After siramesine exposure of spheroid-brain slice co-cultures, these were fixed, paraffin embedded, sectioned (3?m) and immunohistochemically stained with anti-human specific CD56 in order to identify the tumor cells. of caspases and cathepsins. Effects of siramesine on migrating tumor cells were investigated by a flat surface migration assay and by implanting spheroids into organotypic rat brain slice cultures followed by confocal time-lapse imaging. Finally the effect of siramesine was investigated in an orthotopic mouse glioblastoma model. Results obtained in vitro and in vivo were confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains. Results The results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal membrane. Co-treatment of the cell lines with inhibitors of caspases and cathepsins suggested differential involvement in cell death. Siramesine caused tumor cell death and reduced secondary spheroid formation of patient-derived spheroid cultures. In the flat surface migration model siramesine caused tumor cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional Verinurad spheroid-brain slice culture model or in the mice xenograft model. Conclusions In conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal destabilizing drugs in killing glioblastoma cells, but siramesine was without effect in the organotypic spheroid-brain slice culture model and the in vivo xenograft model. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3162-3) contains supplementary material, which is available to authorized users. which accumulates in acidic cellular compartments, primarily in lysosomes resulting in staining in the glioma cell lines appeared as dot-like staining corresponding to the presence of intact lysosomes (0?M siramesine). Confocal imaging identified loss of red Verinurad fluorescence in the lysosomes upon siramesine exposure in all of the glioma cell lines after only 1 1?h of exposure to siramesine (5C30?M). This suggested that siramesine exposure lead to compromised/ruptured lysosomal membranes. Scalebar 100?m (a), Scalebar 50?m (e). Data are displayed as mean values??SEM, and *- overlap between DiO (green) and PI (red)). Results were confirmed by histology in Fig.?6. Control cells received culture medium or DMSO (images not shown) both without siramesine. Scalebar 600?m (a), Scalebar 100?m (cCd). Data are displayed as mean values??SEM, and **P?P?Verinurad in implanted spheroids where due Verinurad to limited diffusion of siramesine through the membrane into brain slice cultures, DiO labelled spheroids were placed directly on the membrane. A significant PI uptake in the spheroids was found confirming the diffusion potential of siramesine across the membrane (Additional file 4: Figure S4). Marker expression in siramesine exposed co-cultures Immunohistochemical staining with anti-human CD56 was used to identify the spheroids and the invasive cells upon implantation of T78 and T86 into the brain slice cultures (Fig.?6a). No differences in the tumor migration area or distance were found (Fig.?6b), however, a tendency towards a change in morphology from cells being elongated to being more rounded cells were seen in cultures exposed to 100?M siramesine (Fig.?6a and Additional file 5: Figure S5 shown for T78). When exposing the co-cultures to 100?M Siramesine, 5 out of 12 cultures implanted Rabbit Polyclonal to OR2B6 with T78 disintegrated upon paraffin embedding and for T86 this number was even higher loosing 10 out of 12 cultures. The surviving cultures were probably less affected by siramesine thus the pictures shown.