In recent years, an exponentially growing number of studies have focused on identifying cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial in controlling and curing cancer: through asymmetric division CSCs supposedly drive tumor growth and evade therapy with the help of traits shared with normal stem cells such as quiescence, self‐renewal ability, and multidrug resistance pump activity. Here, we give a brief overview of techniques used to confirm the stem cell‐like behavior of putative CSCs and discuss markers and methods for identifying, isolating, and culturing them. We touch on the limitations of each marker and why the combined use of CSC markers, in vitro and in vivo assays may still fail to identify all relevant CSC populations. Finally, the various experimental findings supporting and contradicting the CSC hypothesis are summarized. The large number of tumor types thus far with a subpopulation of uniquely tumorigenic and therapy resistant cells suggests that despite the unanswered questions and inconsistencies, the CSC hypothesis has a legitimate role to play in tumor biology. At the same time, experimental evidence supporting the established alternative theory of clonal evolution can be found as well. Therefore, a model that describes cancer initiation and progression should combine elements of clonal evolution and CSC theory. © 2008 International Society for Advancement of Cytometry

Background: Fluorescence resonance energy transfer applied in flow cytometry (FCET) is an excellent tool for determining supramolecular organization of biomolecules at the cell surface or inside the cell. Availability of new fluorophores and cytometers requires the establishment of fluorophore dye pairs most suitable for FCET measurements.Methods: A gastric tumor cell line (N87) was labeled for major histocompatibility complex class I heavy chain and β2‐microglobulin with antibodies conjugated with fluorescein‐ and indocarbocyanine‐like fluorophores and analyzed in FCET measurements on a cell‐by‐cell basis using three flow cytometers: FACSCalibur, FACSDiVa, and FACSArray.Results: Normalized fluorescence intensity values were measured and normalized energy transfer efficiencies, spectral overlap integrals, and crucial dye‐ and instrument‐dependent parameters were calculated for all matching pairs of seven fluorophores on the three commercial cytometers. The most crucial parameter in determining the applicability of the donor‐acceptor pairs was the normalized fluorescence intensity and the least important one was the spectral overlap.Conclusions: On the basis of available laser lines, the optimal dye pair for all three cytometers is the Alexa546‐Alexa647 pair, which produces high energy transfer efficiency values and has the best spectral characteristics with regard to laser excitation, detection of emission, and spectral overlap.

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, β1‐integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two‐sided FRET), the relationship of β1‐integrin–ErbB2 heteroassociation to ErbB2 homoassociation and of β1‐integrin–ErbB2 heteroassociation to ErbB2–CD44 heteroassociation was studied by correlating pixel‐by‐pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between β1‐integrin–ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK‐BR‐3 cells, while modest positive correlation was found between β1‐integrin–ErbB2 and ErbB2–CD44 heteroassociation on trastuzumab resistant MKN‐7 cells. The FRET efficiency values of β1‐integrin–ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set‐up termed two‐sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with β1‐integrins. © 2007 International Society for Analytical Cytology