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Protein Expression and Cell Signaling Quantified in Rare Cells (ISSCR 2008)

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The discovery of tumor stem cells in acute myeloid leukemia a decade ago initiated a field of research has accelerated growth in the past few years. Researchers are now describing tumor stem cells in a variety of hematopoietic and solid tumors. The impetus for much of this research is the desire to identify targets for drug intervention in these critical tumor populations. The molecular pathways that functionally define these cells are important therapeutic targets. Tumor stem cells are rare and provide insufficient material to use standard assay methods. Although DNA µarray and/or qPCR are used to study tumor stem cells, their rare nature limits quantitative protein analysis. This creates a gap in our knowledge since many proteins, such as β-catenin or MAPK signaling proteins, are not regulated at the transcriptional level, but through post-translational modifications (phosphorylation, ubiquitination, etc.). Here we describe a technique utilizing a nanoimmunoassay platform (Firefly?) to measure tumor stem cell proteins. Transitional tumor stem cells (TCC+) were sorted from a patient tumor and lysed for analysis. A lysate of 400 cells was subjected to isoelectric focusing and immobilization. Immunodetection was performed and quantitation of signal was measured using HRP-labeled secondary chemiluminescence reagents. Here we report β-catenin protein concentrations of 192 ng/mg of total protein in the tumor stem cells, which was undetectable in 'non-stem' tumor cells. Comparisons of protein levels and the degree of phosphorylation are made between these samples, other tumor cell lines and hematopoetic stem cells.

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