Replication and Virus-Induced Transcriptome of HAdV-5 in Normal Host Cells versus Cancer Cells - Differences of Relevance for Adenoviral Oncolysis

Adenoviruses (Ads), especially HAdV-5, have been genetically equipped with tumor-restricted replication potential to enable applications in oncolytic cancer therapy. Such oncolytic adenoviruses have been well tolerated in cancer patients, but their anti-tumor efficacy needs to be enhanced. In this regard, it should be considered that cancer cells, dependent on their tissue of origin, can differ substantially from the normal host cells to which Ads are adapted by complex virus-host interactions. Consequently, viral replication efficiency, a key determinant of oncolytic activity, might be suboptimal in cancer cells. Therefore, we have analyzed both the replication kinetics of HAdV-5 and the virus-induced transcriptome in human bronchial epithelial cells (HBEC) in comparison to cancer cells. This is the first report on genome-wide expression profiling of Ads in their native host cells. We found that E1A expression and onset of viral genome replication are most rapid in HBEC and considerably delayed in melanoma cells. In squamous cell lung carcinoma cells, we observed intermediate HAdV-5 replication kinetics. Infectious particle production, viral spread and lytic activity of HAdV-5 were attenuated in melanoma cells versus HBEC. Expression profiling at the onset of viral genome replication revealed that HAdV-5 induced the strongest changes in the cellular transcriptome in HBEC, followed by lung cancer and melanoma cells. We identified prominent regulation of genes involved in cell cycle and DNA metabolism, replication and packaging in HBEC, which is in accord with the necessity to induce S phase for viral replication. Strikingly, in melanoma cells HAdV-5 triggered opposing regulation of said genes and, in contrast to lung cancer cells, no weak S phase induction was detected when using the E2F promoter as reporter. Our results provide a rationale for improving oncolytic adenoviruses either by adaptation of viral infection to target tumor cells or by modulating tumor cell functions to better support viral replication

Biochemical characterization of the Helicobacter pylori bactofilin-homolog HP1542.

The human pathogen Helicobacter pylori is known for its colonization of the upper digestive system, where it escapes the harsh acidic environment by hiding in the mucus layer. One factor promoting this colonization is the helical cell shape of H. pylori. Among shape determining proteins are cytoskeletal elements like the recently discovered bactofilins. Bactofilins constitute a widespread family of polymer-forming bacterial proteins whose biology is still poorly investigated. Here we describe the first biochemical analysis of the bactofilin HP1542 of H. pylori reference strain 26695. Purified HP1542 forms sheet-like 2D crystalline assemblies, which clearly depend on a natively structured C-terminus. Polymerization properties and protein stability were investigated. Additionally, we also could demarcate HP1542 from amyloid proteins that share similarities with the bactofilin DUF domain. By using zonal centrifugation of total H. pylori cell lysates and immunfluorescence analysis we revealed peripheral membrane association of HP1542 mostly pronounced near mid-cell. Interestingly our results indicate that H. pylori bactofilin does not contribute to cell wall stability. This study might act as a starting point for biophysical studies of the H. pylori bactofilin biology as well as for the investigation of bactofilin cell physiology in this organism. Importantly, this study is the first biochemical analysis of a bactofilin in a human pathogen

Performance of Streck cfDNA Blood Collection Tubes for Liquid Biopsy Testing

<div><p>Objectives</p><p>Making liquid biopsy testing widely available requires a concept to ship whole blood at ambient temperatures while retaining the integrity of the cell-free DNA (cfDNA) population and stability of blood cells to prevent dilution of circulating tumor DNA (ctDNA) with wild-type genomic DNA. The cell- and DNA-stabilizing properties of Streck Cell-Free DNA BCT blood collection tubes (cfDNA BCTs) were evaluated to determine if they can be utilized in combination with highly sensitive mutation detection technologies.</p><p>Methods</p><p>Venous blood from healthy donors or patients with advanced colorectal cancer (CRC) was collected in cfDNA BCTs and standard K₂EDTA tubes. Tubes were stored at different temperatures for various times before plasma preparation and DNA extraction. The isolated cfDNA was analyzed for overall DNA yield of short and long DNA fragments using qPCR as well as for mutational changes using BEAMing and Plasma Safe-Sequencing (Safe-SeqS).</p><p>Results</p><p>Collection of whole blood from healthy individuals in cfDNA BCTs and storage for up to 5 days at room temperature did not affect the DNA yield and mutation background levels (n = 60). Low-frequency mutant DNA spiked into normal blood samples as well as mutant circulating tumor DNA in blood samples from CRC patients collected in cfDNA BCTs were reliably detected after 3 days of storage at room temperature. However, blood samples stored at ≤ 10°C and at 40°C for an extended period of time showed elevated normal genomic DNA levels and an abnormally large cellular plasma interface as well as lower plasma volumes.</p><p>Conclusion</p><p>Whole blood shipped in cfDNA BCTs over several days can be used for downstream liquid biopsy testing using BEAMing and Safe-SeqS. Since the shipping temperature is a critical factor, special care has to be taken to maintain a defined room temperature range to obtain reliable mutation testing results.</p></div

Experimental study cohorts.

