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The discovery of tumor stem cells in acute myeloid leukemia a decade ago initiated a field of research that has seen accelerated growth in the past few years. Now researchers are describing tumor stem cells in a variety of hematopoietic and solid tumors. The impetus for much of this research has been the desire to identify targets for molecular medicine in these critical tumor populations. The molecular pathways that functionally define these cells are of particular interest as therapeutic targets. Techniques describing these pathways in stem cells are different than those used in conventional biology as these cells are rare and often generate insufficient material to use standard methods. Quantitative measurements, where comparisons and contrasts are made between tumor stem cells and their surrounding progeny, are made using DNA microarray and qPCR approaches. Unfortunately, the rare nature of stem cells in these samples limits or prevents quantitative protein analysis. Here we describe a technique utilizing a nano-scale proteomic platform (Firefly) to measure a number of prototypical tumor stem cell proteins. Transitional tumor stem cells (TCC+) were sorted from transitional tumor cells (TCC-) and lysed in HNTG (50 mM HEPES, 150 mM NaCl, 0.1% Triton X 100, 10% Glycerol). Following addition of electrophoresis cocktail, the contents of 400 cells in 400 nl were subjected to isoelectric focusing and immobilization. Immunodetection was performed and quantitation of signal was measured by HRP-labeled secondary chemiluminescence. Quantitation of proteins that are not regulated at the transcriptional level, such as β-catenin and MAPK signaling proteins, could not be differentiated at the RNA levels between TCC+ and TCC- cells. However, protein levels and post-translational modifications were strikingly different. A panel of RNA/proteins which define transitional tumor stem cells will be described along with examples that emphasize the importance of protein measurements in addition to nucleic acid measurements.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA