Single-cell Phosphoprotein Assay to Evaluate Brain Tumor Therapeutic Resistance

Although signal transduction inhibitors occasionally offer clinical benefit for cancer patientssignal flux emanating from oncogenes is often distributed through multiple pathwayspotentially underlying the resistance which causes failure of most such inhibitorsMeasuring signal flux through multiple pathwaysin response to signal transduction inhibitorsmay help uncover network interactions that contribute to therapeutic resistance and that are not predicted by analyzing pathways in isolationProtein protein interactions within signaling pathways are often elucidated by assessing the levels of relevant pathway proteins in model and tumor derived cell lines and with various genetic and molecular perturbationsSuch interactionsand the implied signaling networksmay also be elucidated via quantitative measurements of multiple pathway related proteins within single cellsAt the single cell levelinhibitory and activating protein protein relationshipsas well as stochasticsingle cellfluctuationsare revealedHowevermost techniques for profiling signaling pathways require large numbers of cellsand bulk measurements have proven insufficient to detect secondary pathways post resistanceSinglecell immunostaining is promisingand some flow cytometry techniques are relevantyet limited in finding possible pathways due to intracellular multiplexing limitationsWe describe quantitativemultiplex assays of intracellular signaling proteins from single cancer cells using a platform called the single cell barcode chipSCBCThe SCBC is simple in conceptA single or defined number of cells is isolated within a microchamber that contains a sensitive antibody array specific for the capture and detection of a panel of proteinsThe SCBC design permits lysis of each individual trapped cellIntracellular staining flow cytometry can assay up tophosphoproteins from single cellsOur SCBC can profile a significantly larger panelup todifferent phosphoproteinswithsingle cells per chip for a statistically representative analysis of the sample populationThis new high multi plexed single cell phosphoproteomics analysis tool provides an analytical approach for detecting changes in signal coordination by monitoring phosphoproteinson a much larger scaleThis approach may identify actionable alterations in signal coordination that underlie adaptive resistancewhich can be suppressed through combination drug therapyincluding nonobvious drug combinationsSPECIFIC AIMDevelop a robust microchamber array flow cell that can be easily incorporated into larger automated workflow device for analysis of intracellular protein targetsSPECIFIC AIMDouble multiplexing capability of high density barcode SCBC chip by monitoring both intracellular proteins and metabolites simultaneouslyPerform single cellplex measurement for more comprehensive GBM pathway analysisSPECIFIC AIMImprove consumable to performflow cellin cartridge lysisdetection and washing capabilities for automationDevelop fully automated device workflowSPECIFIC AIMbDemonstrate utility of device in patient clinical trials as a commercial tool Although signal transduction inhibitors occasionally offer clinical benefit for cancer patientssignal flux emanating from oncogenes is often distributed through multiple pathwayspotentially underlying the resistance which causes failure of most such inhibitorsWe developed a new high multi plexed single cell intracellular analysis tool that provides an analytical approach for detecting changes in signal coordination by monitoring phosphoproteins and metaboliteson a much larger scaleWe propose to further developstandardize and scale this technology for commercialization as a clinical tool for developing combination drug therapies for glioblastoma patients