Abstract
Intercellular communication within complex biological and pathological systems via extracellular vesicles (EVs) and secreted factors is a highly attractive area of research. However, cell models enabling investigation of such communication in vitro are limited. Commonly utilized is the supplementation of hyper-concentrated EVs or other extracellular factors to the recipient cell cultures. This approach requires purification of the secreted complexes and is confounded by the contamination of media components. Two-chamber co-cultures of donor and recipient cells separated by a pore membrane may represent a more physiological and better-controlled system for the investigation of intercellular communication. Yet, distinct culture conditions for different neural cell types often make them incompatible for co-culturing. Here we optimized short-term co-cultures of patient-derived low-passage glioma-initiating stem cells with normal cells of the brain microenvironment, such as primary neurons, astrocytes, microglia, and brain endothelial cells. We demonstrate the culture compatibility of these cell types and internalization of glioma-derived extracellular RNA by the normal recipient cells. The presented protocols are valuable for the investigation of intercellular communication between glioma brain tumor and cells of its microenvironment, including but not limited to the EVs-mediated communication.RESEARCH IN CONTEXTCell-to-cell communication is essential in normal physiology and implicated in disease; however, experimental systems for its modeling in vitro are limited. Particularly, the investigation of communication between brain tumors and normal cells of the brain microenvironment has been challenged by the lack of adequate culture models. Here we developed co-cultures of glioma stem cells with various types of normal brain cells, including primary neurons, astrocytes, microglia, and brain endothelial cells, and demonstrated their utility for the study of intercellular communication. Detection of proposed markers in the recipient cells confirmed RNA transfer in these co-cultures.
Highlights
Intercellular communication is critical for all biological and pathological processes
Additional routes of transmission, such as those mediated by extracellular vesicles (EVs) (Valadi et al, 2007; Skog et al, 2008) and extravesicular ribonucleoproteins (RNPs) (Vickers et al, 2011) carrying RNA, proteins, lipids and other cargo molecules, have been extensively explored
Investigation of these modes of communication is an especially active area of research in the field of cancer biology, as they are thought to contribute to the invasion, metastasis, angiogenesis, chemo-resistance, and immune tolerance of many malignancies (Hanahan and Weinberg, 2011)
Summary
Intercellular communication is critical for all biological and pathological processes. Comparing to the previous strategy, co-culture experiments may better model the complex and persistent communication between the cells, physiological release-uptake dynamics, and enable assessment of multiple mediators of communication between the donor and recipient cultures Such experimental design has been utilized in many studies, including those reporting miRNA transfer from cardiac fibroblast to cardiomyocytes (Bang et al, 2014) and between dendritic cells (Alexander et al, 2105), ncRNA transfer from cardiospherederived cells to macrophages (Cambier et al, 2107), mRNA transfer from acute myelogenous leukemia to stromal niche (Huan et al, 2103), DNA transfer between parasites (RegevRudzki et al, 2013), protein transfer from mesenchymal stromal cells to haematopoietic cells (Bauer et al, 2011), and virus transfer from infected hepatoma cells to plasmacytoid dendritic cells (Feng et al, 2014). The established cell systems may provide a simple and physiologically relevant model for studying the bidirectional communication in malignant gliomas
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