Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin.

BACKGROUND: We analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers. METHODS: Primary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA. RESULTS: Ionizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF. CONCLUSIONS: Our results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.

The Triple-negative Breast Cancer Cell Line MDA-MB 231 Is Specifically Inhibited by the Ionophore Salinomycin.

BACKGROUND/AIM: Tumour cells of the profile CD44+/CD24low/- have a high tumorigenic potential. Salinomycin can specifically inhibit the growth of these cells. Herein, we investigated the effects of salinomycin on the viability and migration of triple negative breast cancer cells. MATERIALS AND METHODS: We analysed two cell lines: i) triple-negative MDA-MB 231 breast cancer cells and ii) a cytokeratin 18-transfected, re-differentiated subclone of the MDA-MB 231 cell line. The viability was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test, and the migration was determined using 24-h videography. The expression of oestrogen receptor was determined using immunohistochemistry. RESULTS: Salinomycin reduces all migration parameters in MDA-MB 231 cells. A significant correlation was found between increasing salinomycin concentrations and loss of cell viability, which was significantly less noticeable in the transfected control cells. CONCLUSION: With salinomycin there is a specific inhibition of MDA-MB 231 cells. Since MDA-MB 231 has over 90% cells with the profile CD44+/CD24low/-, these might represent a possible point of attack for salinomycin.

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.

Influence of vascular endothelial growth factor and radiation on gap junctional intercellular communication in glioblastoma multiforme cell lines.

Glioblastoma multiforme (GBM) is a highly aggressive glial brain tumor with an unfavorable prognosis despite all current therapies including surgery, radiation and chemotherapy. One characteristic of this tumor is a strong synthesis of vascular endothelial growth factor (VEGF), an angiogenesis factor, followed by pronounced vascularization. VEGF became a target in the treatment of GBM, for example with bevacizumab or the tyrosine kinase inhibitor axitinib, which blocks VEGF receptors. To improve patients' prognosis, new targets in the treatment of GBM are under investigations. The role of gap junctions in GBM remains unknown, but some experimental therapies affect these intercellular channels to treat the tumor. Gap junctions are composed of connexins to allow the transport of small molecules between adjacent cells through gap junctional intercellular communication (GJIC). Based on data derived from astrocytes in former studies, which show that VEGF is able to enhance GJIC, the current study analyzed the effects of VEGF, radiation therapy and VEGF receptor blockade by axitinib on GJIC in human GBM cell lines U-87 and U-251. While VEGF is able to induce GJIC in U-251 cells but not in U-87 cells, radiation enhances GJIC in both cell lines. VEGF receptor blockade by axitinib diminishes radiation induced effects in U-251 partially, while increases GJIC in U-87 cells. Our data indicate that VEGF and radiation are both modifying components of GJIC in pathologic brain tumor tissue.

Vascular Endothelial Growth Factor, Irradiation, and Axitinib Have Diverse Effects on Motility and Proliferation of Glioblastoma Multiforme Cells.

Glioblastoma multiforme (GBM) is the most common primary brain tumor. It is highly aggressive with an unfavorable prognosis for the patients despite therapies including surgery, irradiation, and chemotherapy. One important characteristic of highly vascularized GBM is the strong expression of vascular endothelial growth factor (VEGF). VEGF has become a new target in the treatment of GBM, and targeted therapies such as the VEGF-receptor blocker axitinib are in clinical trials. Most studies focus on VEGF-induced angiogenesis, but only very few investigations analyze autocrine or paracrine effects of VEGF on the tumor cells. In this study, we examined the impact of VEGF, irradiation, and axitinib on cell proliferation and cell motility in human GBM cell lines U-251 and U-373. VEGF receptor 2 was shown to be expressed within both cell lines by using PCR and immunochemistry. Moreover, we performed 24-h videography to analyze motility, and a viability assay for cell proliferation. We observed increasing effects of VEGF and irradiation on cell motility in both cell lines, as well as strong inhibiting effects on cellular motility by VEGF-receptor blockade using axitinib. Moreover, axitinib diminished irradiation induced accelerating effects. While VEGF stimulation or irradiation did not affect cell proliferation, axitinib significantly decreased cell proliferation in both cell lines. Therefore, the impairment of VEGF signaling might have a crucial role in the treatment of GBM.

