To test the ability of TMV to interact with autologous
(parental) cancer cells, GC1415 cells were incubated with
PKH26-labelled TMV. FACS analysis indicated that after
2 h of culture about 70 % of GC1415 cells were fluorescent,
which increased to 99 % after 24 h (Fig. 3b, d). To discriminate,
whether TMV only adhered to the cell surface or
were engulfed by the cells, crystal violet was added. After
quenching, the percentage of positive cells decreased to
16.8 and 37.7 % at 2 and 24 h, respectively (Fig. 3f, h), thus
indicating intracellular localization of TMV.
To confirm localization of TMV in GC1415 cells confocal
microscopy was applied. When GC1415 cells were
incubated with PKH26-labelled TMV for 2 h the red
fluorescence of the vesicles was associated with plasma
membranes (Fig. 4a), however following a 24 h incubation
the majority of TMVs were detected inside the cells (Fig. 4b). TMV localized intracellularly were of different size. AFM analysis of control and TMV-treated GC1415 cells showed at 24 h, but not 2 h, significant differences in their surface outlook. The cells not exposed to TMV (Fig. 5a) showed smooth surface and sharp contour. After
incubation with TMV (Fig. 5b) the cell surface was rough,uneven with visible protuberations. The cells edges were thicker and diffused of torus shape.