Multiparameter Nanoparticle Detection of Phosphoproteins

Stanford University, Stanford CA

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Abstract

There is a critical need to measure the complex behavior of cancer cells, the manner in which they interact[unreadable] with immune system cells, and to phenotype both cell sets not only for their cell lineage status but also for[unreadable] the relative activation state of the signaling networks that drive their biology. It is thought that signaling[unreadable] network status is reflective of a cancer's aggressiveness, its response to chemotherapy, as well how it[unreadable] influences or evades the immune response. We will extend the number of parameters that can be measured[unreadable] simultaneously for cytometric approaches by creating a new class of Raman Scatter measurable reagents,[unreadable] using Composite Organic-Inorganic Nanoparticles (COINs). The technique we will use adapt our recently[unreadable] developed procedure applying monoclonal and polyclonal antibodies against phosphorylation sites [1-10][unreadable] (and Preliminary Results). We will apply the COINS technology, in collaboration with the creators of these[unreadable] unique nanoparticles from the Berlin group at Intel, to first validate a series of Raman spectroscopic probes[unreadable] for important kinase phosphoproteins in microarray formats. With validated sets of reagents we will refine[unreadable] simultaneous, multi-parameter staining and visualization of cells using Raman signatures as validated with[unreadable] fluorophore-based staining to extend the number of cytometrically determined parameters per cell. Finally,[unreadable] we will use the additional, simultaneous measurements to construct larger signaling networks using[unreadable] Bayesian computation; this approach will be applied to data from normal B cells and lymphomas from[unreadable] selected patients. Taken together, this concerted approach will allow for us to create, on a patient by patient[unreadable] basis, inference maps of signaling system architecture across multiple, highly informative, signaling nodes[unreadable] for established and novel signaling systems in normal cells and cancer. These techniques have the potential[unreadable] to create rich biomarkers that are informative not only of patient status, but provide detailed information[unreadable] about the signaling systems against which chemotherapies act as well as provide mechanistic conclusions[unreadable] about individual cancers and patient immune status.