16 m beads: p = 0.0012, TRAMP-C2-luc vs. to the microvasculature of different organs. Their dissemination was compared to biologically passive microbeads. The spine and additional organs were resected three hours after intraarterial injection of tumor cells or microbeads. Ex lover vivo homogenization and fluorescence analysis allowed quantification of tumor cells or microbeads in different organs. Interestingly, tumor cell distribution to the spinal bone was comparable to dissemination of microbeads independent of the tumor cell type (melanoma: 5.646% 7.614%, lung: 6.007% 1.785%, prostate: 3.469% 0.602%, 7 m beads: 9.884% 7.379%, 16 m beads: 7.23% 1.488%). Tumor cell seeding differed significantly between tumor cells and microbeads in Rabbit polyclonal to Smac all smooth cells organs. Moreover, there were significant differences between the different tumor cell lines in their dissemination behaviour to soft cells organs only. These findings demonstrate that metastatic dissemination of tumor cells to spinal bone and SB 239063 additional osseous organs is definitely mediated by passive entrapment of tumor cells much like passive plugging of microvasculature observed after intraarterial microbeads injection. Introduction Increasing incidence of spinal bone metastasis leading to epidural spinal cord compression and devastating neurological deficits is becoming a major medical challenge for neurooncological individuals [1]. Despite improvements in metastasis study, the development of spinal bone metastasis represents a prognosis limiting manifestation of the underlying oncological disease [2]. Currently, we are still challenged to develop strategies to suppress spinal bone metastasis [2]. Therefore, it is crucial to understand the underlying biological principles. In SB 239063 terms of metastasis formation, the key methods tumor cell intravasation, tumor-cell endothelial-cell connection, extravasation and SB 239063 subsequent metastasis formation have been explained (seed and dirt hypothesis). Tumor cell surface markers and SB 239063 organ specific surface / growth factors actively mediate tumor cell endothelial cell relationships in order to prepare the extravasation process [3]. However, passive entrapment of tumor cells in microvessels (microemboli) is also involved in the seeding process [4]. Up to today it remains unknown to what degree passive entrapment or active homing mechanisms contribute to spinal metastasis. In order to address this problem we targeted to compare metastatic seeding of tumor cells in the spinal bone to the perfusion-dependent dissemination pattern of biologically inert microparticles after intraarterial injection [5C7]. Material and Methods Cell collection cultivation B16-F1 (ATCC? CRL-6323?) and LLC1 (ATCC? CRL-1642?) cells were routinely managed at 37C with 5% CO2 in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FCS, 50 devices / ml penicillin and 50 g/ml streptomycin. For B16-luc and LLC1-luc cells, infected with FFLUC-eGFP-Puro vector construct explained previously [8], the medium was supplemented 5 g / ml puromycin. TRAMP-C2 (ATCC? CRL-2731?) cells were routinely managed in DMEM with 4 mM L-glutamine modified to contain 1.5 g/L sodium bicarbonate and 4.5 g/L glucose supplemented with 0.005 mg/ml bovine insulin and 10 nM dehydroisoandrosterone, 90%; fetal bovine serum, 5%; Nu-Serum IV, 5%. TRAMP-C2-luc medium was supplemented with 5 g / ml puromycin. MTT-Viability assay MTT assay has been explained previously [9]. In brief, cells were plated to 96 well plates in different densities (2500, 5000, 10000 cells / well). After 24 h medium was changed and MTT reagent was added to the medium. The cells were incubated with MTT for 4 h. Supernatant was softly discarded and cells were lysed in 100 l isopropanol / DMSO (1:1). A Tecan 200M spectrometer (Tecan, M?nnedorf, Switzerland) was used to measure absorbance at 560 nm. Correction for protein precipitate interference was conducted having a 630 nm reading research. Cell migration Scuff assay was performed to measure migration [10]. Cells were seeded with 200000 cells / well in 6 well plates and a scuff was performed 12 h post seeding having a 2 ml pipet tip (Falcon, # 13-675-16). Photos of the same area were taken 24 hours post scuff. SB 239063 Cell protection was measured in % of total area photographed. Analysis was performed instantly using CellProfiler 2.1 [11]. Retrograde carotid artery injection This study was carried out in stringent accordance with.