An In Vitro Model of Glioma Development

Abstract

Gliomas are the prevalent forms of brain cancer and derive from glial cells. Among them, astrocytomas are the most frequent. Astrocytes are fundamental for most brain functions, as they contribute to neuronal metabolism and neurotransmission. When they acquire cancer properties, their functions are altered, and, in addition, they start invading the brain parenchyma. Thus, a better knowledge of transformed astrocyte molecular properties is essential. With this aim, we previously developed rat astrocyte clones with increasing cancer properties. In this study, we used proteomic analysis to compare the most transformed clone (A-FC6) with normal primary astrocytes. We found that 154 proteins are downregulated and 101 upregulated in the clone. Moreover, 46 proteins are only expressed in the clone and 82 only in the normal cells. Notably, only 11 upregulated/unique proteins are encoded in the duplicated q arm of isochromosome 8 (i(8q)), which cytogenetically characterizes the clone. Since both normal and transformed brain cells release extracellular vesicles (EVs), which might induce epigenetic modifications in the neighboring cells, we also compared EVs released from transformed and normal astrocytes. Interestingly, we found that the clone releases EVs containing proteins, such as matrix metalloproteinase 3 (MMP3), that can modify the extracellular matrix, thus allowing invasion.

Keywords: astrocyte cell lines; astrocytomas; chromosome alterations; epigenetic alterations; extracellular vesicles (EVs); metalloproteinases; proteomics.

Figure 1

Comparative proteomic analysis of cell lysates from primary astrocytes and A-FC6 cells. (A) Venn diagram showing the proteins (unique, common, up/downregulated and total) identified in primary astrocytes and A-FC6 cells. (B) Volcano plot of significant proteins differentially expressed between primary astrocytes and the A-FC6. On the X axis, the differences in the intensity of “label-free quantification” (LFQ) are reported, for the proteins identified in primary astrocytes and A-FC6 cells; on the Y axis, the log of the p-value, corresponding to significance, is shown.

Figure 2

Enrichment pathway analysis of differentially expressed proteins in A-FC6. (A) Proteins upregulated in A-FC6. (B) Proteins downregulated in A-FC6. Significantly enriched biological pathways were ranked by p-value using the FunRich 3.0 software. Red boxes highlight significant pathways in which identified proteins are involved.

Figure 3

Venn diagram showing proteins expressed by chromosome #8 genes and identified in primary astrocytes and in A-FC6.

Figure 4

Comparative proteomic analysis of EVs from primary astrocytes and the A-FC6 cells. (A) Venn diagram showing the identified proteins (unique, common, up/downregulated and total) from primary astrocytes and the A-FC6 clone. (B) Volcano plot of significant proteins differentially expressed in primary astrocytes and A-FC6 cells. On the X axis, the differences in the intensity of “label-free quantification” (LFQ) of the proteins identified in primary astrocytes and A-FC6 cells are reported; on the Y axis, the log of the p-value, corresponding to significance, is reported.

Figure 5

Enrichment pathway analysis of differentially expressed proteins in A-FC6. (A) Unique proteins identified in A-FC6-derived EVs. (B) Unique proteins identified in EVs derived from primary astrocytes. Significantly enriched pathways were ranked by p-value using the FunRich 3.0 software. Red boxes highlight the most significant pathways in which identified proteins are involved.

Figure 6

Western blot analysis of proteins present in EVs released from normal astrocytes (nA) and A-FC6 cells. After blotting, the membrane was immunostained with a mouse monoclonal anti-MMP3 antibody (Calbiochem, CA, USA). The secondary anti-mouse antibody was from Promega (St. Louis, MO, USA).