RNA sequencing and functional studies of patient-derived cells reveal that neurexin-1 and regulators of this pathway are associated with poor outcomes in Ewing sarcoma

Abstract Purpose The development of biomarkers and molecularly targeted therapies for patients with Ewing sarcoma (ES) in order to minimise morbidity and improve outcome is urgently needed. Here, we set out to isolate and characterise patient-derived ES primary cell cultures and daughter cancer stem-like cells (CSCs) to identify biomarkers of high-risk disease and candidate therapeutic targets. Methods Thirty-two patient-derived primary cultures were established from treatment-naïve tumours and primary ES-CSCs isolated from these cultures using functional methods. By RNA-sequencing we analysed the transcriptome of ES patient-derived cells (n = 24) and ES-CSCs (n = 11) to identify the most abundant and differentially expressed genes (DEGs). Expression of the top DEG(s) in ES-CSCs compared to ES cells was validated at both RNA and protein levels. The functional and prognostic potential of the most significant gene (neurexin-1) was investigated using knock-down studies and immunohistochemistry of two independent tumour cohorts. Results ES-CSCs were isolated from all primary cell cultures, consistent with the premise that ES is a CSC driven cancer. Transcriptional profiling confirmed that these cells were of mesenchymal origin, revealed novel cell surface targets for therapy that regulate cell-extracellular matrix interactions and identified candidate drivers of progression and relapse. High expression of neurexin-1 and low levels of regulators of its activity, APBA1 and NLGN4X, were associated with poor event-free and overall survival rates. Knock-down of neurexin-1 decreased viable cell numbers and spheroid formation. Conclusions Genes that regulate extracellular interactions, including neurexin-1, are candidate therapeutic targets in ES. High levels of neurexin-1 at diagnosis are associated with poor outcome and identify patients with localised disease that will relapse. These patients could benefit from more intensive or novel treatment modalities. The prognostic significance of neurexin-1 should be validated independently.

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Morphological features of primary cultures of adrenal cells of neonatal animals of different species

V N Karazin National University Series “Biology” ◽

10.26565/2075-5457-2019-33-9 ◽

2019 ◽

Keyword(s):

Adrenal Gland ◽

Cell Cultures ◽

Adrenal Glands ◽

Primary Cultures ◽

Cell Monolayer ◽

Calf Serum ◽

Morphological Features ◽

Primary Cell ◽

Endocrine Gland ◽

Primary Cell Cultures

The adrenal gland is an endocrine gland, which in the process of organogenesis is formed from ecto- and mesoderm derivatives. The mechanisms that make cell types of different origins unite, migration routes, and cell interactions are still not fully understood. One of the tools for studying these mechanisms is the primary cell culture obtained from the adrenal gland. The aim of our work was to compare the morphological features of primary cell cultures of model animals belonging to different orders – pigs, rabbits and mice in vitro under various cultivation conditions (growth surface pattern, presence of growth factors), as well as developing methodological approaches for obtaining and maintaining primary cultures of adrenal cell of neonatal animals. Cultivation was performed under standard conditions of temperature and humidity, carbon dioxide concentration, on culture surfaces with normal and reduced adhesiveness in a nutrient medium DMEM enriched with 10% fetal calf serum (FTS) or growth supplements B-27 and FGF. It was established that cell cultures of adrenal neonatal rabbits and piglets that were cultured under conditions of normal adhesion and using FCS had a heterogeneous composition, and were presented as a monolayer consisting of cells of several morphological types, and multicellular spheroids (MS). When cultivated on the surface with reduced adhesive properties in cultures of adrenal glands of piglets and rabbits, a cell monolayer was not formed, but flotation MCs were formed. After transferring MCs of both species to the adhesive culture surface on day 14, cell eviction, their migration from the MCs and formation of a monolayer are observed. Similar stages in the development of primary cell cultures derived from rabbits and piglets suggest the existence of a universal cellular composition in the neonatal adrenal glands of these species and allow applying the same approaches to the primary cultures derived from them. Unlike other studied species, monolayer and MS formation does not occur in cell cultures of mouse neonatal adrenal glands. Cultures consist of single attached and floating cells and small cell aggregates.

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Review Article. Technical aspects of oxygen level regulation in primary cell cultures: A review

Interdisciplinary Toxicology ◽

10.1515/intox-2016-0011 ◽

2016 ◽

Vol 9 (3-4) ◽

pp. 85-89 ◽

Cited By ~ 7

Author(s):

Mazyar Yazdani

Keyword(s):

Cell Culture ◽

Cell Cultures ◽

Mammalian Cells ◽

Primary Cultures ◽

Essential Element ◽

Primary Cell ◽

Atmospheric Conditions ◽

Atmospheric Concentration ◽

Primary Cell Cultures

Abstract Oxygen (O2) is an essential element for aerobic respiration. Atmospheric concentration of O2 is approximately 21%. Mammalian cells, however, are generally adapted to O2 levels much lower than atmospheric conditions. The pericellular levels of O2 must also be maintained within a fairly narrow range to meet the demands of cells. This applies equally to cells in vivo and cells in primary cultures. There has been growing interest in the performance of cell culture experiments under various O2 levels to study molecular and cellular responses. To this end, a range of technologies (e.g. gas-permeable technology) and instruments (e.g. gas-tight boxes and gas-controlled incubators) have been developed. It should be noted, however, that some of these have limitations and they are still undergoing refinement. Nevertheless, better results should be possible when technical concerns are taken into account. This paper aims to review various aspects of O2 level adjustment in primary cell cultures, regulation of pericellular O2 gradients and possible effects of the cell culture medium.

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