Astrocytes enhance the invasion potential of glioblastoma stem-like cells

Abstract

Glioblastomas (GBMs) are characterized as highly invasive; the contribution of GBM stem-like cells (GSCs) to the invasive phenotype, however, has not been completely defined. Towards this end, we have defined the invasion potential of CD133+ GSCs and their differentiated CD133- counterparts grown under standard in vitro conditions and in co-culture with astrocytes. Using a trans-well assay, astrocytes or astrocyte conditioned media in the bottom chamber significantly increased the invasion of GSCs yet had no effect on CD133- cells. In addition, a monolayer invasion assay showed that the GSCs invaded farther into an astrocyte monolayer than their differentiated progeny. Gene expression profiles were generated from two GSC lines grown in trans-well culture with astrocytes in the bottom chamber or directly in contact with astrocyte monolayers. In each co-culture model, genes whose expression was commonly increased in both GSC lines involved cell movement and included a number of genes that have been previously associated with tumor cell invasion. Similar gene expression modifications were not detected in CD133- cells co-cultured under the same conditions with astrocytes. Finally, evaluation of the secretome of astrocytes grown in monolayer identified a number of chemokines and cytokines associated with tumor cell invasion. These data suggest that astrocytes enhance the invasion of CD133+ GSCs and provide additional support for a critical role of brain microenvironment in the regulation of GBM biology.

Figure 1. Trans-well invasion assay.

A) CD133+ B) CD133− NSC11 or GBAM1 cells were seeded onto trans-well membranes with the specified conditions in the corresponding bottom well, invasion was determined 48 h later (CM: astrocyte-conditioned medium, FBS: fetal bovine serum, MRC9: normal human fibroblasts). Values shown represent the mean ± SE of 3–4 independent experiments. *p<0.05 versus control.

Figure 2. Monolayer-invasion assay.

CD133+ GSCs or CD133- cells stained with PKH67 (green) were grown on a cover-slip, inverted and placed on a confluent astrocyte monolayer stained with PKH26 (red) for 48 h. A) Representative image of CD133+ NSC11 cells on astrocyte monolayer. B) Representative image of CD133− differentiated NSC11 cells on astrocyte monolayer (scale bar: 500 µm). C) Invasion of NSC11 and GBAM1 CD133+ and CD133− cells into astrocyte monolayer was defined by image analysis. Values shown represent the mean ± SE of 3–4 independent experiments. *p<0.05.

Figure 3. GSC gene expression changes induced by indirect co-culture with astrocytes.

A) Venn diagram comparing affected genes in CD133+ NCS11 and GBAM1 cells. B) Commonly increased genes (117) were subjected to IPA and the top five molecular and cellular function categories are shown. C) Interconnecting network formed by 42 of the 117 genes whose expression was commonly increased in NSC11 and GBAM1 CD133+ cells as a result of indirect co-culture with astrocytes. D) Immunoblots generated from GSCs 48 hours after seeding onto po/ln coated trans-well membranes without (control) and with astrocytes (co-culture) in the bottom well. Blots are representative of 2 independent experiments.

Figure 4. GSC gene expression changes induced by direct co-culture with astrocytes.

A) Venn diagram comparing affected genes in CD133+ NCS11 and GBAM1 cells. B) Commonly increased genes (229) were subjected to IPA; the top five molecular and cellular function categories are shown.

Figure 5. Comparison of GSC gene expression changes induced by indirect and direct co-culture with astrocytes.

A) Venn diagram comparing genes whose expression was increased in both CD133+ NCS11 and GBAM1 cells after indirect and direct co-culture with astrocytes. B) Genes commonly affected only in direct co-culture with astrocytes (169) were subjected to IPA; the top five molecular and cellular function categories are shown. C) Interconnecting network formed by 58 of the 169 genes whose expression was increased in both NSC11 and GBAM1 CD133+ cells only as a result of direct co-culture with astrocytes.

Figure 6. Chemokine/Cytokine profile from astrocyte conditioned media.

Waterfall diagram comparing chemokines/cytokines detected in astrocyte conditioned media as compared to stem cell medium. Values shown represent the level of proteins in astrocyte conditioned media with a greater >2-fold-change compared to stem cell medium alone.