02 BOOK OF ABSTRACTS .indd

Tumor proteolysis: a multidimensional approach
Kamiar Moin 1,2 , Donald Schwartz 2 , Mansoureh Sameni 1 ,
Stefanie R. Mullins 1,2 , Bruce Linebaugh 1 , Deborah Rudy 1 , Ching Tung 3
and Bonnie F. Sloane 1,2
1
Department of Pharmacology and 2 Karmanos Cancer Institute, Wayne State University
School of Medicine, Detroit, Michigan, USA; 3 Center for Molecular Imaging Research,
Massachusetts General Hospital, Harvard University, Boston, Massachusetts, USA
Despite the awareness that proteolysis is essential for cancer progression, and
that proteases represent potential drug targets, clinical trials for cancer treatment
with inhibitors of matrix metalloproteases have failed. Moreover, a broad and
comprehensive strategy to identify potential protease targets has not been employed.
We hypothesize that proteases are valid therapeutic and prevention targets in cancer
and that imaging of protease activity and its inhibition in vivo will provide a means
to confirm this hypothesis. In our laboratories we have developed functional optical
imaging techniques to monitor tumor progression and tumor-host interactions based
on proteolytic activity, both in vitro in live cells and in vivo.
In vitro, we have used a 3-dimensional assay system to study tumor-stromal
interactions in real time, utilizing confocal and multiphoton microscopy to document
our observations. Using this system, we have found that both pericellular and
intracellular proteolysis occur during tumor invasion. Furthermore, there is a high
degree of interaction between tumor and stromal cells. Our results indicate that
tumor cells actively recruit stromal cells and that these cells contribute significantly
to the proteolytic events occurring in the tumor environment.
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In order to identify the potential proteases, their endogenous inhibitors, and genes
that interact with proteases in the tumor micronenvironment, we developed a dual
species custom oligonucleotide microarray in conjunction with Affymetrix, Inc.
The Hu/Mu ProtIn array contains 516 and 456 custom probes sets that survey 426
and 390 unique human and mouse genes of interest, respectively. Our goal is to
determine tumor (human) and host (murine) contributions to the degradome in
orthotopic xenograft models of cancer and demonstrate the utility, versatility and
specificity of the custom probe sets. To validate the utility of the array, we profiled
human MDA231 and MDA435 breast carcinoma cells derived from in vitro cultures
or orthotopically implanted xenografts, as well as normal mouse mammary fat pads.
We also profiled MCF-10A breast cell lines, grown in the 3D Matrigel overlay model,
as well as xenografts of the MCF-10DCIS.com line, a line that forms DCIS lesions that
progress to invasive carcinomas in mice. We have identified genes that were either
significantly up or down regulated in DCIS.com as compared with MCF10A or were
derived from host cells that have infiltrated into the DCIS.com xenografts. We are
currently validating genes of interest by both Q-RT-PCR and immunohistochemistry
in human breast samples.
In vivo, we have utilized quenched fluorescent probes that are activated by proteases.
We have found that upon injection of these probes into tumor-bearing mice, the