Visiomorph

After superimposing the confocal z-stacks, the area of the spheroids was measured using the software Visiomorph (Visiopharm, Hørsholm, Denmark). The spheroids were outlined at the spheroid boarder identifying the beginning of the invasion zone. The area of the invasive cells was also measured in Visiomorph using a classifier identifying the area of DiI staining representing only the invasive cells and not the spheroids. The invasion distance in the confocal images was not measurable because of the small field of view.

The glioma cell lines were incubated in 12 well glass bottom plates until approximately 80% confluence and exposed to siramesine (5–30 μM). After 1 h, Acridine orange (5 μg/ml, Invitrogen) was added to the cells and incubated for 15 min at 37 °C. Images were recorded using confocal microscopy (Nikon, Inverted Microscope, ECLIPSE TE2000-E). Analysis was made using the image analysis tool Visiomorph™ (Visiopharm, Hørsholm, Denmark). The results represent the proportion of red/green + yellow area, accounting for the loss of red staining and gain of green and yellow staining in the cells.

Image analysis was performed on whole slide images using Visiomorph (Visiopharm, Denmark). Hematoxylin and eosin (H&E)-stained sections were used to determine tissue areas (mm²), and section thicknesses (μm) were then used to calculate tissue volume (μL). Cells per μL of tissue were determined by dividing cell equivalents calculated from total DNA yield by the volume of each tissue slice analyzed by PCR. (At the highest viral concentrations in our set of 9 sections, viral DNA was <2% of total DNA, allowing total DNA to be used as an estimate of cellular DNA; calculation not shown.) JCV capsid concentrations were calculated by dividing moles of VP1 per sample by 360 (72 pentamers per virion × 5 VP1 molecules per pentamer) and by the tissue volume (in μL/mm³). Linear regression, significance, and graphic display were generated in Prism 5 (GraphPad Software, Inc., La Jolla, CA).