<p>Supplementary Figure S1: Optimization of the triple immunostaining on different cell lines with known status of cytokeratins, TF and vimentin expression. Supplementary Figure S2: TF expression and pro-coagulant activity of EMT inducible cell lines. Supplementary Figure S3: Impact of TF blocking antibody on coagulant activity. Supplementary Figure S4: Regulation of TF by EMT transcription factors. Supplementary Figure S5: Induction of TF after de novo expression of Snail in MDA-MB-468. Supplementary Figure S6: Validation of TF shRNA. Supplementary Figure S7: Presence of fibrin fibers around lung colonizing MDA-MB-231 cells as revealed by transmission electron microscopy.</p>
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Epithelial-mesenchymal transition (EMT) is prominent in circulating tumor cells (CTC), but how it influences metastatic spread in this setting is obscure. Insofar as blood provides a specific microenvironment for tumor cells, we explored a potential link between EMT and coagulation that may provide EMT-positive CTCs with enhanced colonizing properties. Here we report that EMT induces tissue factor (TF), a major cell-associated initiator of coagulation and related procoagulant properties in the blood. TF blockade by antibody or shRNA diminished the procoagulant activity of EMT-positive cells, confirming a functional role for TF in these processes. Silencing the EMT transcription factor ZEB1 inhibited both EMT-associated TF expression and coagulant activity, further strengthening the link between EMT and coagulation. Accordingly, EMT-positive cells exhibited a higher persistance/survival in the lungs of mice colonized after intravenous injection, a feature diminished by TF or ZEB1 silencing. In tumor cells with limited metastatic capability, enforcing expression of the EMT transcription factor Snail increased TF, coagulant properties, and early metastasis. Clinically, we identified a subpopulation of CTC expressing vimentin and TF in the blood of metastatic breast cancer patients consistent with our observations. Overall, our findings define a novel EMT-TF regulatory axis that triggers local activation of coagulation pathways to support metastatic colonization of EMT-positive CTCs. Cancer Res; 76(14); 1-13. (c)2016 AACR.
<p>Supplementary Figure S1: Optimization of the triple immunostaining on different cell lines with known status of cytokeratins, TF and vimentin expression. Supplementary Figure S2: TF expression and pro-coagulant activity of EMT inducible cell lines. Supplementary Figure S3: Impact of TF blocking antibody on coagulant activity. Supplementary Figure S4: Regulation of TF by EMT transcription factors. Supplementary Figure S5: Induction of TF after de novo expression of Snail in MDA-MB-468. Supplementary Figure S6: Validation of TF shRNA. Supplementary Figure S7: Presence of fibrin fibers around lung colonizing MDA-MB-231 cells as revealed by transmission electron microscopy.</p>
Abstract Background/Aims Patients with rheumatoid arthritis (RA) are at increased risk of developing osteoporosis (OP) and have a twofold increased risk of vertebral fracture compared with the general population. There is increasing evidence that teriparatide (TPTD) is superior to anti-resorptive medication in patients with severe spinal OP.The aim of this study was to compare the efficacy of TPTD with anti-resorptive treatment (ART) in RA patients with severe spinal OP. Methods Observational study of RA patients and controls with severe OP who were referred to a specialist osteoporosis clinic. Patients with a history of two vertebral fractures or a spinal BMD Tscore < -4 were offered either TPTD or standard care with either oral or parenteral ART. After completion of TPTD treatment patients were advised to commence ART. DEXA re-evaluation was usually performed after 1, 2 and 5 years. Results We studied 59 postmenopausal women with RA who had severe spinal OP. In the RA group 29 patients received TPTD treatment and 30 patients were started on ART (12 Zoledronic acid, 11 Alendronate, 3 Risedronate, 2 Denosumab, 1 Etidronate and 1 unknown).RA patients who were started on TPTD were on average 