RESUMO
Personalized tumor chemotherapy depends on reliable assay methods, either based on molecular "predictive biomarkers" or on a direct, functional ex vivo assessment of cellular chemosensitivity. As a member of the latter category, a novel high-content platform is described monitoring human mamma carcinoma explants in real time and label-free before, during and after an ex vivo modeled chemotherapy. Tissue explants are sliced with a vibratome and laid into the microreaction chambers of a 24-well sensor test plate. Within these ~23 µl volume chambers, sensors for pH and dissolved oxygen record rates of cellular oxygen uptake and extracellular acidification. Robot-controlled fluid system and incubation are parts of the tissue culture maintenance system while an integrated microscope is used for process surveillance. Sliced surgical explants from breast cancerous tissue generate well-detectable ex vivo metabolic activity. Metabolic rates, in particular oxygen consumption rates have a tendency to decrease over time. Nonetheless, the impact of added drugs (doxorubicin, chloroacetaldehyde) is discriminable. Sensor-based platforms should be evaluated in explorative clinical studies for their suitability to support targeted systemic cancer therapy. Throughput is sufficient for testing various drugs in a range of concentrations while the information content obtained from multiparametric real-time analysis is superior to conventional endpoint assays.
Assuntos
Antineoplásicos/farmacologia , Técnicas Biossensoriais/métodos , Neoplasias da Mama/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Medicina de Precisão/métodos , Feminino , Humanos , Terapia de Alvo Molecular/métodos , Consumo de Oxigênio/fisiologia , Células Tumorais CultivadasRESUMO
Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time.