Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes

Rhabdomyosarcomas (RMS) are mesenchyme-derived tumors and the most common childhood soft tissue sarcomas. Treatment is intense, with a nevertheless poor prognosis for high-risk patients. Discovery of new therapies would benefit from additional preclinical models. Here, we describe the generation of a collection of 19 pediatric RMS tumor organoid (tumoroid) models (success rate of 41%) comprising all major subtypes. For aggressive tumors, tumoroid models can often be established within 4-8 weeks, indicating the feasibility of personalized drug screening. Molecular, genetic, and histological characterization show that the models closely resemble the original tumors, with genetic stability over extended culture periods of up to 6 months. Importantly, drug screening reflects established sensitivities and the models can be modified by CRISPR/Cas9 with TP53 knockout in an embryonal RMS model resulting in replicative stress drug sensitivity. Tumors of mesenchymal origin can therefore be used to generate organoid models, relevant for a variety of preclinical and clinical research questions

Carbon nanoparticles in lateral flow methods to detect genes encoding virulence factors of Shiga toxin-producing Escherichia coli

The use of carbon nanoparticles is shown for the detection and identification of different Shiga toxin-producing Escherichia coli virulence factors (vt1, vt2, eae and ehxA) and a 16S control (specific for E. coli) based on the use of lateral flow strips (nucleic acid lateral flow immunoassay, NALFIA). Prior to the detection with NALFIA, a rapid amplification method with tagged primers was applied. In the evaluation of the optimised NALFIA strips, no cross-reactivity was found for any of the antibodies used. The limit of detection was higher than for quantitative PCR (q-PCR), in most cases between 104 and 105 colony forming units/mL or 0.1–0.9 ng/μL DNA. NALFIA strips were applied to 48 isolates from cattle faeces, and results were compared to those achieved by q-PCR. E. coli virulence factors identified by NALFIA were in very good agreement with those observed in q-PCR, showing in most cases sensitivity and specificity values of 1.0 and an almost perfect agreement between both methods (kappa coefficient larger than 0.9). The results demonstrate that the screening method developed is reliable, cost-effective and user-friendly, and that the procedure is fast as the total time required is <1 h, which includes amplification

High-Efficiency Gene Transfer into Adult Fish : a New Tool to Study Fin Regeneration

Zebrafish represents an excellent model to study the function of vertebrate genes (e.g., well-developed genetics, large number of mutants, and genomic sequencing in progress), inasmuch as we have tools to manipulate gene expression. Recent use of injected morpholinos in eggs provides a good method to " knockdown " gene expression in early development (Nasevicius and Ekker, 2000), and the "caged" RNA injected in eggs allows to overexpress a gene in a specific set of cells (Ando et al., 2001). However, a method to specifically modify gene expression in the juvenile or in the adult is still missing. Such a method would be a very powerful tool to understand gene function in differentiated tissues. We describe here an electroporation-based approach, which allows gene transfer in adult tissues. Its efficiency was assessed using a GFP (green fluorescent protein) dependent assay. We then used this method to disrupt the Fgf signalling pathway during the process of regeneration

Growth and differentiation of C2 myogenic cells are dependent on serum response factor.

In order to study to what extent and at which stage serum response factor (SRF) is indispensable for myogenesis, we stably transfected C2 myogenic cells with, successively, a glucocorticoid receptor expression vector and a construct allowing for the expression of an SRF antisense RNA under the direction of the mouse mammary tumor virus long terminal repeat. In the clones obtained, SRF synthesis is reversibly down-regulated by induction of SRF antisense RNA expression by dexamethasone, whose effect is antagonized by the anti-hormone RU486. Two kinds of proliferation and differentiation patterns have been obtained in the resulting clones. Some clones with a high level of constitutive SRF antisense RNA expression are unable to differentiate into myotubes; their growth can be blocked by further induction of SRF antisense RNA expression by dexamethasone. Other clones are able to differentiate and are able to synthesize SRF, MyoD, myogenin, and myosin heavy chain at confluency. When SRF antisense RNA expression is induced in proliferating myoblasts by dexamethasone treatment, cell growth is blocked and cyclin A concentration drops. When SRF antisense RNA synthesis is induced in arrested confluent myoblasts cultured in a differentiation medium, cell fusion is blocked and synthesis of not only SRF but also MyoD, myogenin, and myosin heavy chain is inhibited. Our results show, therefore, that SRF synthesis is indispensable for both myoblast proliferation and myogenic differentiation