A new live-cell biobank workflow efficiently recovers heterogeneous melanoma cells from native biopsies.

Fibroblast contamination can make establishing primary melanoma cell cultures from native biopsies a major challenge, due to fibroblasts overgrowing the melanoma cells. Standard protocols therefore enrich for highly proliferative melanoma cells that grow well in vitro but may not represent the full range of in vivo tumor heterogeneity. Here we apply conditional methods that more effectively retrieve melanoma cells by differential trypsinization or by inducing fibroblast senescence through contact inhibition, serum starvation or deprivation of adhesion. Simple mixing experiments of melanoma and fibroblast cells demonstrated the efficacy of the new protocols in retrieving slow-growing melanoma cells. Applying our protocols to 20 cultures that had failed to grow by conventional methods, we could retrieve 12 (60%) validated melanoma cell cultures. Further application of the protocols in the live-cell biobank of 124 early passage cultures significantly improved recovery rates from 13% using standard protocols to 70% overall for the new workflow.

Bacterially produced recombinant influenza vaccines based on virus-like particles.

Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

Glycerophospholipid profile in oncogene-induced senescence.

Alterations in lipid metabolism and in the lipid composition of cellular membranes are linked to the pathology of numerous diseases including cancer. However, the influence of oncogene expression on cellular lipid profile is currently unknown. In this work we analyzed changes in lipid profiles that are induced in the course of ERBB2-expression mediated premature senescence. As a model system we used MCF-7 breast cancer cells with doxycycline-inducible expression of NeuT, an oncogenic ERBB2 variant. Affymetrix gene array data showed NeuT-induced alterations in the transcription of many enzymes involved in lipid metabolism, several of which (ACSL3, CHPT1, PLD1, LIPG, MGLL, LDL and NPC1) could be confirmed by quantitative realtime PCR. A study of the glycerophospholipid and lyso-glycerophospholipid profiles, obtained by high performance liquid chromatography coupled to Fourier-transform ion cyclotron resonance-mass spectrometry revealed senescence-associated changes in numerous lipid species, including mitochondrial lipids. The most prominent changes were found in PG(34:1), PG(36:1) (increased) and LPE(18:1), PG(40:7) and PI(36:1) (decreased). Statistical analysis revealed a general trend towards shortened phospholipid acyl chains in senescence and a significant trend to more saturated acyl chains in the class of phosphatidylglycerol. Additionally, the cellular cholesterol content was elevated and accumulated in vacuoles in senescent cells. These changes were accompanied by increased membrane fluidity. In mitochondria, loss of membrane potential along with altered intracellular distribution was observed. In conclusion, we present a comprehensive overview of altered cholesterol and glycerophospholipid patterns in senescence, showing that predominantly mitochondrial lipids are affected and lipid species less susceptible to peroxidation are increased.