Ines Block

Evaluation of somatostatin and nucleolin receptors for therapeutic delivery in non-small cell lung cancer stem cells applying the somatostatin-analog DOTATATE and the nucleolin-targeting aptamer AS1411

Cancer stem cells represent the putative tumor-driving subpopulation thought to account for drug resistance, relapse, and metastatic spread of epithelial and other cancer types. Accordingly, cell surface markers for therapeutic delivery to cancer stem cells are subject of intense research. Somatostatin receptor 2 and nucleolin are known to be overexpressed by various cancer types, which have elicited comprehensive efforts to explore their therapeutic utilization. Here, we evaluated somatostatin receptor 2 targeting and nucleolin targeting for therapeutic delivery to cancer stem cells from lung cancer. Nucleolin is expressed highly but not selectively, while somatostatin receptor 2 is expressed selectively but not highly by cancer cells. The non-small cell lung cancer cell lines A549 and H1299, displayed average levels of both surface molecules as judged based on analysis of a larger cell line panel. H1299 compared to A549 cells showed significantly elevated sphere-forming capacity, indicating higher cancer stem cell content, thus qualifying as suitable test system. Nucleolin-targeting 57Co-DOTA-AS1411 aptamer showed efficient internalization by cancer cells and, remarkably, at even higher efficiency by cancer stem cells. In contrast, somatostatin receptor 2 expression levels were not sufficiently high in H1299 cells to confer efficient uptake by either non-cancer stem cells or cancer stem cells. The data provides indication that the nucleolin-targeting AS1411 aptamer might be used for therapeutic delivery to non-small cell lung cancer stem cells.

Microarray R-based analysis of complex lysate experiments with MIRACLE

Motivation: Reverse-phase protein arrays (RPPAs) allow sensitive quantification of relative protein abundance in thousands of samples in parallel. Typical challenges involved in this technology are antibody selection, sample preparation and optimization of staining conditions. The issue of combining effective sample management and data analysis, however, has been widely neglected. Results: This motivated us to develop MIRACLE, a comprehensive and user-friendly web application bridging the gap between spotting and array analysis by conveniently keeping track of sample information. Data processing includes correction of staining bias, estimation of protein concentration from response curves, normalization for total protein amount per sample and statistical evaluation. Established analysis methods have been integrated with MIRACLE, offering experimental scientists an end-to-end solution for sample management and for carrying out data analysis. In addition, experienced users have the possibility to export data to R for more complex analyses. MIRACLE thus has the potential to further spread utilization of RPPAs as an emerging technology for high-throughput protein analysis. Availability: Project URL: http://www.nanocan.org/miracle/ Contact: mlist@health.sdu.dk Supplementary information: Supplementary data are available at Bioinformatics online.

Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes. This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment, a library of 108 isogenic melanoma cell lines was constructed and 8 genes were identified that significantly reduced viability in a discovery screen and in an independent validation screen. Here, we demonstrate the broad applicability of this recombination-based method and we proved its potential to identify new drug targets via the identification of the tumor suppressor DUSP6 as potential synthetic lethal target in melanoma cell lines with BRAF V600E mutations and high DUSP6 expression.

Association of miR-548c-5p, miR-7-5p, miR-210-3p, miR-128-3p with recurrence in systemically untreated breast cancer

Current prognostic markers allocate the majority of lymph node (LN) negative and estrogen receptor (ER) positive breast cancer patients into the high-risk group. Accordingly, most patients receive systemic treatments although approximately 40% of these patients may have been cured by surgery and radiotherapy alone. Two studies identified seven prognostic microRNAs in systemically untreated, LN negative and ER positive breast cancer patients which may allow more precise patient classification. However, six of the seven microRNAs were analyzed in both studies but only found to be prognostic in one study. To validate their prognostic potential, we analyzed microRNA expression in an independent cohort (n = 110) using a pair-matched study design minimizing dependence of classical markers. The expression of hsa-miR-548c-5p was significantly associated with abridged disease-free survival (hazard ratio [HR]:1.96, p = 0.027). Contradicting published results, high hsa-miR-516-3p expression was associated with favorable outcome (HR:0.29, p = 0.0068). The association is probably time-dependent indicating later relapse. Additionally, re-analysis of previously published expression data of two matching cohorts (n = 100, n = 255) supports an association of hsa-miR-128-3p with shortened disease-free survival (HR:2.48, p = 0.0033) and an upregulation of miR-7-5p (p = 0.0038; p = 0.039) and miR-210-3p (p = 0.031) in primary tumors of patients who experienced metastases. Further analysis may verify the prognostic potential of these microRNAs.

Protein-based nanotoxicology assessment strategy

The nanomaterial community calls for standardized in vitro assays to determine nanoparticle toxicity in the effort to reduce the number of in vivo validation experiments. We demonstrate that chip-based protein detection is suitable for assessing toxicity and may complement traditional assays to improve selection of primary hits for subsequent analysis. As nanodrug mimics, we analyzed the effect of transiently transfected siRNAs in MCF7 breast cancer cells and normal MCF12A breast cells, resembling a differential screen. As a measure of cytotoxicity, we determined cell viability as well as protein expression of glyceraldehyde-3-phosphate dehydrogenase, transferrin receptor, and the proliferation marker Ki67. The evaluation of cell lethality and protein expression unraveled cellular effects overseen by one method alone.

Corrigendum to “Protein-based nanotoxicology assessment strategy” [Nanomed Nanotechnol Biol Med. 2017;13(3):1229–1233]

O F Corrigendum to “Protein-based nanotoxicology assessment strategy” [Nanomed Nanotechnol Biol Med. 2017;13(3):1229–1233] Marlene Pedersen Elnegaard, MSc⁎, Markus List, PhD , Helle Christiansen, PhD, Steffen Schmidt, PhD, Jan Mollenhauer, PhD, Ines Block, PhD Lundbeckfonden Center of Excellence in Nanomedicine NanoCAN, University of Southern Denmark, Odense, Denmark Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark Institute of Clinical Research, University of Southern Denmark, Odense, Denmark O R P

Nanomedicine Approaches for Cancer Stem Cell Targeting and Personalized Cancer Treatment

Nanotechnology is on the route to providing novel breakthroughs in medicine. While there are already examples of the success of the new field of nanomedicine in cancer therapy, it remains open how nanomedicine can appropriately address future demands for advanced cancer therapy, such as personalization of treatment and elimination of cancer stem cells. This article introduces the particular challenges associated with these goals, which relate to the discussion about the definition of nanodrugs. Interdisciplinary sites of intersection, by which nanomedicine, i.e. research at the small nanoscale, can profit from the most recent developments in ‘omics’ technologies, i.e. research at the very large scale, are discussed. We propose strategies for the systematic identification and assembly of advanced nanodrugs, to which functional genomics and synthetic biology approaches could make important future contributions. Combining these disciplines can potentially lead to the design of advanced nanodrugs that may meet the challenges of the future.