Jayne M. Silver

Virtual screening targeting the urokinase receptor, biochemical and cell-based studies, synthesis, pharmacokinetic characterization, and effect on breast tumor metastasis.

Virtual screening targeting the urokinase receptor (uPAR) led to (±)-3-(benzo[d][1,3]dioxol-5-yl)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-4-phenylbutan-1-amine 1 (IPR-1) and N-(3,5-dimethylphenyl)-1-(4-isopropylphenyl)-5-(piperidin-4-yl)-1H-pyrazole-4-carboxamide 3 (IPR-69). Synthesis of an analogue of 1, namely, 2 (IPR-9), and 3 led to breast MDA-MB-231 invasion, migration and adhesion assays with IC(50) near 30 μM. Both compounds blocked angiogenesis with IC(50) of 3 μM. Compounds 2 and 3 inhibited cell growth with IC(50) of 6 and 18 μM and induced apoptosis. Biochemical assays revealed leadlike properties for 3, but not 2. Compound 3 administered orally reached peak concentration of nearly 40 μM with a half-life of about 2 h. In NOD-SCID mice inoculated with breast TMD-231 cells in their mammary fat pads, compound 3 showed a 20% reduction in tumor volumes and less extensive metastasis was observed for the treated mice. The suitable pharmacokinetic properties of 3 and the encouraging preliminary results in metastasis make it an ideal starting point for next generation compounds.

Differential Secondary Reconstitution of In Vivo-Selected Human SCID-Repopulating Cells in NOD/SCID versus NOD/SCID/γ chainnull Mice

Humanized bone-marrow xenograft models that can monitor the long-term impact of gene-therapy strategies will help facilitate evaluation of clinical utility. The ability of the murine bone-marrow microenvironment in NOD/SCID versus NOD/SCID/γ chainnull mice to support long-term engraftment of MGMTP140K-transduced human-hematopoietic cells following alkylator-mediated in vivo selection was investigated. Mice were transplanted with MGMTP140K-transduced CD34+ cells and transduced cells selected in vivo. At 4 months after transplantation, levels of human-cell engraftment, and MGMTP140K-transduced cells in the bone marrow of NOD/SCID versus NSG mice varied slightly in vehicle- and drug-treated mice. In secondary transplants, although equal numbers of MGMTP140K-transduced human cells were transplanted, engraftment was significantly higher in NOD/SCID/γ chainnull mice compared to NOD/SCID mice at 2 months after transplantation. These data indicate that reconstitution of NOD/SCID/γ chainnull mice with human-hematopoietic cells represents a more promising model in which to test for genotoxicity and efficacy of strategies that focus on manipulation of long-term repopulating cells of human origin.

Longitudinal Bioluminescence Imaging of Primary Versus Abdominal Metastatic Tumor Growth in Orthotopic Pancreatic Tumor Models in NSG Mice.

Objectives The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring primary and abdominal metastatic tumor growth in mouse orthotopic models of pancreatic cancer. Methods A semiautomated maximum entropy segmentation method was implemented for the primary tumor region of interest, and a rule-based method for manually drawing a region of interest for the abdominal metastatic region was developed for monitoring tumor growth in orthotopic models of pancreatic cancer. The 2 region-of-interest methods were validated by having 2 observers independently segment Panc-1 tumors, and the results were compared with the number of mesenteric lymph node nodules and histopathologic assessment of liver metastases. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where separate groups of animals were implanted with different numbers of cells. Results The results demonstrated that the segmentation methods were highly reliable, reproducible, and robust and allowed statistically significant discrimination in the growth rates of primary and abdominal metastatic tumors of different cell lines implanted with different numbers of cells. Conclusions The present results demonstrate that primary tumors and abdominal metastatic foci in orthotopic pancreatic cancer models can be reliably quantified separately and noninvasively over time with bioluminescence imaging.

Abstract 4961: Longitudinal bioluminescence imaging of primary versus abdominal metastatic tumor growth in orthotopic pancreatic tumor models in NOD/SCIDγ(-/-) mice

Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis and is currently the fourth leading cause of cancer death in the United States. The lethal nature of PDAC is strongly associated with metastases to distant organs. The purpose of the present study was to develop and validate noninvasive bioluminescence imaging methods for differentially monitoring the kinetics of primary and abdominal metastatic tumor growth in mouse orthotopic models of pancreatic cancer. A semiautomated maximum entropy segmentation method was implemented for the primary tumor region-of-interest (ROI), and a rule-based method for manually drawing an ROI for the abdominal metastatic region also was developed. The two ROI methods were first validated by having two observers independently construct ROIs for the tumors of animals implanted orthotopically with Panc-1 cells, and the results compared with the number of mesenteric lymph node metastatic nodules counted upon necropsy. The findings were extended to orthotopic tumors of the more metastatic MIA PaCa-2 and AsPC-1 cells where different groups of animals were implanted with different numbers of cells. When the data were expressed as the total photon flux (Ph/sec) in the ROIs for the primary tumor and metastases, the total flux within the metastasis ROI was larger in magnitude than the total flux from the primary tumor ROI, at times by as much as several orders of magnitude. However, when the data were expressed as the average flux density (Ph/sec*mm2) within the ROIs, the density of the flux within the smaller primary tumor ROI was larger in magnitude than the density of the flux from the larger metastasis ROI, by as much as several orders of magnitude. Interobserver assessments for total flux and flux density from ROIs for both the primary tumors and metastatic region were highly concordant, with correlation coefficients of r > 0.98, coefficients of variation of ≤ 0.02, and limits of agreement within Citation Format: Harlan E. Shannon, Melissa L. Fishel, Jingwu Xie, Dongsheng Gu, Brian P. McCarthy, Amanda A. Riley, Anthony L. Sinn, Jayne M. Silver, Mark R. Kelley, Helmut Hanenberg, Murray Korc, Karen E. Pollok, Paul R. Territo. Longitudinal bioluminescence imaging of primary versus abdominal metastatic tumor growth in orthotopic pancreatic tumor models in NOD/SCIDγ(-/-) mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4961. doi:10.1158/1538-7445.AM2014-4961

