Background Low-dose photon irradiation has repeatedly been suspected to increase a
Background Low-dose photon irradiation has repeatedly been suspected to increase a risk of promoting local recurrence of disease or even systemic dissemination. co-administration of photon irradiation and the CXCR4-antagonist AMD3100 or the use of carbon ions instead Ezetimibe of photons may be possible solutions to reduce the risk of locoregional tumor recurrence after radiotherapy for MPM. study, which may not accurately reflect the situation. Moreover, the effect of photon irradiation on the microenvironment surrounding the tumor should not be ignored. In order to confirm Ezetimibe this mechanism and to address the contribution of microenvironmental modifications through radiation, radiation experiments in animal models with human MPM xenografts would be a promising approach for a subsequent corroborating study . As another limitation, SDF-1/CXCR4 signaling is not the only pathway to promote tumor cell migration. Other pathways may contribute simultaneously, and the SDF-1/CXCR4 pathway may work indirectly through further up- and downstream signaling pathways, such as the interaction of other chemokines and chemokine receptors. In this study, we only examined the SDF-1/CXCR4 pathway and the possible role of integrins, as they have repeatedly CDKN1B been identified to be responsible for radiation-increased cell motility and as there is a pharmacological agent, FDA-approved AMD3100, that can be administered to reverse the observed events. In order to exclude the potential contribution of further pathways, comprehensive analysis, for example through DNA microarray, RNA-ChiP, or Proteomic analysis, would be required. In the present manuscript, we decided to not perform such experiments, as the detected phenotype was successfully inhibited by adding AMD3100. In conclusion, low doses of photon irradiation promoted MPM cell migration through the increased expression of CXCR4 with subsequently increased SDF-1/CXCR4 signaling. Clinically, this might enhance the risk of tumor cell spread and infiltration and, therefore, explain prior disappointing results from clinical trials investigating photon radiotherapy in MPM patients. The administration of the CXCR4 antagonist AMD3100 effectively inhibited this increased migration. As an alternative to photon irradiation, carbon ion irradiation did not significantly promote migration. Therefore, our finding suggest that the co-administration of the clinically already available CXCR4 antagonist AMD3100 concurrently to photon irradiation or, alternatively, the replacement of photon irradiation with carbon ion irradiation may be two possible solutions to establish and enhance the clinical benefit of radiation treatments in MPM patients. MATERIALS AND METHODS Reagents and cell lines H28 and H226 mesothelioma cells were purchased from ATCC Ezetimibe Ezetimibe and maintained at 37C and 5% CO2 in RPMI 1640 medium supplemented with 1% Penicillin/Streptomycin and 10% fetal bovine serum (FBS; Biochrom, Berlin, Germany). Twenty-four hours before the migration assay, cells were serum starved in RPMI 1640 medium containing 1% Penicillin/Streptomycin and 0.5% FBS. Cell passaging was performed every week. Stromal cell-derived factor 1 (SDF-1) was purchased from Gibco (Eggenstein, Germany). The CXC chemokine receptor 4 (CXCR4) antagonist AMD3100 was purchased from Sigma Aldrich (Munich, Germany). To block CXCR4, cells were exposed to AMD3100 at a concentration of 25g/ml 24 hours before the migration assay. For FACS analysis, PE-labeled anti-human CXCR4 antibody (555974), PE-labeled anti-human 1 antibody (556049), PE-labeled anti-human 3 antibody (556025), PE-labeled anti-human 5 antibody (555617), and isotope controls corresponding to these antibodies were purchased from BD Bioscience (Heidelberg, Germany). For Western blot analysis, anti- CXCR4 antibody (clone 12G5), and anti- -actin (clone BA3R) were purchased from Thermo Fisher Scientific (Darmstadt, Germany). Anti- Na+-K+ ATPase (clone EP1845Y) was purchased from Abcam (Cambridge, UK). Migration assays using membranes coated with extracellular matrix proteins For migration assays, polycarbonate membranes with 8-m pores were coated with 0.5 g/cm2 Collagen I (Corning, Bodenheim, Germany) and 0.5 g/cm2 collagen IV (Corning, Bodenheim, Germany) and stored overnight at.