Biomedical Engineering Reference
In-Depth Information
7.2 STAGES IN THE DEVELOPMENT OF IN VIVO TESTING FOR
NOVEL AGENTS
7.2.1 Developing Assays from First Principles
Most of the applications reviewed in this chapter can be defined in terms of cellular
biochemistry. There is an unusual breadth in the range of applications used to monitor
drug effects at the single-cell level, relative to other fields of flow cytometry. This
reflects the scope of drug development in the era of molecular medicine. Although
some applications can be regarded as “generic,” for example, identifying and
optimizing an appropriate antibody readout for drug effect, other techniques that
have been published are completely novel and built up from first principles. It
therefore follows that success in this field requires a multidisciplinary approach,
involving flow cytometry technology, pharmacology, and cell biology.
In addition, for the biochemical changes to be measured, the type of material to be
studied needs to be considered. This is particularly important when the intent is to use
flow cytometry to monitor drug effects in patients rather than laboratory models.
Obtaining sequential samples from solid tumor patients is clearly problematic.
Although there are many recent reports where this has been successfully done using
needle biopsies, very few studies have applied flow cytometry to these samples.
However, after years of relative neglect, an appreciation of the analytical power of
high dimensional flow cytometry to study solid tumors has resurfaced, in large
measure due to interest in cancer stem cells [1, 2]. Further developmental work to
study pharmacodynamic effects therefore appears warranted, perhaps initially based
on primary human cancer xenografts rather than samples obtained directly from the
patient.
7.2.2 Cell Signaling: Introduction
Cells respond to environmental cues, including growth factors and cell/cell or cell/
extracellular matrix contact, by activating (or sometimes inhibiting) signal transduc-
tion pathways. These pathways involve protein interactions that result in posttransla-
tional modifications, the commonest of which is phosphorylation at serine, threonine,
or tyrosine sites. They are highly regulated by inhibitory and stimulatory feedback
loops and through pathway interactions. In addition to regulating genes involved in
cell growth, survival, and differentiation, signaling pathways also control cellular
metabolism and motility. Cell signaling is disrupted in the early stages of cancer
development, and it plays a major role in tumor progression toward more aggressive
phenotypes. Therefore, there is intense interest in the modulation of these pathways as
a novel approach to cancer treatment [3-10]. Most of the agents in current use are
either monoclonal antibodies directed toward extracellular components of signaling
pathways or small molecules that typically inhibit kinases through ATP competition.
The Her2 inhibitor trastuzumab (Herceptin), used to target Her2 overexpressing
breast cancers, and the bcr/abl kinase inhibitor imatinib (Gleevec), used to treat
chronic myeloid leukemia (CML), are among the best known examples [11, 12].
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