Abstract 4309: Whole slide image analysis quantification in a mouse lung metastasis model

Digital whole slide imaging is the technique of digitizing a microscope slide at the highest resolution to produce a “digital virtual microscope slide”. This digital image can be viewed in three or four fields, from low to high power fields, which can be commonly used to evaluate the tissue. Many of these systems have a whole slide software image analysis system. The goal of this study was to determine if the Aperio positive pixel algorithm (image analysis) could effectively quantitate metastatic mouse lung tumors in a lung section using a HE 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4309. doi:10.1158/1538-7445.AM2014-4309

Abstract 1954: Human circulating progenitor cells of hematopoietic origin promote tumor growth in melanoma xenograft models

The tumor microenvironment contains various cell sub-populations of hematopoietic and endothelial origin. These cells are recruited by the tumor and can play an integral role in regulating tumor growth and metastasis. However, the heterogeneity of these cells has made it difficult to study their specific function in tumorigenesis. We recently developed a detailed polychromatic flow cytometric (PFC) protocol to isolate and characterize the phenotype of the hematopoietic versus endothelial circulating progenitor cells (CPCs) found in peripheral blood (PB). Transplantation of CPC populations into NOD/SCID mice indicated that a purported endothelial CPC subset largely consisted of in vivo engrafting hematopoietic stem cells and myeloblasts, and not endothelial cells. Additionally, our recent PFC studies indicate that hematopoietic CPCs can be subdivided into two cell populations based upon AC133 expression (ViViD − CD14 − glyA − CD34 + AC133 + CD45 dim CD31 + and ViViD − CD14 − glyA − CD34 + AC133 − CD45 dim CD31 + cells), and will be referred to as primitive CPCs and mature CPCs respectively. To date, the functional role of hematopoietic CPCs in tumor growth is not known. We hypothesize that primitive CPCs home to the tumor bed and secrete pro-angiogenic factors to promote tumor growth. To address this hypothesis, we have developed two in vivo modeling approaches. In the first approach, primitive and mature CPCs were isolated from umbilical cord blood CD34 + cells and intravenously injected into NOD/SCID mice harboring C32 human melanoma xenografts that secrete high levels of vascular endothelial growth factor. Tumor growth was monitored for 7 weeks and a statistically significant increase in melanoma growth and weight was observed in mice injected with the primitive CPCs compared to control mice (p + cells). Histological analyses did not detect human CD34 + , AC133 + , or CD31 + cells in the tumor xenograft tissue at week 7. Time-course studies are in progress to determine if human hematopoietic cells can be found in the tumor bed during the early stages of tumor growth. In the second approach, sub-lethally irradiated NOD/SCID mice were first transplanted with human CD34 + cells and detectable levels of primitive CPCs in the PB were observed at one month post-transplantation. C32 melanoma cells were then injected subcutaneously into the flank and tumor growth monitored. At 3-4 weeks post-tumor implantation, melanoma xenografts began to grow more rapidly in mice previously transplanted with human CD34 + cells compared to melanoma xenografts in sub-lethally irradiated, non-transplanted mice. Taken together, these data indicate that primitive human CPCs of hematopoietic origin play a functional role in tumorigenesis and the in vivo models developed here can be used to delineate how hematopoietic CPCs regulate tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1954.

Abstract 5373: Modulation of temozolomide-mediated DNA damage in glioblastoma multiforme by the HDM2 antagonist, nutlin3

Development of efficacious strategies for eradication of glioblastoma multiforme (GBM) remains a significant challenge. While treatment of GBM with radiation and temozolomide (TMZ) has improved clinical outcome, lack of long-term efficacy can be due to the ability of GBM to acquire resistance to therapy by modulation of signaling pathways that control DNA repair and cell survival. The HDM2 antagonist, Nutlin3, blocks interactions of HDM2 with key signaling molecules such as p53, p73α, and HIF1α, and modulates their downstream effector function. A panel of human GBM cell lines is currently being used to monitor the extent to which Nutlin3 can sensitize cells to TMZ. In vitro survival assays indicate that Nutlin3 potentiates TMZ-mediated cell death; analysis of cell survival data using Dose Effect CalcuSyn software indicates that the combination index value was 6 -methylguanine DNA methyltransferase (MGMT) and proteins involved in base-excision repair (BER). In vivo target-validation studies using GBM.10 xenografts confirmed that repair of TMZ-mediated DNA damage is modulated in the presence of Nutlin3. GBM.10 xenografts were treated with vehicle, TMZ, nutlin3, or TMZ/Nutlin3 and modulation of critical targets determined by Western analyses. Increases in total p53, p53 phosphorylated at residue serine16, p73α, and substantial decreases in MGMT and the BER protein, APE-1, were observed following treatment with Nutlin3/TMZ compared to vehicle or single-agent exposure. Busso et al (Oncogene 2009) recently reported that HDM2 can monoubiquitinate APE-1 leading to proteosomal-mediated degradation. In addition, recombinant activity assays in our laboratory indicate that HDM2 directly monoubiquitinates recombinant MGMT and may play a role in its degradation. These data suggest that DNA repair proteins required for repair of TMZ-mediated DNA damage can be downregulated by altering HDM2-mediated signaling. Combination therapy that targets the p53-HDM2 E3 ligase network and DNA repair represents a novel approach towards improving treatment of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5373.