays can potentially be applied to other biomarkers beyond C-reactive proteins.
Asunto(s)
Proteína C-Reactiva/análisis , Inmunoensayo/instrumentación , Dispositivos Laboratorio en un Chip , Límite de Detección , Espectrometría de FluorescenciaRESUMEN
One of greatest challenges to the successful treatment of cancer is drug resistance. An exciting approach is the eradication of cancer stem cells (CSCs). However, little is known about key signals regulating the formation and expansion of CSCs. Moreover, lack of a reliable predictive preclinical model has been a major obstacle to discover new cancer drugs and predict their clinical activity. Here, in ovarian cancer, a highly chemoresistant tumor that is rapidly fatal, we provide the first evidence demonstrating the causal involvement of mechanical stimulus in the CSC phenotype using a customizable microfluidic platform and three-dimensional spheroids, which most closely mimic tumor behavior. We found that ovarian cancer cells significantly acquired the expression of epithelial-to-mesenchymal transition and CSC markers and a remarkable chemoresistance to clinically relevant doses of frontline chemotherapeutic drugs cisplatin and paclitaxel when grown under fluid shear stress, which corroborates with the physiological attainable levels in the malignant ascites, but not under static condition. Furthermore, we uncovered a new link of microRNA-199a-3p, phosphatidylinositol 3-kinase/Akt, and multidrug transporter activation in shear stress-induced CSC enrichment. Our findings shed new light on the significance of hydrodynamics in cancer progression, emphasizing the need of a flow-informed framework in the development of therapeutics.
Asunto(s)
Carcinoma/patología , Resistencia a Antineoplásicos , Hidrodinámica , Células Madre Neoplásicas/citología , Neoplasias Ováricas/patología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/fisiología , Animales , Ascitis/patología , Transición Epitelial-Mesenquimal , Femenino , Xenoinjertos , Humanos , Dispositivos Laboratorio en un Chip , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , MicroARNs/fisiología , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/trasplante , ARN Neoplásico/fisiología , Resistencia al Corte , Transducción de Señal , Esferoides Celulares , Células Tumorales CultivadasRESUMEN
Accelerating imaging speed in optical microscopy is often realized at the expense of image contrast, image resolution, and detection sensitivity--a common predicament for advancing high-speed and high-throughput cellular imaging. We here demonstrate a new imaging approach, called asymmetric-detection time-stretch optical microscopy (ATOM), which can deliver ultrafast label-free high-contrast flow imaging with well delineated cellular morphological resolution and in-line optical image amplification to overcome the compromised imaging sensitivity at high speed. We show that ATOM can separately reveal the enhanced phase-gradient and absorption contrast in microfluidic live-cell imaging at a flow speed as high as ~10 m/s, corresponding to an imaging throughput of ~100,000 cells/sec. ATOM could thus be the enabling platform to meet the pressing need for intercalating optical microscopy in cellular assay, e.g. imaging flow cytometry--permitting high-throughput access to the morphological information of the individual cells simultaneously with a multitude of parameters obtained in the standard assay.