tbuckley/cabot-search huggingface dataset:
170. TransfeX and TransIT-LT1 Mediated Gene Delivery to Cervical and Oral Squamous Cell Carcinoma Cells
Authors: Jennifer Cheung, Krystyna Konopka, Takahiro Chino, Nejat Düzgünes
Journal: Molecular Therapy (2015)

Cancer gene therapy involves the introduction of therapeutic genes into malignant cells. The use of lipid-based nonviral vectors is a promising approach to gene delivery since it avoids potential immune effects presented with the use of viral vectors.. However, the transfection efficiency of non-viral vectors needs to be improved to achieve adequate transgene expression in most cancer cells. In particular, oral squamous cell carcinoma cells are highly resistant to transfection by non-viral vectors. We investigated the ability of two novel reagents to deliver the gene for firefly luciferase into cervical and oral squamous cell carcinoma cells. HeLa cervical carcinoma, and HSC-3 and H357 human oral squamous cell carcinoma cells were seeded in 48-well culture plates one day prior to transfection, and used at ~85% confluence. The cells were transfected with either TransfeX (ATCC, Manassas, VA) or TransIT-LT1 (Mirus, Madison, WI). Each transfection reagent was used at ratios of 1, 2, 4, or 8 μl per 1 μg plasmid DNA (pCMV.Luc). Toxicities of the reagents were measured with the Alamar Blue cell viability assay. Transfection activity was evaluated by measuring luciferase expression 48 h after transfection, using the Luciferase Assay System (Promega) and a Turner Designs TD-20/20 luminometer. Data were expressed as relative light units (RLU) per ml of cell lysate. In HeLa cells, luciferase expression with 4 μl TransfeX/1 μg DNA reached 50,620,00±13,195,000 RLU/ml, the highest activity ever seen in our laboratory. TransIT-LT1 at 4 μl/1 μg DNA achieved 10,221,000±1,616,000 RLU/ml. In HSC-3 cells, luciferase expression obtained with TransfeX was 1,035,000±37,000 RLU/ml, and 35,000±6,000 RLU/ml with TransIT-LT1. In H357 cells that are highly resistant to transfection with previously available reagents, TransfeX showed maximum transfection efficiency at 2 μl TransfeX/1 μg DNA, with 2,514,000±250,000 RLU/ml. While TransIT-LT1 is not quite as effective, TransfeX facilitates unusually high levels of transgene expression at levels comparable to that achieved by adenoviral transduction (our unpublished data). Transfection of HeLa cells with TransfeX exhibited the highest transfection activity obtained in our laboratory, and was almost 5 times higher than that achieved with TransIT-LT1. In HSC-3 cells, transfection efficiency of TransfeX was 29 times higher than that of TransIT-LT1. Very high levels of luciferase activity were obtained in H357 cells with TransfeX. TransfeX is likely to be beneficial in various cancer gene therapy approaches and other gene therapies involving non-viral vectors.
467. Assay Conditions Significantly Alter AAV Neutralizing Antibody Determination
Authors: Heikki T. Turunen, Eric Zinn, Luk H. Vandenberghe
Journal: Molecular Therapy (2016)

A major limitation of vectors based on current AAVs is the wide prevalence of memory B and T cells reactive to the AAV capsid in human populations arisen following natural AAV infection. The Neutralizing Antibody (NAB) assay guides experimentation and determines inclusion or exclusion of subjects in clinical studies in order to predict in vivo neutralization. Furthermore, evidence of a memory B-cell response to AAV may correlate with anti-capsid memory T-cell populations implicated in the safety and efficacy outcome of clinical AAV liver studies. Based on previously reported findings, NAB assay protocols in current use are unable to robustly predict in vivo neutralization in monkeys (Zinn et al. Cell Reports, 2015 & Wang et al. Mol Ther, 2010) leading to false negatives. In a clinical setting, the enrollment of a seropositive individual exposes the subject to undue risk, and generates outcome variability of the often small clinical studies. Here, we sought to permutate several of the NAB assay conditions in order to evaluate their impact on the NAB titer readout. Specifically, we varied the following variables: cell line, transgene detection, use of adenovirus, and AAV amount. Prior to evaluating these assay permutation, we sought to increase the sensitivity of the readout for transgene expression in the assay in order to evaluate at all conditions both high and low transducing AAV serotypes. This optimization increased our sensitivity by ~200-fold. With this increased sensitivity of readout, we first sought to evaluate the compliance of standard AAVNAB assays with the percentage law, formulated by Andrewes and Elford in 1933, a standard for virological NAB assays, which seeks stoichiometric conditions of NAB and viral load that enables measurement of NAB titers independent of viral input in the assay. Our data shows that at the conditions in common use in the field the percentage law is not upheld, and NAB titers are highly dependent on input viral load of the assay, directly impacting sensitivity and specificity, and leading to the occurrence of false negatives. Indeed, quantitative assessment of seroprevalence in primate populations with assays that reduce the amount of AAV to within percentage law range affects assay outcome qualitatively and quantitatively. We also independently assessed other assay parameters to improve sensitivity, robustness and reproducibility, and to study whether they may also have qualitative effects on titer outcomes. Our studies highlight the importance of pre-existing immunity in AAV gene therapy and limitations of the current methodologies to measure it in a robust and predictive manner. Data indicates the potential for false positive and negative readout for commonly used assay protocols. An optimized protocol was developed for further validation and evaluation. These studies may impact translational and clinical AAV gene therapy studies.