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.

Zoledronic Acid Inhibits the Motility of Cancer Stem-like Cells from the Human Breast Cancer Cell Line MDA-MB 231.

BACKGROUND/AIM: Various effects on tumor cells have been described for zoledronic acid (ZOL). However, only limited data exist regarding its influence on the motility of tumor cells. Since migration of tumor stem cells is a decisive step in metastasis, we examined whether ZOL reduces their motility. MATERIALS AND METHODS: We investigated the effects of ZOL on stem-like progenitor cells obtained via the formation of spheroids from the human breast cancer cell line MDA-MB 231. Stem cell properties were verified by measurement of high CD44 expression and absence of CD24 expression. Motility was explored by time-resolved videography, protein expression by western blotting. RESULTS: ZOL strongly reduced the migration of stem-like progenitor cells. Cellular velocity was reduced by 61% following exposure to 1 µM ZOL and by 82% after exposure to 10 µM ZOL. Accumulated distance traveled by the cells was reduced by 60% and 79% after exposure to 1 µM and 10 µM ZOL, respectively. The remaining cellular motility led to very little change in distance, with cellular activity appearing more as "stepping on the spot". The reduced motility might be due to reduced phosphorylation of focal adhesion kinase (FAK), an important enzyme in cellular migration. CONCLUSION: ZOL reduces the motility and cellular velocity of breast cancer cells in vitro. The reduced mobility might slow down or even stop metastasis, which is a known result of adjuvant ZOL therapy in breast cancer patients. In vivo studies are warranted to evaluate the impact of the reduced motility on the metastatic cascade.

Survival and Symptom Relief after Palliative Radiotherapy for Esophageal Cancer.

PURPOSE: The aim of this study was to assess the 6-months dysphagia-free survival, improvement in swallowing function, complication rate, and overall survival in patients with incurable esophageal cancer treated with palliative radiotherapy. METHODS: We retrospectively reviewed data from 139 patients (median age 72 years) with advanced/recurrent incurable esophageal cancer, who were referred to 3 German radiation oncology centers for palliative radiotherapy between 1994 and 2014. Radiotherapy consisted of external beam radiotherapy (EBRT) with 30 - 40.5 Gy/2.5 - 3 Gy per fraction, brachytherapy alone (BT) with 15 - 25 Gy/5 - 7Gy per fraction/weekly and EBRT + BT (30 - 40.5 Gy plus 10 - 14 Gy with BT) in 65, 46, and 28 patients, respectively. Dysphagia-free survival (Dy-PFS) was defined as the time to worsening of dysphagia for at least one point, a new loco-regional failure or death of any cause. RESULTS: Median follow-up time was 6 months (range 1-6 months). Subjective symptom relief was achieved in 72 % of patients with median response duration of 5 months. The 1-year survival rate was 30%. The 6-months Dy-PFS time for the whole group was 73 ± 4%. The 6-months Dy-PFS was 90 ± 4% after EBRT, 92 ± 5% after EBRT + BT and 37 ± 7% after BT, respectively (p<0.001). Five patients lived for more than 2 years, all of them were treated with EBRT ± BT. Ulceration, fistula and stricture developed in 3, 6 and 7 patients, respectively. CONCLUSIONS: Radiotherapy leads to symptom improvement in the majority of patients with advanced incurable esophageal cancer. The present results favor EBRT ± BT over BT alone. Due to the retrospective nature of this study, imbalances in baseline characteristics might have contributed to this finding, and further trials appear necessary.

Treatment Results of MammoSite Catheter in Combination with Whole-breast Irradiation.

