In 2006, Ovar’coming Together awarded $30,000 in research grants. The proposals were reviewed by a national review board with final determination being made by the Ovar’coming Together Executive Committee. In addition, $25,000 was gifted to Indiana University Cancer Center for ovarian cancer research. Proceeds form the Old Bags Luncheon provided the gift award, and was donated in memory of Susie Hazelett.

Research Title	Researcher and Institution	Amount Awarded
Chemo sensitization of Platinum-Resistant Ovarian Tumors by Combined Epigenetic Therapies	Submitted by Curtis Balch, Ph.D. Medical Sciences Program Indiana University	$10,000
Probing the Metastic Ovarian Cancer Cells by IN Vivo Two-Photon Flow Cytometry	Submitted by Philip S. Low, Ph.D. Department of Chemistry Purdue University	$20,000
Ovarian Cancer Research Gift in memory of Susie Hazelett	Indiana University Cancer Center	$25,000
Total Funded for 2006 Research		$55,000

Chemo sensitization of Platinum-Resistant Ovarian Tumors by Combined Epigenetic Therapies – Dr. Curt Balch

Ovarian cancer is asymptomatic in its early stages and thus diagnosis is generally restricted to advanced disease.  While tumor resection and a post surgical platinum/taxane regimen are effective in achieving clinical remission, the emergence of drug-resistant tumor phenotypes usually occurs, at which point the disease is fatal.  Consequently, strategies aimed at avoiding or reversing chemo resistance would be highly beneficial in prolonging remission, and possibly, eliminating recalcitrant tumor subpopulations.

One contributing factor strongly implicated in tumor progression and the development of drug resistance is silencing of tumor suppressors by epigenetic (non-DNA coding) modifications.  Subsequently, agents capable of reversing such modifications could likely restore sensitivity to standard therapies: two such   reversible epigenetic modifications are DNA methylation and histone deacetylation.  As both of these phenomena are transcriptionally repressive, their coordinated inhibition (vs. either alone) has shown maximal potential for tumor suppressor re-expression.  In this proposal, preclinical studies will be performed to optimize a regimen combining a novel methylation inhibitor, zebularine, with a rationally designed histone deacetylase inhibitor, OSU-42, for resensitization of primary (patient-derived ascites) and secondary ovarian cancer lines and tumors.  Resensitization will be assessed by cell viability assays, following carboplatin treatment, with and without pretreatment with various zebularine/OSU-42 combinations.

To determine possible mechanisms of chemo sensitization by this combined regimen, demethylation of key tumor suppressor genes and inhibition of deacetylation of histone and non histone proteins will be examined.  Following optimization of a combined treatment used to resensitize carboplatin-resistant ovarian cancer cell lines. A pilot study will be performed to assess the feasibility of chemo sensitization in whole animals, using chemo resistant human ovarian cancer cells as xenografts in mice.  Following tumor formation, carboplatin will be administered with or without pretreatment with zebularine/OSU-42. To assess chemo sensitization, tumor volumes will be recorded biweekly.  Additionally, bioactivity of the regimen will be examined by blood draws and examination of global DNA methylation and histone acetylation in peripheral blood lymphocytes.  Tumor volumes will be directly assessed and compared between mice treated with carboplatin in the presence or absence of zebularine/OSU-42; tumors will also (similar to lymphocytes) be examined for epigenetic changes and gene re-expression.

The results of this proposed undertaking will lay the foundation for an R01 or an equivalent grant proposal from federal or private agencies.  Such a large scale study would comprehensively examine pharmacological parameters such as clearance rates, bioavailability, and toxicity of the successful treatment regimens, establishing feasibility for human studies.  In summary, theses therapies, with further study, possess considerable potential for clinical translation.

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Probing the Metastatic Ovarian Cancer Cells by In Vivo Two-Photon Flow Cytometry - Dr. Phillip S. Low           

Melanoma cell adhesion molecule (MCAM) is a cell-surface adhesion molecule expressed on over 70% of metastatic melanoma cells but not expressed in normal melanocytes in vivo.  Protein levels of MCAM correlate with the aggressive invasive behavior of melanoma cells in vitro and in vivo.  Folate receptor positive (FR+) metastatic disease can arise from malignant masses in which FR expression cannot be detected, suggesting that FR expression might constitute another marker of cells with elevated metastatic potential. In short, markers of metastatic cells are becoming available that will allow fluorescent labeling of malignant cells during circulation in vivo.  We have developed a method called in vivo two-photon flow cytometry (IVTPFC) that is able to detect and quantify the circulating leukemia cells through near surface blood vessels. In this specific aim, we propose to exploit the application of this method for the analysis of circulating ovarian cancer cells in vivo because over 90% of ovarian cancer cells are FR positive. The two-photon scope scans transversely across the blood vessel to pick up the fluorescence signals emitting from the blood circulation.

Previous studies by our group have shown that folate conjugates are rapidly cleared from the blood (t ½ = 3.5min for folate-FITC) following intravenous administration, if they are not captured by a FR-expressing cancer cell.  Another study on the plasma depletion of folate-rhodamine show similar depletion kinetics to folate-FITC.  Thus, folate conjugates should not cause significant interference from background fluorescence if sufficient time is allowed for unbound conjugates to clear.  More importantly, there are no signals captured by two-photon scanner for at least 40 minutes on the mice without tumors with injection of fluorescent folate conjugates.  This strongly indicates that high selectivity and specificity of the probes rendering no false positives.

We have initially tested the feasibility of using IVTPFC to quantitate circulating leukemia cells by intravenously injecting L1210A cells mimicking circulating leukemia cancer cells in vivo and then intravenously injecting fluorescent folate conjugates. The imaging procedure is carried out on mice using two-photon excitation fluorescence and the optimal imaging method for deep tissue penetration together with programmable software.  Cancer cells were readily detected as they flowed past the two-photon scanner.  Assuming the mouse model is representative of the human situation, these data would suggest that efficient quantitation of circulating tumor cells by in vivo flow cytometry is feasible.

Antibodies are widely used in biological assay such as western blots, flow cytometry and etc.  The disadvantages for antibody targeting are longer plasma circulation time, immuno-triggered scavenging of target cells thus lower detection sensitivity etc.  For further exploration on the choice between folate conjugates and antibody conjugates on this technology, three home-made polyclonal antibodies from anti-rabbit serums against folate receptor were conjugated and tested by in vivo flow cytometry.  However, no signals can be detected by the two-photon scanner.  Ex vivo affinity binding assay on cell culture by FACS (Fluorescence-Activated Cell Sorter) indicates that the labeling efficiency of the three antibodies is about 10 folds lower than EC17.  The weaker binding of the antibody may be attributed to the special hindrance caused by bulky hydration volume compared to the small ligand conjugates.  Consequently, both lower binding efficiency of antibodies and fast depletion of the tumor cells triggered by the phagacytosis of macrophage contribute to the low sensitivity of antibody labeling.

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