LUME-Colon 1: a randomized phase III study of nintedanib vs placebo in patients with advanced colorectal cancer
Authors: Takayuki Yoshino, Josep Tabernero, Zohra Oum'Hamed, Matus Studeny, Mouna Sassi, Heinz-Josef Lenz, Eric Van Cutsem
Journal: Annals of Oncology (2015)
108. Versican G1 Domain Stimulates Adenoviral Transgene Expression in a JAK/STAT Dependent Manner
Authors: Patricia Akinfenwa, Wesley S. Bond, Cristian J. Ildefonso, Mary Y. Hurwitz, Richard L. Hurwitz
Journal: Molecular Therapy (2015)

Gene therapy using either adeno-associated (AAV) or adenoviral (AdV) vectors has been shown to be effective in the ocular environment. Understanding the mechanisms of this enhancement could benefit gene therapy protocols in general. Vitreous, the gelatinous material filling the posterior eye, enhances the expression of transgenes delivered by AdV and AAV. Vitreous has no effect on vector internalization but is associated with increased transgene mRNA levels and mediates enhancement in hyaluronan-dependent and independent-manners (Chaudhuri et al. Mol Therapy 2007). We hypothesize that versican, a hyaluronan-binding proteoglycan expressed in vitreous, has a role in both mechanisms. Furthermore, we define an intracellular pathway through which the increased gene expression occurs. Versican-containing supernatant (VCS) was produced by culturing versican-secreting ACHN cells or HepG2 cells transfected to transiently express the recombinant versican G1 domain either lacking (G1-) or containing (G1+) the hyaluronan-binding region. Y79 or Weri retinoblastoma cells, or SKNDZ neuroblastoma cells that lack a cellular hyaluronan binding receptor were transduced in the presence of bovine vitreous, VCS, or either G1 domain with AdV vectors delivering a luciferase reporter transgene. Incubation of target cells with vitreous enhanced AdV or AAV gene expression greater than 4-fold (p<0.001) when Y79 cells, Weri cells, or SKNDZ cells were used as targets. Boiling vitreous prevented this enhancement. Thus, heat-stable hyaluronan alone was not responsible. Incubation with dasatanib (a Src kinase inhibitor), VCS, G1+, or G1- mimicked the effects of vitreous. Small-molecule inhibition of JAK1/2 or STAT3/5 using ruxolitinib or C188-9 mitigated the enhancement of transgene expression mediated by vitreous, dasatanib, VCS or either G1 domain, while inhibition of the mTOR pathway using rapamycin or everolimus showed no effect on enhancement. Vitreous or VCS treatment enhanced STAT3 phosphorylation in comparison to untreated cells. These results support a model in which Src kinase activity inhibits AdV or AAV viral vector mediated transgene expression. This inhibition can be overcome by hyaluronan-independent and dependent mechanisms mediated by the G1 domain of versican through activation of the JAK/STAT signaling pathway. Therefore, the versican G1 domain could be used as an adjuvant to enhance gene therapy protocols.
Efficacy and Tolerability of Liposomial Cytosine Arabinoside (DepoCyte®) for the Treatment of Lymphomatous and Leukemic Meningitis.