5 years younger (65.4 ±10.6) than patients who were started on ART (70.6±8.2; p = 0.041). Slightly more than half of TPTD RA patients (55.2%) had previously received bisphosphonates and 10.3% received low dose Prednisolone (mean± SD dose = 5.5 ± 3.3 mg). Baseline lumbar spine T-score was -4.25±0.57 in the RA TPTD group. Patients with RA who elected to have ART as opposed to TPTD had higher BMD values as compared with those who chose to have TPTD (T-score = -3.39±1.09; p = 0.001, from RA TPTD group).We found that increase in BMD with TPTD treatment was superior to ART in the RA group at increasing spine BMD after 2 years (+17.59% ± 9.63% vs 3.19 % ± 4.99% , p value<0.001). Assessment after 5 years following commencement of ART showed that spine BMD remained higher in the RA TPTD group than in the RA ART group alone (18.0% ± 11.6% vs 6.36 % ± 8.95%; p = 0.019). However, there was no significant difference between TPTD and ART on hip BMD change at 2 years (+ 3.6% ± 12.2 % vs -0.57% ± 5.96%, p = 0.237) or 5 years (+1.0% ± 10.9% vs -0.3% vs ± 7.8%, p = 0.724). Conclusion This real-world study confirms that TPTD treatment is more effective in treating severe spinal OP in RA patients than antiresorptive medication alone. Despite the fact that the majority of RA patients had been pre-treated with bisphosphonates the TPTD treatment effect in RA patients was robust. Anabolic treatment with TPTD is a good option for RA patients with severe spinal OP. Disclosure B. Hauser: Other; Dr Hauser has received fees for a promotional article from Gedeon Richter. K.M. Berg: None. J.A. Lambert: None. S.H. Ralston: Grants/research support; Prof Ralston has received grant funding from Lilly for an observational study and donation of Teriparatide for the TOPAZ trial in Osteogenesis Imperfecta.
Parkinson's disease is characterized by neurodegeneration of the dopaminergic neurons in the substantia nigra pars compacta. The 6-hydroxydopamine (6-OHDA) rat model has been used to study neurodegeneration in the nigro-striatal dopaminergic system. The goal of this study was to evaluate the reliability of diffusion MRI and resting-state functional MRI biomarkers in monitoring neurodegeneration in the 6-OHDA rat model assessed by quantitative histology. We performed a unilateral injection of 6-OHDA in the striatum of Sprague Dawley rats to produce retrograde degeneration of the dopamine neurons in the substantia nigra pars compacta. We carried out a longitudinal study with a multi-modal approach combining structural and functional MRI together with quantitative histological validation to follow the effects of the lesion. Functional and structural connectivity were assessed in the brain of 6-OHDA rats and sham rats (NaCl injection) at 3 and 6 weeks post-lesioning using resting-state functional MRI and diffusion-weighted. Our results showed (i) increased functional connectivity in ipsi- and contra-lesioned regions of the cortico-basal ganglia network pathway including the motor cortex, the globus pallidus, and the striatum regions at 3 weeks; (ii) increased fractional anisotropy (FA) in the ipsi- and contralateral striatum of the 6-OHDA group at 3 weeks, and increased axial diffusivity (AD) and mean diffusivity in the ipsilateral striatum at 6 weeks; (iii) a trend for increased FA in both substantia nigra of the 6-OHDA group at 3 weeks. Optical density measurements of tyrosine-hydroxylase (TH) staining of the striatum showed good correlations with the FA and AD measurements in the striatum. No correlations were found between the number of TH-stained dopaminergic neurons and MRI measurements in the substantia nigra. This study suggested that (i) FA and AD were reliable biomarkers to evaluate neurodegeneration in the cortico-basal ganglia network of the 6-OHDA model, (ii) diffusion MRI and resting-state functional MRI (rsfMRI) were not sensitive enough to detect changes in the substantia nigra in this model.
<p>Supplementary Table 1: list of siRNA used. Supplementary Table 2: list of primers used. Supplementary Table 3: list of antibodies used in different applications. Supplementary Table 4: list of patients and donors information</p>
