Metabolic Fingerprinting of Murine L929 Fibroblasts as a Cell-Based Tumour Suppressor Model System for Methionine Restriction

Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level.

Authentication of Primary Murine Cell Lines by a Microfluidics-Based Lab-On-Chip System

The reliable authentication of cell lines is a prerequisite for the reproducibility and replicability of experiments. A common method of cell line authentication is the fragment length analysis (FLA) of short-tandem repeats (STR) by capillary electrophoresis. However, this technique is not always accessible and is often costly. Using a microfluidic electrophoresis system, we analyzed the quality and integrity of different murine cell lines by STR profiling. As a proof of concept, we isolated and immortalized hematopoietic progenitor cells (HPC) of various genotypes through retroviral transduction of the fusion of the estrogen receptor hormone-binding domain with the coding sequence of HoxB8. Cell lines were maintained in the HPC state with Flt3 ligand (FL) and estrogen treatment and could be characterized upon differentiation. In a validation cohort, we applied this technique on primary mutant Kras-driven pancreatic cancer cell lines, which again allowed for clear discrimination. In summary, our study provides evidence that FLA of STR-amplicons by microfluidic electrophoresis allows for stringent quality control and the tracking of cross-contaminations in both genetically stable HPC lines and cancer cell lines, making it a simple and cost-efficient alternative to traditional capillary electrophoresis.

Molecular Cytogenomic Characterization of the Murine Breast Cancer Cell Lines C-127I, EMT6/P and TA3 Hauschka

Background: To test and introduce effective and less toxic breast cancer (BC) treatment strategies, animal models, including murine BC cell lines, are considered as perfect platforms. Strikingly, the knowledge on the genetic background of applied BC cell lines is often sparse though urgently necessary for their targeted and really justified application. Methods: In this study, we performed the first molecular cytogenetic characterization for three murine BC cell lines C-127I, EMT6/P and TA3 Hauschka. Besides fluorescence in situ hybridization-banding, array comparative genomic hybridization was also applied. Thus, overall, an in silico translation for the detected imbalances and chromosomal break events in the murine cell lines to the corresponding homologous imbalances in humans could be provided. The latter enabled a comparison of the murine cell line with human BC cytogenomics. Results: All three BC cell lines showed a rearranged karyotype at different stages of complexity, which can be interpreted carefully as reflectance of more or less advanced tumor stages. Conclusions: Accordingly, the C-127I cell line would represent the late stage BC while the cell lines EMT6/P and TA3 Hauschka would be models for the premalignant or early BC stage and an early or benign BC, respectively. With this cytogenomic information provided, these cell lines now can be applied really adequately in future research studies.

Prion infection, transmission, and cytopathology modeled in a low-biohazard human cell line

Transmission of prion infectivity to susceptible murine cell lines has simplified prion titration assays and has greatly reduced the need for animal experimentation. However, murine cell models suffer from technical and biological constraints. Human cell lines might be more useful, but they are much more biohazardous and are often poorly infectible. Here, we describe the human clonal cell line hovS, which lacks the human PRNP gene and expresses instead the ovine PRNP VRQ allele. HovS cells were highly susceptible to the PG127 strain of sheep-derived murine prions, reaching up to 90% infected cells in any given culture and were maintained in a continuous infected state for at least 14 passages. Infected hovS cells produced proteinase K–resistant prion protein (PrPSc), pelletable PrP aggregates, and bona fide infectious prions capable of infecting further generations of naïve hovS cells and mice expressing the VRQ allelic variant of ovine PrPC. Infection in hovS led to prominent cytopathic vacuolation akin to the spongiform changes observed in individuals suffering from prion diseases. In addition to expanding the toolbox for prion research to human experimental genetics, the hovS cell line provides a human-derived system that does not require human prions. Hence, the manipulation of scrapie-infected hovS cells may present fewer biosafety hazards than that of genuine human prions.