Invasion of primary glioma- and cell line-derived spheroids implanted into corticostriatal slice cultures

Abstract

Gliomas are highly invasive tumors and the pronounced invasive features of gliomas prevent radical surgical resection. In the search for new therapeutics targeting invasive glioma cells, in vivo-like in vitro models are of great interest. We developed and evaluated an in vivo-like in vitro model preserving the invasive features and stem cell features of glioma cells. Fluorescently labelled primary glioma spheroids and U87MG cell line-derived spheroids were implanted into organotypic rat corticostriatal slice cultures and the invasion was followed over time by confocal microscopy. The invasion was validated immunohistochemically with paraffin sections using a human-specific vimentin antibody. Moreover, the preservation of immature stem cell features was evaluated immunohistochemically using the stem cell markers CD133, Sox2, Bmi-1 and nestin. The confocal and immunohistochemical results showed that the primary glioma spheroid area was constant or decreasing after implantation, with a clear increase in the number of invading cells over time. In contrast, the U87MG spheroid area increased after implantation, with no convincing tumor cell invasion. High levels of Bmi-1 and nestin were found in all spheroids, whereas high levels of Sox2 and low to moderate levels of CD133 were only found in the primary spheroids. In conclusion, the invasion of gliomas is preserved using primary glioma spheroids. Some stem cell features are preserved as well, making this model useful in drug development elucidating both invasion and cancer stemness at the early in vitro level.

Keywords: Glioma; invasion; organotypic slice cultures; spheroids.

Figure 1

Tumor cell invasion was followed over time using confocal microscopy. Images were taken every 20 μm down through the co-culture, visualizing invasive tumor cells in the individual layers (A-E). When investigating the individual images in the z-stack obtained from glioblastoma-1 at day 6, invading cells were seen in almost all layers (A-E) with only few invading cells at the top (A) and at the bottom (E) of the spheroids suggesting pronounced tumor cell invasion inside the brain slice cultures (B-D). The images in the z-stack were superimposed into one image (F), visualizing all invasive tumor cells. Scalebar 100 μm.

Figure 2

The images in the confocal z-stacks were superimposed into one image, visualizing all invasive tumor cells. Hereafter the total tumor cell invasion was assessed using the program EZ-C1 FreeViewer (Nikon). Invasive tumor cells were counted in a 200 μm zone around the spheroid, and the diameter of the spheroids was estimated.

Figure 3

The U87MG cell line-derived spheroids (A-C) and primary spheroids (D-L) were labelled with the vital fluorescent dye DiI and implanted into 4 days old brain slice cultures between the cortex and striatum into or close to the corpus callosum. The tumor cell invasion was followed using confocal microscopy at the day of implantation (A, D, G, J), after three days (B, E, H, K) and after six days (C, F, I, L). For the U87MG cell line-derived spheroids only a few fluorescent cells were detected close to the spheroid at day three (B) and day six (C). In contrast to U87MG spheroids pronounced invasion was observed for the primary spheroids derived from the anaplastic astrocytoma (E, F) and glioblastoma-2 (K, L), whereas invasion appeared to be limited for glioblastoma-1 (H, I). Scalebar 100 μm.

Figure 4

Spheroid area was measured (A, C, E, G) and invasive cells were counted (B, D, F, H) on superimposed confocal z-stack images of U87MG spheroids (A, B) and primary spheroids (C-H). The area of the U87MG spheroids increased rapidly over time (A). When implanting primary spheroids derived from an anaplastic astrocytoma (C) and from two glioblastomas (E, G) the area of the spheroids did not increase significantly (C, E, G). In fact, a small decrease was seen for glioblastoma-1 spheroids (E). For the U87MG spheroids only a few invading cells were detected (B). In contrast to U87MG pronounced invasion was observed for the anaplastic astrocytoma (D) and glioblastoma-2 (H), whereas invasion appearently was limited for glioblastoma-1 (H, I). Scalebar 100 μm.

Figure 5

Hematoxylin and eosin-stained paraffin sections of co-cultures revealed viable brain slice cultures with a well preserved cytoarchitecture and viable tumor spheroids with a high tumor-like cell density (A, C, E, G). Immunohistochemical staining with a human specific vimentin antibody (B, D, F, H) confirmed the invasion observed by confocal microscopy. Only few invasive tumor cells were seen around the implanted U87MG cell line-derived spheroids (B) in contrast to a more pronounced invasion seen around the primary spheroids (D, F, H), where small round tumor cells (F) as well as long fusiform tumor cells (H) were seen. Spheroids are marked with *(A, C, E, G). Scalebar 200 μm.

Figure 6

Ki-67 immunohistochemically stained paraffin sections revealed a high level of proliferating cells in all U87MG spheroids (A). The Ki-67 expression in the primary spheroids (B-D) was considerably lower compared to U87MG (A). Scalebar 200 μm.

Figure 7

The co-cultures were investigated immunohistochemically using the putative cancer stem cell marker CD133 (A, E, I, M), the early transcription factors Sox2 (B, F, J, N) and Bmi-1 (C, G, K, O) as well as nestin (D, H, L, P). Using paraffin sections the expression of CD133 was in general not detected in U87MG spheroids (A) whereas some CD133 staining was detected in the primary spheroids (E, I, M). Sox2 was not detected in the U87MG spheroids (B), whereas high levels of Sox2 were seen in all primary spheroids (F, J, N). All tumor cells expressed Bmi-1 in the U87MG cell line-derived spheroids (C) as well as in the primary spheroids (G, K, O). The majority of the tumor cells in all spheroids were nestin positive (D, H, L, P), though differences in staining intensity were seen between spheroids from the same tumor. In general, the lowest staining intensity was seen in U87MG derived spheroids (D), whereas higher staining intensities were seen in the primary glioblastoma spheroids (H, L, P). Scalebar 200 μm.