233 Designing Therapeutic Strategy By Studying Interactions and Effects of Hypoxia, HIF 1α, Surrounding Fibroblasts, and Ectopic p16 Expression on Breast Cancer Mobility and Invasion Molecular Ther[.]
Cancer – Targeted Gene and Cell Therapy I candidate breast cancer metastasis genes including WWTR1 (TAZ), a known and recently validated breast cancer metastasis gene Identifying genes that drive the metastatic process can identify targets for improved therapy 231 Test A Stem Cell Approach for Cancer Blood Linan Liu,1,2,3,4,5 Shirley X Zhang,1,2,3,4,5 Rangoli Aeran,1,2,3,4,5 Wenbin Liao,1,2,3,4,5 Mengrou Lu,1,2,3,4,5 George Polovin,1,2,3,4,5 Egest J Pone,1,2,3,4,5 Weian Zhao.1,2,3,4,5 Department of Pharmaceutical Sciences, University of California, Irvine, CA; 2Department of Biomedical Engineering, University of California, Irvine, CA; 3Sue and Bill Gross Stem Cell Research Center, University of California, Irvine, CA; 4Chao Family Comprehensive Cancer Center, University of California, Irvine, CA; 5Edwards Lifesciences Center for Advanced Cardiovascular Technology, University of California, Irvine, CA Mesenchymal stem cells (MSC) are adult multipotent stem cells that possess regenerative and immunomodulatory properties They have been widely investigated as therapeutic agents for a variety of disease conditions, including tissue repair, inflammation, autoimmunity and organ transplantation Importantly, systemicallyinfused MSC selectively home to primary and metastatic tumors, though the molecular mechanisms of tumor tropism of MSC remain incompletely understood We have exploited the active and selective MSC homing to cancer microenvironments to develop a rapid and selective blood test for the presence of cancer Here we present the concept of using transplanted MSC as the basis for a simple cancer blood test MSC engineered to express humanized Gaussia luciferase (hGluc) co-localize with and persist in cancerous tissue, secreting hGluc into circulation that can be assayed in a minimally invasive fashion as a reporter for cancer presence In vitro, hGluc secreted by engineered MSC can be detected stably over a period of days in the presence of serum In vivo imaging showed that MSC homed to breast cancer lung metastases and persisted longer in tumor-bearing mice than in tumor-free mice ( P < 0.05) hGluc activity in blood of tumor-bearing mice was significantly higher than in their tumor-free counterparts ( P < 0.05) Both in vitro and in vivo data show that MSC expressing hGluc can identify and report small tumors or metastases in a simple blood test format Our novel and simple stem cell-based blood test can potentially be used to screen, detect and monitor cancer and metastasis at early-stages and during treatment Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy 232 High Levels of iCasp9 Expression Are Needed for Suicide Gene Transfer Sabrina Donnou,1,2,3 Catherine Poinsignon,1,2,3 Anne Galy,1,2,3 Sylvain Fisson.1,2,3 Généthon, “Integrare” Research Unit, Evry, France; 2Inserm, U951, Evry, France; 3Université Evry Val d’Essonne (UEVE), UMR_S951, Evry, France Primary central nervous system lymphomas are aggressive tumors with no efficient treatment In this study, we wanted to take advantage of a lentiviral vector (LV) specifically designed to target murine MHC II expressing cells (eg lymphoma B-cells) to evaluate the feasibility of an in vivo targeted suicide gene therapy in a murine model of primary intraocular lymphoma The inducible caspase (iCasp9) (*) strategy used efficiently in T cells was tested as a suicide gene for this approach As a first step, we showed that 15-20% of A20 IIA lymphoma tumor cells were transduced in vitro with the MHC II targeted LV expressing iCasp9, however, apoptosis could not be induced in these cells even after enrichment and cloning None of the stably-expressing clones could be induced to apoptose by the inductor of dimerization These clones had integrated only copy of the transgene suggesting that levels of iCasp9 were insufficient or that cells were resistant to treatment The same results were obtained with LV expressing iCasp9 from the cellular promoters (PGK or EF1α) In contrast, clones transduced with only copy of a gammaretroviral LTR-driven-iCasp9 vector were efficiently induced to apoptose The MLV clones expressed higher levels of transgene than LV clones and 90% of the cells died 24 hours after iCasp9 activation showing that A20.IIA tumor cells were not resistant to the mechanism Clearly, a threshold of iCasp9 expression must be achieved to induce apoptosis by gene transfer In other cells such as 3T3 cells, Jurkat T cells, HCT116 cells and Raji cells, more than copies of the LV PGK-iCasp9 must be integrated to induce apoptosis since cells expressing only copy survive Whereas this threshold of LV transduction may be easily attained in