Pediatric glioblastoma cells inhibit neurogenesis and promote astrogenesis, phenotypic transformation and migration of human neural progenitor cells within cocultures

Abstract

Neural progenitor cell (NPC) fate is influenced by a variety of biological cues elicited from the surrounding microenvironment and recent studies suggest their possible role in pediatric glioblastoma multiforme (GBM) development. Since a few GBM cells also display NPC characteristics, it is not clear whether NPCs transform to tumor cell phenotype leading to the onset of GBM formation, or NPCs migrate to developing tumor sites in response to paracrine signaling from GBM cells. Elucidating the paracrine interactions between GBM cells and NPCs in vivo is challenging due to the inherent complexity of the CNS. Here, we investigated the interactions between human NPCs (ReNcell) and human pediatric GBM-derived cells (SJ-GBM2) using a Transwell^® coculture setup to assess the effects of GBM cells on ReNcells (cytokine and chemokine release, viability, phenotype, differentiation, migration). Standalone ReNcell or GBM cultures served as controls. Qualitative and quantitative results from ELISA^®, Live/Dead^® and BrdU assays, immunofluorescence labeling, western blot analysis, and scratch test suggests that although ReNcell viability remained unaffected in the presence of pediatric GBM cells, their morphology, phenotype, differentiation patterns, neurite outgrowth, migration patterns (average speed, distance, number of cells) and GSK-3β expression were significantly influenced. The cumulative distance migrated by the cells in each condition was fit to Furth's formula, derived formally from Ornstein-Uhlenbeck process. ReNcell differentiation into neural lineage was compromised and astrogenesis promoted within cocultures. Such coculture platform could be extended to identify the specific molecules contributing to the observed phenomena, to investigate whether NPCs could be transplanted to replace lesions of excised tumor sites, and to elucidate the underlying molecular pathways involved in GBM-NPC interactions within the tumor microenvironment.

Keywords: Differentiation; GSK-3β; Migration; Neural progenitor cells; Pediatric glioblastoma cells.

Figure 1

Heat map representing cytokine and chemokine amounts released by ReNcells alone (presence or absence of bFGF), SJ-GBM2 cells alone, ReNcell cocultures, and SJ-GBM2 cocultures, as measured via laser bead immunoassay. Each row corresponds to one culture condition, and each column corresponds to one analyte. Analytes were clustered based on their type – cytokines, chemokines and cancer markers. Cocultures were performed using a Transwell^® setup. The actual analyte concentration levels were shown in Supplementary Figure 4. No detectable levels of IL-10, IL-3, MIP1α, Myeloperoxidase, and Fibroblast Activation Protein were found.

Figure 2

Cellular proliferation was measured via BrdU uptake and expressed as optical density (O.D) in various culture conditions. Measurements were taken immediately after seeding (4 h), and at days 1, 5 and 10. Error bars represent mean ± SEM for all cases.

Figure 3

Representative immunofluorescence images of ReNcells and GBMs, in standalone and cocultures, on day 10. For comparison, in select cases, ReNcells cultures in the presence of bFGF were also stained and imaged after 24 h in culture. Cultures were counterstained with DAPI for cell identification. Primary antibodies for TUJ1, GFAP, Nestin, CD133, EGFR, MAP2, and β-actin were used, with appropriate secondary antibodies. Double-immunolabeling was performed under select conditions. Scale bar: 50 μm.

Figure 4

Quantification of ReNcell and GBM phenotype in standalone and cocultures on days 5 and 10, staining for (A) CD-133, (B) Nestin, (C) GFAP and (D) TUJ1. Values are expressed as a percentage of the number of cells stained positively for the respective marker (normalized to total number cells staining positive for DAPI). * indicates significant differences (p < 0.05) between day 5 vs. day 10, while # indicates significance in differences (p < 0.05) between standalone and cocultures of ReNcells or GBMs, at a given time point. Error bars represent mean ± SEM for all cases (n = 3/case).

Figure 5

(A) Cell shape index (CSI) was measured using a particle analyzer plugin in ImageJ software, from images captured on days 5 and 10. CSI equal to one indicates infinitely elongated cell shape and a value of zero indicates perfectly rounded shape. (B) Average neurite outgrowth (μm) on days 5 and 10 was measured from TUJ1-stained cells within ReNcell cultures, in both standalone and coculture conditions. A NeuronJ plugin in ImageJ software was used to measure average outgrowth. * indicates significant differences (p < 0.05) between day 5 vs. day 10, while # indicates significant differences (p < 0.05) between coculture vs. standalone cultures at a given time point. Error bars represent mean ± SEM for all cases (n = 3/case). (C) Semi-quantitative western blot analysis of GSK-3β protein expression in ReNcells and GBM cells, cultured alone or in cocultures, at the end of 10 day culture period. Undifferentiated ReNcells (i.e., the presence of bFGF) were used as controls. Multi-fold increases in GSK-3β expression were noted in all the cases, with significantly higher levels in cocultures, specifically within GBM cells. Inset shows representative protein bands under respective culture conditions. ** indicates p < 0.05 vs. controls.

Figure 6

(A) Representative bright-field images at selected time points, obtained from a scratch test assay. ReNcells or GBMs were cultured in 24-well plates, either standalone or in coculture (Transwell^® setup) with the other cell type. Images were taken at regular intervals over an 80 h period to visualize cell migration. Yellow dotted lines in each image represent the starting position of the cells (at t = 0) when the scratch was initially made. Scale bar is 100 μm. (B) The number of ReNcells or GBMs, in standalone or cocultures, which migrated from their initial starting point into the cell-free zone created by scratch were quantified at various time points. * indicates significant differences in coculture vs. standalone cultures. Error bars represent mean ± SEM for all cases (n = 3/case). (C) The average cell speed (μm/h) was quantified in these cultures at various time points (n = 3 wells/time point). (D) The average cumulative distance (μm) covered by the cells was quantified at various time points (n = 3 wells/time point). Lines indicate the Ornstein-Uhlenbeck process model fit to the data shown in symbols. Error bars represent mean ± SEM for all cases.

Figure 7

From scratch test assay, cell migration within standalone and cocultures of ReNcells and GBMs was divided into two zones – frontline (first 100 μm from scratch interface) and midline (next 100 μm behind frontline). Cumulative distance migrated by individual frontline cells over 56 h was tracked in GBMs cultured alone (A-1), GBM cocultures (B-1), ReNcells cultured alone (C-1), and ReNcell cocultures (D-1). Similarly, distance migrated by individual midline cells was tracked within GBMs cultured alone (A-2), GBM cocultures (B-2), ReNcells cultured alone (C-2), and ReNcell cocultures (D-2). Each symbol corresponds to a cell tracked over the test duration, and at least 15 individual cells were randomly selected and plotted for each culture condition.