CHARACTERIZATION OF TRANSFORMED CELL LINES OBTAINED FROM PRIMARY RAT CORTICAL ASTROCYTES

Abstract

Brain cancers are complex and heterogeneous; most of them derive from glial cells[1], and are called gliomas, further subdivided into astrocytomas, oligodendrogliomas, ependymomas and glioastrocytomas[2]. The malignant cells undergo modifications of their metabolism and behaviour, and acquire the ability to migrate along the blood vessels in small groups (model of the guerrilla war)[3], thus invading the surrounding brain parenchyma. Most important, they have the capacity to affect the surrounding microenvironment, by altering both the extracellular matrix and the properties of the normal cells present in the brain, including glial-, endothelial-, and immune-cells, further promoting cancer cell growth and migration. Most of these effects are probably due to molecules (nucleic acids, proteins, lipids etc.) released from cancer cells into their environment via extracellular vesicles (EVs)[1]. Although a lot of efforts have been done, over the years, in order to acquire a better understanding of gliomas at the molecular level, and to set combined therapeutic approaches, brain cancers are mostly incurable. In order to gain a better knowledge of the cellular and molecular events that accompany their transformation, we started from primary cultures of rat cortical astrocytes, and selected three clones that showed increasingly high cell division rates. Then we analyzed both normal cells and their clones at the cytogenetic, cytogenomic and epigenetic level, and found that the most modified astrocytes (A-FC6 clone) have epigenetic and chromosomal alterations typical of cancer, such as an isochromosome (i8q) and that the other two clones (A-GS1 and A-VV5) have intermediate properties. We also analyzed the expression of the linker histone H1.0, normally expressed in differentiated cells[4]. Surprisingly, we found that the somatic histone H1.0 steadily increases from normal astrocytes to the A-FC6 clone. These results suggested that the normal cell cultures together with their three clones may constitute a potential model for studying glioma development. We are now analyzing the expression of other proteins normally expressed in higher amount in cancer cells (e.g. PDI and FABP7), and we are studying how much different is the ability of transformed cells to release EVs respect to normal astrocytes. One of the aim of this study is also to identify proteins and RNAs specifically sorted to EVs from both normal and transformed astrocytes.