T cells, it is very difficult to integrate more than copies of LV per B cell in spite of repeated cycles of infection coupled with various preactivation stimuli We are currently exploring solutions to augment B cell transduction and to increase iCasp9 expression from LV Altogether, our results provide benchmark values for iCasp9 efficacy and show that highly expressing vectors must be developed to apply iCasp9 suicide gene therapy strategies in B-lymphoma but also with other target cells, to avoid escape to treatment 233 Designing Therapeutic Strategy By Studying Interactions and Effects of Hypoxia, HIF-1α, Surrounding Fibroblasts, and Ectopic p16 Expression on Breast Cancer Mobility and Invasion Yi Lu,1 Jun Zhang.1 Pathology and Laboratory Medicine, University of Tennessee Health Science Center, Memphis, TN Cancer cell migration and invasion play essential roles in the metastatic cascade that transforms the local, noninvasive confined tumor cells to the motile, metastatic cancer cells moving through the extracellular matrix and basement into the circulation Accumulated evidences suggest that intratumoral hypoxia, a characteristic of fastgrowing solid tumors, promotes cancer cell motility and invasive abilities In this study, we investigated the effects of hypoxia and HIF-1α, surrounding fibroblasts, and p16 expression on the migration and invasion of breast cancer cells We found that in general hypoxia promoted breast cancer cell migration and invasion, and cocultured fibroblasts stimulated invasiveness of breast cancer cells Interestingly, HIF-1α from cancer cells and surrounding S91 Cancer – Targeted Gene and Cell Therapy I fibroblasts has respectively profound effects on the invasive ability of breast cancer cells By applying both HIF-1α wild-type (WT) and knockout (KO) fibroblasts and breast cancer cells and using an in vitro invasion assay through commercially precoated invasion inserts, we found that fibroblasts, regardless their HIF-1α status, stimulated cell invasion of breast cancer cells that have intact HIF-1α In contrast, only HIF-1α-expressing fibroblasts, not HIF-1α-nonexpressing ones, stimulated HIF-1α KO breast cancer cell invasion These results suggest the complicated and respective HIF-1α contribution from breast cancer per se and surrounding fibroblasts to the tumor invasion and malignancy Moreover, by using a Tet-on inducible system, we found that ectopic p16 gene transfer is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer cells These results suggest that p16, in addition to its well-known anti-tumor proliferation function, has novel anti-cancer properties capable of suppressing hypoxia/HIF-1α-mediated cancer cell migration and invasion Furthermore, our current ongoing study implies an interaction between p16 and HIF-1α that may elucidate a novel p16-mediated tumor suppression pathway via HIF-1α-regulated signaling These studies together may provide new information of the convergence of several important cell-growth regulation pathways on tumor invasion that would yield potential novel targets for effective cell- or gene-therapy strategy for treatment of breast cancer 234 Hydrodynamic Cell Delivery for Assessment of Tumor Growth and Drug Sensitivity in Different Organs and Microenvironment Mohammad H Alsaggar,1 Qian Yao,1 Dexi Liu.1 Pharmaceutical & Biomedical Sciences, University of Georgia, Athens, GA Tumor metastasis often confers poor prognosis for cancer patients because successful treatment of metastatic tumors remains as an unmet need in clinic Currently used subcutaneous and orthotopic models have failed to truly recapitulate metastatic tumor biology, particularly tumor-stroma interactions within tumor microenvironment in different organs This certainly accounts for insufficient therapeutic outcomes of current antimetastatic therapies that were designed assuming metastatic tumors behave the same in all organs Therefore, relevant tumor models are urgently needed for reliable anticancer drug screening and development, and assessment of tumor-environment interactions in different metastatic sites Toward this end, we have developed a multi-organ tumor growth model using hydrodynamic cell delivery method to establish simultaneous growth in liver, lung and kidney in mice by hydrodynamic tail vein injection of cell suspension We hypothesized that tumors growing in different organs are biologically heterogeneous in growth characters, as well as in tumor response to a given anti-cancer therapy Using luciferase-expressing murine melanoma cells that can be quantified by luciferase assay, our results showed that the liver is the primary organ to be seeded with tumor cells upon hydrodynamic injection, with 35% of tumor cells in liver, 21% in the lungs, and 8% in kidneys Growth rate assessment revealed that liver microenvironment was the most