AIM: To report the initial outcomes of patients treated with the MammoSite brachytherapy device (MSBT) as a boost followed by external whole-breast irradiation (WBI). PATIENTS AND METHODS: From June 2011 to March 2014, 107 patients (typically with pT1-2, pN0-1, M0 disease) were treated with breast-conserving therapy and adjuvant radiotherapy with MSBT (15 Gy in 2.5-Gy fractions) followed by WBI (median=50.4 Gy). Toxicity was classified according to the Common Terminology Criteria for Adverse Events v3.0. The median follow-up was 21 months. RESULTS: To date, no ipsilateral breast-tumor recurrences have been observed; 102 patients (95%) were alive at last follow-up. Two patients (2%) developed distant metastases. Five patients (5%) died during follow-up, only one as a result of breast cancer. The 2-year disease-free survival was 95±3%. The incidence of asymptomatic and symptomatic seroma in 90 days after MSBT was 28% and 10%, respectively. Infectious mastitis was observed in three patients (3%), who were treated successfully with antibiotics. Only three patients (3%) developed RT-induced dermatitis greater than grade 2 after WBI. CONCLUSION: The boost technique used in this study seems to provide excellent local control with acceptable toxicity, similar to the results observed with other forms of interstitial accelerated partial-breast irradiation as a boost. Long-term follow-up is necessary to refine the patient selection criteria and to assess efficacy and late toxicities.

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.

Generation of neuronal progenitor cells in response to tumors in the human brain.

Data from transgenic mouse models show that neuronal progenitor cells (NPCs) migrate toward experimental brain tumors and modulate the course of pathology. However, the pathways whereby NPCs are attracted to CNS neoplasms are not fully understood and it is unexplored if NPCs migrate toward brain tumors (high-grade astrocytomas) in humans. We analyzed the tumor-parenchyma interface of neurosurgical resections for the presence of (NPCs) and distinguished these physiological cells from the tumor mass. We observed that polysialic acid neural cell adhesion molecule-positive NPCs accumulate at the border of high-grade astrocytomas and display a marker profile consistent with immature migratory NPCs. Importantly, these high-grade astrocytoma-associated NPCs did not carry genetic aberrations that are indicative of the tumor. Additionally, we observed NPCs accumulating in CNS metastases. These metastatic tumors are distinguished from neural cells by defined sets of markers. Transplanting murine glioma cells embedded in a cell-impermeable hollow fiber capsule into the brains of nestin-gfp reporter mice showed that diffusible factors are sufficient to induce a neurogenic reaction. In vitro, vascular endothelial growth factor (VEGF) secreted from glioma cells increases the migratory and proliferative behavior of adult human brain-derived neural stem and progenitor cells via stimulation of VEGF receptor-2 (VEGFR-2). In vivo, inhibiting VEGFR-2 signaling with a function-blocking antibody led to a reduction in NPC migration toward tumors. Overall, our data reveal a mechanism by which NPCs are attracted to CNS tumors and suggest that NPCs accumulate in human high-grade astrocytomas.

Epidermal growth factor receptor changes during breast cancer metastasis.

BACKGROUND: Since the anti-HER2 monoclonal antibody trastuzumab made its triumphant advance into breast cancer therapy, new selective agents, including pan-HER inhibitors are entering clinical practice. MATERIALS AND METHODS: This study investigates the expression of the four HER-family members, HER1-4, in 48 primary breast carcinomas (PBC) and corresponding distant metastases (CDM) by immunohistochemistry and fluorescence in situ hybridisation. RESULTS: Concordance rate between PBC and CDM was 79% for HERI and HER2, 67% for HER3 and 56% or HER4. Expression of HER1-3 was associated with poor prognosis compared to HER-negative disease (p = 0.036). HER4 overexpression was associated with a better outcome (p = 0.003). CONCLUSION: Though most tumours demonstrate a stable HER expression pattern, both loss and acquisition of HER receptor overexpression can occur during metastasis. HER4 overexpression predicts prolonged survival compared to receptor negative disease, while the opposite is true for HER1-3. Consequences for modem antibody therapy are discussed.

Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo.

This study shows that high keratin 18 (K18) expression in tumor cells is associated with reduced invasiveness in vitro and lack of tumorigenicity in nude mice. We previously showed that high K18 expression correlated with a good prognosis and that reducing K18 expression increased the aggressiveness of established breast cancer cell lines. To confirm these observations, we transfected the human K18 gene into the human breast cancer cell line MDA-MB-231 and isolated a stable overexpressing clone. The forced K18 expression was associated with a complete loss of the previously strong vimentin expression in the parent cell line, induction of the K18 dimerization partner K8, and up-regulation of adhesion proteins. These changes were accompanied by a dramatic reduction in the aggressiveness of the K18 transfectants in vitro and in vivo. We conclude that forced reexpression of K18 causes at least partial redifferentiation of the tumor cell, followed by a corresponding regression of malignant phenotype.