Project description:The Prostate Cancer Medically Optimized Genome-Enhanced Therapy (PROMOTE) study uses genetic clues in castration-resistant prostate cancer that may identify an individualized treatment approach for men with the disease. Understanding the molecular biology behind castration-resistant prostate cancer has led to more treatment options, but there are still no definite conclusions about which specific drug best treats patients - maximum suppression of cancer growth while minimizing side effects. The PROMOTE study explores the genetic characteristics of each tumor to predict these treatment paradigms for the future, resulting in more effective and less toxic options for patients. Our long-term goal is to improve treatments for men with advanced prostate cancer by using genomic sequencing to increase life span and quality of life. We also will uncover novel vulnerable targets in the cancer genome that may provide new drug therapies. PARTICIPATION. Eligible... (for more see dbGaP study page.)
Project description:Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-induced pluripotent stem cells (iPSCs), day4 mesodermal cells, and day8, day20, and day30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day20 CMs was significantly higher compared to other cells. Transplantation of day20 CMs into the infarcted hearts of immunodeficient mice showed significant functional improvement. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. Differentiated cells, N=10 Undifferentiated pluripotent stem cells, N=1 Heart samples, N=6
Project description:Drug resistance invariably limits the clinical efficacy of targeted therapy with kinase inhibitors against cancer. We found that targeted therapy with BRAF, ALK, or EGFR inhibitors induces a complex network of secreted signals in drug-stressed melanoma and lung adenocarcinoma cells. This therapy-induced secretome (TIS) stimulates the outgrowth, infiltration and metastasis of drug-resistant cancer clones in the tumour. Additionally, the TIS supports the survival of drug-sensitive cells, contributing to incomplete tumour regression. We used transcriptomic analysis of sensitive tumour cells and xenograft tumours treated with vehicle, vemurafenib, or crizotinib to identify the transcriptional drivers and to dissect the TIS in melanoma (A375, Colo800, UACC62) and lung adenocarcinoma (H3122). In addition, we utilize cell type–specific mRNA purification by translating ribosome affinity purification (TRAP) to identify pathways that are up-regulated in resistant cells (A375R) in response to the regressing tumour microenvironment. Overall design: Analysis of the response of drug sensitive melanoma and lung adenocarcinoma cells to pharmacological inhibition of their driver oncogene and gene expression analysis of drug resistant cancer cells responding to different tumor microenvironments.
Project description:BACKGROUND: Array Comparative Genomic Hybridization (aCGH) is a rapidly evolving technology that still lacks complete standardization. Yet, it is of great importance to obtain robust and reproducible data to enable meaningful multiple hybridization comparisons. Special difficulties arise when aCGH is performed on archival formalin-fixed, paraffin-embedded (FFPE) tissue due to its variable DNA quality. Recently, we have developed an effective DNA quality test that predicts suitability of archival samples for BAC aCGH. METHODS: In this report, we first used DNA from a cancer cell-line (SKBR3) to optimize the aCGH protocol for automated hybridization, and subsequently optimized and validated the procedure for FFPE breast cancer samples. We aimed for highest throughput, accuracy, and reproducibility applicable to FFPE samples, which can also be important in future diagnostic use. RESULTS: Our protocol of automated array-CGH on archival FFPE ULS-labeled DNA showed very similar results compared with published data and our previous manual hybridization method. CONCLUSION: This report combines automated aCGH on unamplified archival FFPE DNA using non-enzymatic ULS labeling, and describes an optimized protocol for this combination resulting in improved quality and reproducibility. In this study, we optimized the BAC araay-CGH protocol for automated hybridization for FFPE breast cancer samples. We have tested hybridization temperature and duration, different hybridization buffer conditions, and post-hybridization washing.
Project description:Men with clinically localized prostate cancer were treated with 0 to 9 months of neoadjuvant hormone suppression prior to prostatectomy. Keywords: microarray, hormone, 9 men with prostate cancer were assigned to neoadjuvant hormone suppression therapy for 3-6, 6, or 0 months.
Project description:Male patients (n=6, mean age 62 years) with NYHA III-IV and an left ventricular ejection fraction of <35% despite pharmacological therapy received 35 hours of enhanced external counterpulsation (EECP) over a period of 7 weeks. Before and after treatment, lateral vastus muscle biopsies were obtained and skeletal muscle gene expression was evaluated using the Affymetrix HuGene 1.0 platform. Skeletal muscle gene expression before and after treatment with enhanced external counterpulsation for 7 weeks in 6 male patients with severe heart failure
Project description:Two shRNAs were placed into expression vectors harboring mir30 microRNA scaffold and an optimized scaffold where the artificial restriction sights in mir30 have been removed. After infection and selection shRNA processing was assessed by small-RNA cloning. For both shRNAs, placement into the optimized scaffold resulted in a ~two-fold increase in processing (based on smallRNA levels). Purpose: Others have reported that the EcoRI site that was introduced to the mir30 scaffold results in decreased smallRNA processing and hence reduced target knockdown. We've developed an alternative scaffold (termed ultramir) where this site is removed. smallRNA cloning was used to determine if the movement of this sight resulted in an increase in shRNA processing. Method: Two shRNAs (one targeting Renilla Luciferase and one targeting Human RPA3) were cloned into the original mir30 cassette the ultramir cassette. Each of the 4 constructs were infected in duplicate at single copy into cells and the cells seltected unitil infection percentages reached >90% (the shRenilla hairpin was infected into HEK293T cells and the shRPA3 construts into the Gallus gallus cell line ERC. After selection smallRNA cloning was perfromed and the amount of smallRNAs corrresponding to the two shRNAs compared to the endogenous microRNA populatlon. Results: smallRNA levels of the two shRNAs doubled relative to the microRNA population when they were placed into the ultramir scaffold.
Project description:Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs, variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2–3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved. Deep sequencing libraries from either a randomized RNA oligo or an equimolar miRNA mix were analyzed for evenness of capture.