Authors: Davide Rossi, Luciana Annino, Velia Bongarzoni, Maide Cavalli, Francesco Di Raimondo, Gianluca Gaidano, Alfredo Gagliardi, Giulio Giordano, Giovanni Martinelli, Eustachio Miraglia, Sergio Storti, Andrea Camera
Journal: Blood (2006)
Plant viral nanoparticles-based HER2 vaccine: Immune response influenced by differential transport, localization and cellular interactions of particulate carriers
Authors: Sourabh Shukla, Jay T. Myers, Sarah E. Woods, Xingjian Gong, Anna E. Czapar, Ulrich Commandeur, Alex Y. Huang, Alan D. Levine, Nicole F. Steinmetz
Journal: Biomaterials (2016)
Abstract 2112: A novel mechanism of NF-κB regulation in bladder cancer
Authors: Neelam Mukherjee, Rita Ghosh
Journal: Cancer Research (2015)

Abstract Protein methyltransferases (PMTs) methylate protein lysine and arginine residues and play an important role in regulating chromatin remodeling and gene transcription. Deregulation of SET-domain-containing PMTs has a crucial role in carcinogenesis (1). SETD6 is a novel methyltransferase that has been shown to monomethylate p65/RelA (a principal subunit of NF-κB) at Lys310. Monomethylated p65 recruits GLP (G9A-like protein), a histone dimethyl transferase to the NF-κB target genes, which results in transcriptional repression (2). The role of SETD6, in carcinogenesis in general and bladder cancer in particular is unknown. We identified SETD6 as a significantly upregulated gene in a screen of chromatin modifying genes that are differentially regulated in bladder cancer cells compared with normal urothelial cells. Subsequently qPCR and immunoblotting showed upregulation of SETD6 at the mRNA and protein level in bladder cancer cells and tissues relative to their normal counterparts. Surprisingly, we found that overexpression of SETD6 increased nuclear level of NF-κB, phosphorylation of NF-κB at Ser536 and the transcriptional activity of NF-κB. Overexpression of wild type SETD6 increased viability and proliferation of non-tumorigenic urothelial cells while overexpression of mutant SETD6 had no effect. Furthermore, knocking down SETD6 transiently with si-SETD6 significantly decreased viability and proliferation of bladder cancer cells suggesting that SETD6 may have a growth promoting role in bladder cancer. Our data shows for the first time that non-histone protein lysine methylation of NF-κB-p65 by SETD6 may be a novel way to regulate NF-κB pathway in bladder cancer. The lack of curative strategies for bladder cancer leads to recurrence rates of 45-70% with &amp;gt;15% of patients developing muscle invasive disease (3). These observations underscore the need to understand survival mechanism in bladder cancer. In this regard our findings are highly significant and could lead to targeting SETD6/ NF-κB axis in bladder cancer in the future. References: 1. Schneider, R., et al. (2002) Unsafe SETs: histone lysine methyltransferases and cancer. Trends in biochemical sciences, 27, 396-402. 2. Levy, D., et al. (2011) Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling. Nature immunology, 12, 29-36. 3. Nativ, O., et al. (2009) Combined thermo-chemotherapy for recurrent bladder cancer after bacillus Calmette-Guerin. The Journal of urology, 182, 1313-1317. Citation Format: Neelam Mukherjee, Rita Ghosh. A novel mechanism of NF-κB regulation in bladder cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2015-2112
Abstract 4128: Extensive morphological heterogeneity of circulating and selected high metastatic variants and differential chemosensitivity of human prostate cancer in mouse models
Authors: Lei Zhang, Chengyu Wu, Robert. M Hoffman
Journal: Cancer Research (2015)

Abstract Circulating PC-3-GFP human prostate cancer cells were collected from the blood from nude mice orthotopically implanted with PC-3-GFP. In parallel experiments, a highly metastatic PC-3-GFP cell line was developed by injecting parental PC-3-GFP to the footpad of nude mice which metastasized to inguinal lymph nodes from which the cancer cells were collected and were re-injected to the footpad. After 6 cycles, the PC-3-GFP cells collected from inguinal lymph nodes (PC-3-GFP-LN) and were injected to the footpad. PC-3-GFP-LN showed 100% metastasis to major lymph nodes (popliteal, inguinal, axillary, and cervical), and 100% metastasis to bone and lung. PC-3-GFP-CTC and PC-3-GFP-LN were cultured in vitro, and their morphology and chemo-sensitivity were comparing to the parental PC-3-GFP cells. Highly pleomorphic subpopulations of prostate cancer cells were identified in PC-3-GFP-CTC