<p>Experimental setup for cfDNA BCT vs K₂EDTA performance experiments. Cohort I: Time point experiments at room temperature including DNA quantification and mutation analysis using BEAMing and Safe-SeqS. Cohort II: BEAMing analysis of blood samples spiked with synthetic double-stranded mutant DNA fragments at different allele frequencies. Cohort III: BEAMing analysis of samples collected from colorectal cancer (CRC) patients. Cohort IV: Experiment evaluating effects of extreme storage temperatures on DNA quantity. Cohort V: Experimental evaluation of recommended temperature range.</p

Effect of extreme storage temperatures on plasma separation and DNA yield in study cohort IV.

<p>(A) Representative image of cfDNA BCTs centrifuged after 3 days of storage at RT, 4°C and 40°C. RT storage resulted in expected plasma separation with defined buffy coat layer and clear yellow plasma fraction. Extreme temperature conditions resulted in an expanded cellular interface layer or hemolytic plasma at 4°C or 40°C, respectively. (B) Effect of extreme temperatures on genomic DNA release (402 bp LINE-1 qPCR fragment). Statistically significant differences between K₂EDTA and cfDNA BCT storage conditions determined by one-way ANOVA are marked with ** (p ≤ 0.01). Shown are box plots with 1.5 x IQR applied to create whiskers. (C) Obtained mean plasma volume with SD for indicated storage conditions.</p

Genomic DNA release and obtained plasma volume after 3 days of storage within temperature range recommended by the manufacturer (study cohort V).

<p>Blood collected in cfDNA BCTs (n = 8) was stored for 3 days at the indicated temperature and subsequently analyzed using the genomic DNA release assay based on the 402:96 bp LINE-1 ratio (A). Shown are box plots with 1.5 x IQR applied to create whiskers. Statistically significant differences from the reference condition (20°C) were determined by one-way ANOVA and are marked with an * (p ≤ 0.05). (B) Obtained mean plasma volume with SD for indicated storage conditions.</p

Analysis of cfDNA yield and genomic DNA release for study cohort I.

<p>(A) DNA yield was assessed for cfDNA from blood samples stored at room temperature (18°C– 22°C) in K₂EDTA tubes vs cfDNA BCTs (healthy donors, n = 60). Plasma was prepared after indicated storage conditions. Extracted DNA was analyzed for overall yield by qPCR amplifying a 96 bp LINE-1 fragment. (B) Illustration of the DNA yield ratio between long (402 bp) and short (96 bp) LINE-1 fragments (n = 60). Increased ratios compared to K₂EDTA reference would indicate genomic DNA release. Shown are box plots with 1.5 × interquartile range (IQR) applied to create whiskers and outliers. Statistical analysis using one-way ANOVA revealed no significant difference between conditions.</p

Detectability of low frequency mutations in study cohort II.

<p>Blood from healthy donors (n = 8) was spiked with synthetic double-stranded mutant DNA fragments of different allele frequencies in a background of 60,000 genome equivalents of fragmented human genomic wild-type DNA and subsequently stored at RT. DNA was extracted and analyzed by BEAMing after the indicated storage time. Mean and standard deviation of the detected mutant fraction is shown. (A) <i>PIK3CA</i> c.3140A>G spike (0.1%), (B) <i>EGFR</i> c.2369C>T spike (0.5%), (C) <i>KRAS</i> c.34G>A spike (1%).</p

<i>In Vivo</i> Activity and Pharmacokinetics of Nemorosone on Pancreatic Cancer Xenografts

<div><p>Pancreatic cancer is one of the leading cancer-related causes of death in the western world with an urgent need for new treatment strategies. Recently, hyperforin and nemorosone have been described as promising anti-cancer lead compounds. While hyperforin has been thoroughly investigated <i>in vitro</i> and <i>in vivo</i>, <i>in vivo</i> data for nemorosone are still missing. Thus, we investigated the growth-inhibitory potential of nemorosone on pancreatic cancer xenografts in NMRI nu/nu mice and determined basic pharmacokinetic parameters. Xenograft tumors were treated with nemorosone and gemcitabine, the current standard of care. Tumor sections were subjected to H&E as well as caspase 3 and Ki-67 staining. Nemorosone plasma kinetics were determined by HPLC and mass spectrometry. Induction of CYP3A4 and other metabolizing enzymes by nemorosone and hyperforin was tested on primary hepatocytes using qRT-PCR. At a dose of 50 mg/kg nemorosone per day, a significant growth-inhibitory effect was observed in pancreatic cancer xenografts. The compound was well tolerated and rapidly absorbed into the bloodstream with a half-life of approximately 30 min. Different metabolites were detected, possibly resembling CYP3A4-independent oxidation products. It is concluded that nemorosone is a potential anti-cancer lead compound with good bioavailability, little side-effects and promising growth-inhibitory effects, thus representing a valuable compound for a combination therapy approach.</p> </div