PORTEFEUILLE

Growing tumors induce the formation of new vessels (by a process referred to as tumor angiogenesis), which in turn provide oxygen and nutrients favoring tumor progression. Inhibition of tumor angiogenesis suppresses tumor growth and recently three anti-angiogenic drugs have been approved for treating human cancer. Anti-angiogenic therapies are expected to significantly improve cancer treatment in the years to come. To date, however, there are no validated tests allowing detection of angiogenesis and monitoring anti-angiogenic drug activity in patients.
Tumors can mobilize specific cell populations from the bone marrow and these cells accumulate at tumor sites to promote angiogenesis and metastasis. Because of their easy accessibility within peripheral blood, tumor-mobilized cells represent an ideal source of surrogate markers of angiogenesis for clinical use.
The goal of this project is to validate the use of tumor-mobilized bone marrow-derived cells as a source of surrogate marker of angiogenesis. We will analyze changes in their gene expression profile by a technique called the single channel quantitative multiplex polymerase chain reaction (multiplex Q-PCR). Candidate genes have been identified in our laboratory by microarray-based analysis.
Multiplex Q-PCR is a technology that allows simultaneous quantitative measurement of several transcripts based on small amounts of starting material. It has been pioneered and developed by the collaborating investigator Dr. Stavros Therianos and represents the core technology underlying Diagnoplex scientific and business development.

The Gebert Rüf Stiftung is providing an essential bridging function between research and a cutting-edge product. The final product will be a „kit“ to use in standard diagnostic laboratories. This assay will be useful in the clinic for diagnostic and monitoring purposes, and in research for new drug development and the study of regulation of angiogenesis.

In this project we have established two cancer models to monitor mobilization of bone marrow derived cells by tumor-secreted factors. The first is based on HCT116 (a colon cancer-derived line) expressing high and low VEGF levels, the second and third consist of breast cancer-derived lines (human MDA-MB-231 and murine 4T1 cells) injected orthotopically in mice mammary fat pad. During tumor growth we monitored the relative frequency of different circulating leukocyte populations by 4-colour flow cytometry. The data obtained so far revealed that growing tumors increase the frequency of CD11b+ cells, comprising both monocytic and granulocytic populations. In addition we have identified a CD11+ cell subpopulation that is highly induced during tumor progression and its frequency correlates with tumor masse. Based on these results we have sorted different peripheral blood cell populations from control and tumor bearing mice and performed a microarray-based gene expression analysis. Unsupervised cluster analysis revealed that the two cell population clustered separately thereby distinguishing tumor-free and tumor-bearing mice. 414 genes were down- or up-regulated > 2 fold. We have now started to validate these changes using a multiplex PCR platform established in the laboratory.
We have recruited 80% of the cancer patients planned for a clinical study in advanced colon cancer treated with chemotherapy (FOLFOX) alone (group 1) or chemotherapy combined with Avastin (an anti-VEGF-A antibody used in the clinic) (group 2). Blood was collected from patients before therapy, and after the first and second treatment cycles. PBMC are being analyzed by multicolor FACS to monitor changes in the frequency of cell populations containing putative angiogenic precursors, and by PCR to monitor changes in gene expression.

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