supportive to melanoma tumor growth compared to lungs and kidneys; where tumors grew minimally at early phase, and aggressively thereafter Tumors in different organs were also heterogeneous in tumor response to chemotherapy and immune gene therapy using dacarbazine and interferon beta gene, respectively While lung tumors responded to chemotherapy better than tumors in liver, lung tumors showed minimal response to interferon beta, with around 25% tumor suppression in lung, compared to more than 80% in liver and kidneys On the other hand, tumors in liver showed superior response to interferon beta compared to dacarbazine (81% vs 45% suppression) Differential behavior of metastatic tumors was S92 also confirmed using metastatic colon cancer model established in different organs These results point out the increasing need for better understanding of differences in tumor microenvironment in different organs, as it greatly determines therapeutic outcomes of antimetastatic therapies Moreover, the biological heterogeneity of metastatic tumors necessitates precluding of “one size fits all” therapies, and rethinking of organ-specific therapies and combined treatments to act globally on metastatic tumors in different organs 235 Anticancer Effectiveness of Mesenchymal Stem Cells Transfected With Thymidine Phosphorylase for Colorectal Cancer Cells Nasrin Salehi,1 Ching-An Peng.1 Michigan Technological University, Houghton, MI Gene-directed enzyme prodrug therapy (GDEPT) consists of targeted delivery to tumor cells with a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites One of the major bottlenecks of GDEPT is to have the therapeutic gene specifically target on cancer cells prior to the administration of prodrug Among various gene delivery methods, mesenchymal stem cells (MSCs) have emerged as potential cellular vehicles for gene delivery They exhibit remarkable tumor-tropic and low immunogenic capacities In this study, MSCs was engineered to express thymidine phosphorylase (TP) which has the capability of converting prodrug 5’-deoxy-5-fluorouridine into toxic 5-fluorouracil TP expression in the MSCs post-transfection of TP cDNA was confirmed by immunoblotting analysis and its activity was determined by spectrophotometric assay Our results showed the anticancer effectiveness of human HT29 colorectal cells co-cultured with TPexpressing MSCs was enhanced with the addition of various dosages of 5’-deoxy-5-fluorouridine 236 Microwave Ablation Assisted Liver Gene Transfection in Rats Xianghui He,1 Ruoyu Jiang,1 Lingkai Meng,1 Xiaoyu Liang,1 Jie Zhang,1 Zhixiang Zhang,1 Liwei Zhu.1 Department of General Surgery, Tianjin Medical University General Hospital, Tianjin, China BACKGROUNDS: Thermal ablation has been widely used to manage liver tumor This study was designed to determine histological change of rat liver after microwave ablation, investigate the effect of microwave ablation on gene transfection METHODS: In this study, microwave ablation was applied to rat liver, tissue demarcation was evaluated Green fluorescent protein (GFP) and Renilla luciferase-expressing plasmids were administrated to the liver by percutaneous or intravenous injection pre-ablation The expression of transgene in the reactive inflammatory zone was evaluated GFP expression was determined with the inverted fluorescence microscope in frozen section, and Renilla luciferase expression in target tissue was determined using Renilla luciferase plasmid kit The effect of hydrodynamic portal vein injection was also evaluated RESULTS: GFP expression was observed in the tissue of reactive inflammatory zone after microwave ablation and plasmid injection Green fluorescence in targeted area was visible on till the seventh day post ablation under inverted fluorescence microscope On the first day postablation and Renilla luciferase plasmid injection, luminescence value of the experimental group was higher than the control group, but lower than the portal hypertension injection group CONCLUSIONS: Direct injection of plasmid vector to inflammatory reaction zone after microwave ablation can achieve effective gene transfection, but the transfection efficiency is lower Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy ... cell invasion of breast cancer cells that have intact HIF- 1α In contrast, only HIF- 1α-expressing fibroblasts, not HIF- 1α-nonexpressing ones, stimulated HIF- 1α KO breast cancer cell invasion These... complicated and respective HIF- 1α contribution from breast cancer per se and surrounding fibroblasts to the tumor invasion and malignancy Moreover, by using a Tet -on inducible system, we found that ectopic. .. ectopic p16 gene transfer is capable of inhibiting hypoxia-induced cell migration and invasion of breast cancer cells, and suppressing cocultured fibroblast-stimulated invasiveness of breast cancer