Biomedical Engineering Reference
In-Depth Information
isotopes is measured on a per cell basis. The mass cytometer has two major
advantages relative to standard fluorescence cytometry: There are 30-50 metal
isotopes that can be used to tag antibodies and there is essentially no compensa-
tion required, enabling simple experimental setup and reducing the “art” behind
high dimensional flow cytometry.
15.2 INSIGHT GAINED THROUGH MULTIPARAMETER
MEASUREMENTS
Diseases affect many cell types and cellular processes. Although this might seem
obvious, its value is often underestimated in drug discovery paradigms. Current
models attempt to distill diseases down to a single drug target. Massive compound
libraries are then screened against this one target to find lead compounds. Once a drug
is identified, its effects on cellular components other than the initial target are often
poorly defined due to the complexity of cellular biochemistry and the inability to
measure drug action on the many cell types of the body.
The multiparameter nature of flow cytometry, and the unique capabilities of
phospho flow to interrogate signaling networks, provides solutions to these difficul-
ties. First, by measuring 10 or more parameters simultaneously, phospho flow enables
us to analyze the phosphorylation levels of multiple signaling proteins to yield a clear
picture of how the relevant signaling network is structured and how it may be
perturbed. In the context of kinase drug discovery, phospho flow can be used to
determine how selective a drug is for a particular pathway of interest. For instance, one
may be interested in inhibiting signaling via PI3 kinase and Akt without affecting
MAP kinase or Jak-Stat signaling. With phospho flow, major players from each of
these signaling pathways can be measured simultaneously in the presence of the drug
to determine how selective the compound is in the context of the cellular system.
Nonspecific effects of potential drugs on metabolic state or ATP metabolism, for
instance, can quickly be identified. In our experience, analyzing more signaling
pathways yields fewer lead compounds that are selective for the signaling pathway of
interest. Therefore, it is of critical importance to analyze drug action in the context
of the cell and in the context of active signaling, not as isolated proteins devoid of
cellular machinery.
Second, measurement of 10 or more parameters enables us tomeasuremultiple cell
surface proteins in addition tomultiple components of the cell signaling network. This
allows work to be done directly in patient samples, effectively describing the
landscape of signaling abnormalities or changes that occur during disease. For
instance, a blood sample from a cancer patient can be taken and signaling via MAP
kinase and Akt pathways can be measured in B cells, T cells, monocytes, neutrophils,
or known disease-specific cell subsets. These “signaling profiles” obtained after
different extracellular ligands are added to the patient samples to determine how the
signaling system responds can be used to identify potential drug targets and to stratify
patients into unique groups. Importantly, such signaling profiles describe both the cell
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