Cx43 in Neural Progenitors Promotes Glioma Invasion in a 3D Culture System

Abstract

The environment that envelops the cancer cells intimately affects the malignancy of human cancers. In the case of glioma, an aggressive adult brain cancer, its high rate of recurrence after total resection is responsible for a poor prognosis. Connexin43 (Cx43) is a gap junction protein with a prominent presence in glioma-associated normal brain cells, specifically in the reactive astrocytes. We previously demonstrated that elimination of Cx43 in these astrocytes reduces glioma invasion in a syngeneic mouse model. To further our investigation in human glioma cells, we developed a scaffold-free 3D platform that takes into account both the tumor and its interaction with the surrounding tissue. Using cell-tracking dyes and 3D laser scanning confocal microscopy, we now report that the elimination of Cx43 protein in neural progenitor spheroids reduced the invasiveness of human brain tumor-initiating cells, confirming our earlier observation in an intact mouse brain. By investigating the glioma invasion in a defined multicellular system with a tumor boundary that mimics the intact brain environment, our findings strengthen Cx43 as a candidate target for glioma control.

Keywords: 3D; Cx43; glioma; human brain tumor-initiating cells; invasion; time-lapse imaging.

Figure 1

Cx43 protein is expressed in human brain tumor-initiating cells GBM4 and GBM8. (a) Schematic diagram illustrating the culture and labeling of spheroids with cell-tracker dyes (CMTPX, CMFDA and CMAC) to distinguish them for live imaging by confocal microscopy. (b) Western analysis of cell lysates of GBM4 and GBM8 with anti-Cx43 antibody showing multiple protein bands corresponding to different phosphorylated isoforms. Cx43 species in mouse GL261 glioma cells used in our intracranial mouse implantation [15] was included as a comparison. GAPDH was used as loading control.

Figure 2

Invasiveness of GBM4 and GBM8 correlate with their pathogenicity in human patients. (a) Co-culture of GBM4 (red), GBM8 (green) and C57BL6 mouse neural progenitor cell derived spheroids (blue) in Noble agar before imaging. Scale bar 100 μm (b) Representative time-lapse images showing GBM8 preferentially invaded as single cells (white arrowheads) while GBM4 invaded in a collective manner as a group (white arrows). Images were taken 10 min apart at a 5 µm step size using confocal microscopy. Scale bar 100 μm (c) Mean velocity of GBM4 and GBM8 single cells in mouse wild-type spheroids. The data shown here are pooled from at least three experiments. Data were analyzed by Student’s t test. * P < 0.05.

Figure 3

Elimination of Cx43 in mouse progenitor cells reduces GBM8 invasion. (a) Wild-type (WT) and Cx43 knockout (KO) spheroids were co-stained with anti-Cx43 and anti-GFAP antibodies. GFAP was detected in both WT and KO spheroids. In contrast, the punctate staining of Cx43 was only detected in WT but not the KO spheroids, confirming successful knockout of Cx43 in the mouse progenitors. Scale bar 50 μm (b) 3D cell tracking was carried out using the Fiji plugin, MTrackJ. Representative images showing cell movement in two 40-min periods. The track of a single cell is denoted by a colored lane in a 3D projection. Each circle represents the x,y,z location of the cell at a time point from 0 to 40 min, and from 0 to 80 min. The difference between 40 and 80 min track illustrates the movement of cells during this time interval. Note that the movement of a cell in the z direction will not be apparent in these 2D representative images. Scale bar 75 μm (c) Mean velocity of GBM8 in WT and KO mouse spheroids. The data shown here are pooled from at least three experiments. Data were analyzed by Student’s t test. * P < 0.05.