Fluorescent Macromolecular Sensors of Enzymatic Activity for In Vivo Imaging - Progress in Molecular Biology and Translational Science - page 372

metastasis, are always associated with coordinated activation of enzymatic
cascades. Enzymes such as MMPs, cathepsins, and uPA have been linked
to the accelerated breakdown of the extracellular matrix and induction of
endothelial and supporting cell migration and tumor angiogenesis. 2,105-107
For example, it is known that tumor cells trigger protease-dependent
extracellular matrix remodeling in response to interactions with
neighboring mesenchymal and hematopoietic stromal cells, which in turn
allow malignant cells to infiltrate the basement membrane and invade
other tissues. 105,108 A number of different fluorescence-based sensors have
been developed and shown to be capable of tumor detection in animal
models of cancer. These differ most significantly in the way in which the
sensor targets the tumor, thus giving specificity to the generated signal.
Our laboratory achieved one of the initial successes in enzyme-mediated
tumor detection using a polymer-based NIRF sensor initially designed for
imaging tumor proteolytic activity 66 ( Fig. 9.1 ). This PGC, which was de-
scribed in more detail in Section 5 , is biocompatible and consists of a
high-molecular-weight carrier of fluorophores with protease sensitivity
and specificity “built in” the structure elements of the macromolecular sen-
sor. The unique chemistry of the PGC-fluorophore sensor allows it to be
retained in the bloodstream (i.e., have long circulation times) and to pass
through highly permeable tumor vasculature after which it accumulates
in tumors. Other inherent strengths of this design are that (1) the “off”
and “on” states of fluorescence emission are directly linked to the activity
of the targeted enzyme; (2) the enzyme is not inactivated by the probe
and thus can continue to free more cleavable Cy5.5 products, thus ampli-
fying the fluorescent signal over time ( 24 h); and (3) when used to target

intracellular enzymes, the cleaved fluorescent products are sequestered in the
cells that comprise the solid tumor (cancer cells and activated stromal cells),
thereby allowing the target-to-background ratio (TBR) to increase as the
free probe (and its associated background signal) is eliminated from the body.
PGC-based probes have also been developed that target extracellar enzymes
such as MMPs, uPA, and thrombin.
Injection of PGC-based fluorescent sensors into tumor-bearing animals
usually resulted in tumor-associated fluorescence intensity which was on
average 12 times higher than the surrounding background and allowed
detection of tumor xenografts in vivo. 67,68 Subsequent studies have
determined that, although taken up and broken down in some tumor
cells, the macromolecular sensor is largely taken up by stromal cells that
are recruited to the site of

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tumor formation (fibroblasts, monocytes/