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Nov 15, 2010 (Vol. 30, No. 20)

Flow Cytometry Expands Application Range

Novel Dimensions and Capabilities Keep Technology Fresh and in Demand at Labs

  • The Cellular Social Networker

    Click Image To Enlarge +
    Functional tumor characterization of blood cells in a patient with chronic myelomonocytic leukemia (CMML): For each cell subset, signaling activity (boxed proteins) is measured in the presence and absence of drug treatment (yellow boxes) across multiple pathways representing diverse biology. During disease progression, changes in the signaling profile and therapeutic sensitivity/resistance can be monitored. [Nodality]

    Single-cell network profiling applications were discussed by Todd Covey, a scientist at Nodality. Nodality developed its platform in response to the lack of technologies that can measure signal-transduction networks in drug development and in clinical medicine.

    The technology uses quantitative flow cytometry to measure phosphoproteins in signaling pathways inside an individual cell. A cancer cell, for example, has a pathway signature unique to its genetic and epigenetic changes.

    A large proportion of drugs in development are kinase inhibitors, but many fail because the drug is not being correctly used in the patient population with the relevant biology, according to Covey.

    The Nodality approach can be used in the drug-development field to identify what pathways a drug is targeting, or in a clinical application to define a patient population. Later, the two approaches merge as patient and drug are matched based on single-cell network profiles, and biology of tumor is matched to mechanism of drug.

    “Each cell is its own separate measurement,” said Covey. “We’re able to look at signaling in a variety of different cell populations at once. We’re able to stimulate multiple pathways in different cell populations and see the activity of a certain drug in different cell pathways.”

    In a case study of an 83-year-old man with chronic myelogenous leukemia being treated with Dacogen (decitabine), Covey tested multiple drugs including a number of types of targeted kinase inhibitors in various combinations and was able to observe the effects on apoptosis and cytostasis within different tumor populations. One of his findings was that decitabine was active against one cell type of the tumor, but not potent against another cell type.

    “Being able to pull apart heterogeneous cell types and see different drug effects is powerful,” Covey remarked, “especially as you monitor the patients along their disease course.”

  • Toxic Assets

    In his presentation, Joseph Tario Jr., predoctoral research affiliate at the Roswell Park Cancer Institute, explored a new way of exploiting the power of flow cytometry in a practical, clinical research setting. Tario and his colleagues developed an assay for assessing cytotoxicity by flow cytometry, using components easily accessible to most laboratories.

    Cytotoxicity assays are useful in cancer research because they can monitor whether a drug or therapeutic intervention can render the immune system capable of killing tumor cells. This contrasts to other readouts such as the measurement of immune cell numbers or their phenotypic activation status. While such investigations are meaningful, they provide little information as to whether the cells are capable of eliminating disease.

    “Cytotoxicity is an important metric in determining the success of an immune response. Conversely, the absence of cytotoxicity when it is expected to occur may indicate that there’s a suppressive environment that is being imparted upon the cell system,” Tario pointed out.

    Restoring the immune system’s ability to kill cancer cells is an important strategy in developing anticancer therapies. Flow cytometric methods of measuring cytotoxicity have been shown to compare favorably to the current prevailing methodology, the chromium release assay, which requires the use of dangerous radioactive reagents, often exhibits high backgrounds, and is entirely uniparametric.

    In the flow cytometric assay, effector and target cell populations were discriminated from one another with two different lipophilic dyes and viability was assessed with a DNA intercalating reagent. Samples were normalized to controls via the use of fluorescent enumeration beads, and data was acquired on a 14-color LSRII flow cytometer from BD.

    The assay gives an accurate readout of cytotoxicity, but its true power comes from the ability to combine cytotoxicity with additional simultaneous and correlated measurements such as antigen-expression profiles and cytokine production in order to more fully elucidate how an intervention affects immune cells.

    “Our work on the improved cytotoxicity assay is a step in a process that represents a paradigm shift toward the increasing role of flow cytometry in the laboratory, Tario concluded. “Due to its multiparametric nature, flow cytometry yields data that is inherently more useful than uniparametric technologies such as chromium release. Furthermore, flow cytometry is logistically more feasible than many of the technologies it is capable of replacing.”

  • Which Way to the Future?

    Trends in flow cytometry favor smaller, more powerful lasers, instruments capable of larger numbers of simultaneous measurements, and increasingly advanced software to handle the resulting data avalanche. In the category of instruments for research analysis, manufacturers are pushing the boundaries of analysis in two directions. Using greater numbers of lasers and dyes allows researchers to carry out experiments that measure many parameters simultaneously. At the same time, smaller, simpler flow cytometers are being developed that can be taken into the field and into developing countries to do CD4 counts for HIV testing.

    These trends could take flow cytometry in a number of different directions, according to Bill Rhodes, president of BD Biosciences.

    “In terms of cutting edge, what you’re looking at is the ability to sort a wide variety of sizes of cells in ways that keep them viable, and to sort them using multiple parameters,” Rhodes said. “A lot of cell sorting now is being looked at for the purposes of investigating disease and perhaps, using induced pluripotent stem cells, dendritic cells, or other types of regulatory cells for the purposes of therapy.”

    For example, in the future, flow cytometry could be used to purify stem cells for bone marrow transplantation. Advanced cell sorting could help find and eliminate extremely rare cells and improve the odds of success by using a more highly purified donor sample.

    “There are probably dozens, if not more, trials now around the world where flow cytometry is being used in conjunction with bulk separation to get to that high level of purity,” Rhodes added.

    For researchers, Rhodes predicted that, in addition to better instruments for power users, there will also be flow cytometry options for researchers who may not be experts at interpreting fluorescence spectra or calibrating optics but who want to use flow cytometry analysis in their work, for example in marine biology and environmental science. Those systems would have one or two lasers, using three or four colors at a time, but would be equipped with advanced software in their user interface.

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