and PC-3-GFP-LN. The different cell subtypes included characteristic high malignancy features such as high nucleus to cytoplasmic (N/C) ratios and giant cell formation as a result of abnormal mitoses. Variations in growth rate, plating efficiency, size, shape, and number of the nuclei and nucleoli were observed. The results suggest extensive tumor heterogeneity in both sublines. The giant cells, contain multiple nuclei, cannot survive by themselves and need small cells around them in order to proliferate. However, giant cells also can produce smaller cells, which divide further. The ratio of giant cells/total cells were significantly higher in PC-3-GFP-LN compared to parental cells. However, the morphology of cells from lung metastasis and bone metastasis showed lung and bone epithelial features such as squamous on shape and tightly packed together. PC-3-GFP-CTC had similar sensitivity to both cisplatinum and docetaxel when compared to PC-3-parental cells. PC-3-GFP-LN was resistant. In contrast, PC-3-GFP-LN was sensitive to the traditional Chinese medicine (TCM) herbal mixture LQ similar to the parental cells. These results demonstrate that circulating tumor cells (CTCs) and selected high metastatic variants can be extensively heterogeneous and have different chemosensitivity profiles, suggesting new approaches to treatment such as with TCM. Citation Format: Lei Zhang, Chengyu Wu, Robert. M Hoffman. Extensive morphological heterogeneity of circulating and selected high metastatic variants and differential chemosensitivity of human prostate cancer in mouse models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4128. doi:10.1158/1538-7445.AM2015-4128
Abstract 4216: Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models
Authors: J. Milburn Jessup, Abid R. Mattoo, Nikolay Korokhov
Journal: Cancer Research (2015)

Abstract Purpose: Since the NANOG family controls stemness and neoplastic progression in human colorectal carcinoma (CRC), our postulate is that targeting the dominant NANOG and NANOGP8 members with viral vector delivered shRNA will inhibit CRC progression in preclinical models and that replicating viral vectors are more active than non-replicating vectors. Methods: Cells were human CRC lines (Clone A, CX-1 and LS174T) and mouse fibroblasts (3T3). shRNA specific to NANOG (shNG-1), NANOGP8 (shNp8-1) or a control (shNEG) were created in lentivirus (LV) and chimeric adenovirus 5/3 (Ad5/3). CRC and 3T3 were cultured in monolayer with serum-containing medium or in suspension in ultra low attachment plates without serum. Nanostring 770 gene cancer panel was analyzed in 72 hr cultures with NMF clustering and GSEA analysis. Annexin V expression and caspase-3, 8 or 9 activation in suspension culture assessed Apoptosis. LV or Ad5/3 shRNAs were added to spheroids in 3-D suspension cultures to test therapy in vitro. Intratumoral injection of LV or Ad5/3 shRNA into 5-10 mm sub cutis nodules in groups of 5-10 NOD/SCID mice measured activity in vivo. Results: LV shNp8-1 in both Clone A and CX-1 caused significant inhibition of TGF-beta, EGFR, NRF2, E2F3 and the Cyclin D1 pathways by NMF and GSEA analysis in monolayer culture. LV shNp8-1 inhibited growth of LS174T, Clone A and CX-1 by 50% in 3 days in 3-D suspension cultures without further decrease. Apoptosis was due to Caspase 9 activation. In contrast, Ad5/3-shNp8-1 decreased spheroid growth by up to 94% at 9 -14 days while Ad5/3 decreases spheroid growth by 80%. Ad5/3-shNp8-1 but nit Ad5/3 was active in co-cultures with 3T3 to a 2:1 CRC:3T3 ratio. Transduction level was ∼50% in LV treated cells but ∼90% in Ad5/3 treated cells. Intratumoral injection of LVshNG-1 or LVshNp8-1, decreased tumor size by 50% compared to LVshNEG or untreated CX-1 tumors for 11 days after which tumor growth rebounded to match controls. Ad5/3-shNp8-1 injection into 5-9 mm nodules inhibited CX-1 and LS14T growth by ∼50% until euthanized. Conclusions: Targeting NANOGP8 more than NANOG inhibits proliferation and activates apoptosis through multiple pathways. Nonreplicating LV delivered shRNA inhibited tumor growth in in vitro and in vivo preclinical models but replicating oncolytic Ad5/3 infects more cells and promotes shRNA efficacy. Replicating vectors targeting NANOG are important for further development. Citation Format: J. Milburn Jessup, Abid R. Mattoo, Nikolay Korokhov. Targeting NANOG: genes, proteins and response to viral RNAi in preclinical models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2015-4216
