The increased adhesion of tumor cells to endothelial cells after irradiation can be reduced by FAK-inhibition.

BACKGROUND: Radiotherapy is administered in more than 60% of all solid tumors. Most patients are cured but a significant number develops local recurrences or distant metastases. The question arises if irradiation might influence the metastatic process. In the present study we examined whether the adhesion of glioblastoma or breast cancer cells to endothelial cells, an important step in metastasis, is affected by photon irradiation. METHODS: U-87 MG, U-373 MG and MDA-MB-231 cancer cells as well as primary human endothelial cells were irradiated with 0, 2, 4, or 8 Gy photons at a dose rate of 5 Gy/min. The adhesion of cancer cells to endothelial cells was tested either with the Vybrant based assay via fluorescent labelling or with an ibidi pump system able to mimic the physiological blood flow in vitro. In addition, the impact of FAK (focal adhesion kinase) inhibitor PF-573, 228 on the adhesion of non-irradiated and irradiated tumor cells was analyzed. Adhesion related and regulated proteins were analyzed by Western blotting. RESULTS: The cellular adhesion was increased after irradiation regardless of which cell type was irradiated. The FAK-inhibitor was able to reduce the adhesion of non-irradiated cells but also the irradiation-induced increase in adhesion of tumor cells to endothelium. Adhesion related proteins were enhanced after irradiation with 4 Gy or 8 Gy in both cells types. The increased adhesion after irradiation is accompanied by the phosphorylation of src (Y416), FAK (Y397) and increased expression of paxillin. CONCLUSION: Irradiation with photons in therapeutic doses is able to enhance the interaction between tumor cells and endothelial cells and by that might influence important steps of the metastatic process.

Robo1 and vimentin regulate radiation-induced motility of human glioblastoma cells.

Glioblastoma is a primary brain tumor with a poor prognosis despite of many treatment regimens. Radiotherapy significantly prolongs patient survival and remains the most common treatment. Slit2 and Robo1 are evolutionarily conserved proteins involved in axon guidance, migration, and branching of neuronal cells. New studies have shown that Slit2 and Robo1 could play important roles in leukocyte chemotaxis and glioblastoma cell migration. Therefore, we investigated whether the Slit2/Robo1 complex has an impact on the motility of glioblastoma cells and whether irradiation with therapeutic doses modulates this effect. Our results indicate that photon irradiation increases the migration of glioblastoma cells in vitro. qPCR and immunoblotting experiments in two different glioblastoma cell lines (U-373 MG and U-87 MG) with different malignancy revealed that both Slit2 and Robo1 are significantly lower expressed in the cell populations with the highest motility and that the expression was reduced after irradiation. Overexpression of Robo1 significantly decreased the motility of glioblastoma cells and inhibited the accelerated migration of wild-type cells after irradiation. Immunoblotting analysis of migration-associated proteins (fascin and focal adhesion kinase) and of the epithelial-mesenchymal-transition-related protein vimentin showed that irradiation affected the migration of glioblastoma cells by increasing vimentin expression, which can be reversed by the overexpression of Slit2 and Robo1. Our findings suggest that Robo1 expression might counteract migration and also radiation-induced migration of glioblastoma cells, a process that might be connected to mesenchymal-epithelial transition.

Automated Multichamber Time-lapse Videography for Long-term In Vivo Observation of Migrating Cells.

AIM: To observe and document the migration of living cells by time-lapse videography, we constructed a low-budget system based on a common inverted microscope. MATERIALS AND METHODS: Long-term observation of six-well plates is enabled through maintenance of cell culture conditions (5% CO2 in air at 37°C). Points of interest can be revisited in definable intervals with <1 µm repositioning error. Digital photographs from each programmed time point are paired with environmental data and combined into a record. RESULTS: We used this new chamber to observe the migration of various cell lines. The design represents a good compromise between low cost and good precision. Detailed analyses verified that the environmental conditions were appropriately maintained, enabling long-term observation of viable cells. The stimulating influence of irradiation with photons (radiotherapy) on cellular motility of glioblastoma cells is presented. CONCLUSION: This study demonstrates that useful videographic systems can be constructed at low cost.

The therapeutic effect of photon irradiation on viable glioblastoma cells is reinforced by hyperbaric oxygen.

BACKGROUND: Hyperbaric oxygen (HBO) seems to intensify the effect of ionising radiation. We investigated whether HBO combined with irradiation decreases the capability of U251 glioblastoma cells for relapse and metastasis. MATERIALS AND METHODS: Cells were treated with O2 at 1.3 bar and then irradiated with 2 Gy photons. Clonogenic survival was tested with colony formation. Motility is an important feature of metastasis and was measured with time-lapse videography. RESULTS: The clonogenic survival diminished by 22% through HBO, by 49% through irradiation, and by 70% through the combination of both. The accumulated distance travelled by cells fell by 3% with HBO, rose by 17% with irradiation, but was reduced by 11% with their combination. The respective values for the Euclidean distance travelled were +8%, +47% and -14%. Compared to normoxic irradiation, additional HBO lowered travel by 41%. CONCLUSION: HBO strengthens the effect of irradiation on clonogenic survival and reverses radiation-induced increase in the mobility of cells.