325. Persistence of hAQP1 Expression in Human Salivary Gland Cells Following AdhAQP1 Transduction Is Associated With a Lack of Methylation of hCMV Promoter
Authors: Changyu Zheng, Bruce J. Baum, Xibao Liu, Corinne M. Goldsmith, Paola Perez, Shyh-Ing Jang, Ana P. Cotrim, Linda McCullagh, Indu S. Ambudkar, Ilias Alevizos
Journal: Molecular Therapy (2015)

In 2012, we reported that five out of 11 subjects in a clinical trial (NCT00372320) of AdhAQP1 to radiation-damaged parotid glands showed increased saliva flow rates and decreased symptoms over the initial 42 days. AdhAQP1 is a first generation, E1-deleted, replication-defective, serotype 5 adenoviral vector encoding human aquaporin-1 (hAQP1). This vector uses the human cytomegalovirus enhancer/promoter (hCMVp). Since subject responses were longer than expected, we hypothesized that the hCMVp may not be methylated in human salivary gland cells to the extent previously observed in rodent salivary gland cells. To test our hypothesis, initially we transduced mouse and rat cell lines in vitro, or submandibular glands in vivo, with the same AdhAQP1 used in the clinical trial. The hCMVp was gradually methylated over time in rodent cells and associated with a decreased functional hAQP1 expression. The hCMVp, however, was not methylated in human salivary gland primary cultures or human salivary gland cell lines after transduction with AdhAQP1. Importantly, hAQP1 maintained its function in those cells. These data suggest that the hCMVp in AdhAQP1is likely not methylated, resulting in an unexpectedly longer functional expression of hAQP1 in transduced human salivary gland cells.
Azacitidine Treatment for Lenalidomide (LEN)-Resistant Myelodysplastic Syndrome (MDS) with Del 5q
Authors: Rami S. Komrokji, Cecile Bally, Raphael Itzykson, Sylvain Thepot, Eric Padron, Najla H Al Ali, Jeffrey E Lancet, Alan F. List, Pierre Fenaux, Lionel Ades
Journal: Blood (2012)
Identification of Molecular Predictors of Response to ZARNESTRA™ (Tipifarnib, R115777) in Relapsed and Refractory Acute Myeloid Leukemia.
Authors: Mitch Raponi, Bob Lowenberg, Jeffrey E. Lancet, Jean-Luc Harousseau, Richard Stone, Wayne Rackoff, Alain Thibault, Yi Zhang, David Atkins, Yixin Wang
Journal: Blood (2004)
Abstract 3223: A true orthotopic ovarian cancer patient-derived xenograft (PDX) model
Authors: Hongmei Cui, Yingyan He, Clemens Krepler, Janos Tanyi, Mark A. Morgan, Robert A. Burger, Sarah Kim, Emily Ko, Tan Ince, Meenhard Herlyn, Fiona Simpkins
Journal: Cancer Research (2015)

Abstract Introduction: Patient derived xenografts (PDXs) show promise as models to study cancer as they recapitulate the principle characteristics of the patients’ original tumor such as histology, mutational status, DNA copy number, gene expression patterns, and clinical behavior while remaining biologically stable through passaged in mice. We used an orthotopic model transplanting tumor directly to the fallopian tube/ovary in order to accurately study the native environment and the progression of the ovarian cancer (OVCA). Experimental Design: Fresh OVCA tumor was transplanted orthotopically to the fallopian tube/ovary of NSG 5-8 week old mice. Tumor growth was followed over time. Tumors were evaluated by IHC. Alu II probe staining was performed to evaluate human stroma content. RPPA and targeted DNA sequencing are in-process. Drug studies are in progress. Primary tumor lines are being generated. Results: To date, we have transplanted tumor from 18 primary, 2 interval, and 5 recurrent ovarian debulking surgeries using an orthotopic ovarian tumor transplant approach with an 85% success rate in generating tumors in mouse passage 1 (MP1). The median time for tumors to reach ∼2 cm is 3 months when tumors are then passaged again (MP2/MP3). We have generated 8 BRCA 1/2 deleterious mutation positive carrier PDXs. Alu staining of PDXs demonstrates human cells in the stroma. We have generated 8 primary tumor cell lines from original tumors/PDXs. Conclusions: We conclude that although technically more challenging, the orthotopic transplantation technique is feasible in generating ovarian cancer PDXs that most closely resemble the natural environment for ovarian cancer progression. Citation Format: Hongmei Cui, Yingyan He, Clemens Krepler, Janos Tanyi, Mark A. Morgan, Robert A. Burger, Sarah Kim, Emily Ko, Tan Ince, Meenhard Herlyn, Fiona Simpkins. A true orthotopic ovarian cancer patient-derived xenograft (PDX) model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3223. doi:10.1158/1538-7445.AM2015-3223