Supplementary MaterialsSupplementary Information

Supplementary MaterialsSupplementary Information. reversely downregulated in PCa. Interestingly, ectopic expression of miR-296-3p in P69 increases the tolerance to NK cells whereas knockdown of miR-296-3p in M12 reduces the resistance to NK cells, which both phenotypes can be rescued by re-expression or silencing of ICAM-1 in P69 and M12, respectively. These results are also manifested by the decrease in the incidence of pulmonary tumour metastasis exhibited by knockdown of miR-296-3p in M12 when injected into athymic nude mice via tail vein, and consistently down-expression of ICAM-1 reverses this to increase extravasation of CTCs into lungs. Above results suggest that this newly recognized miR-296-3p-ICAM-1 axis has a pivotal role in mediating PCa metastasis by possible enhancing survival of NK cell-resistant CTC. Our findings provide novel potential targets for PCa therapy and prognosis. by escaping from NK cell lysis remains unclear. In this study, we try to solution above questions and to explore why the metastatic potential of PCa is usually associated with their susceptibility to destruction of NK cells.7 We identify a new Angiotensin (1-7) miRNA-296-3p-ICAM-1 axis has crucial functions in avoidance of CTC destruction by NK cells, thereby enhancing CTC survival and concomitantly promoting PCa metastatic extravasation. Results Characterization of human PCa cell lines P69 and M12 P69 is an immortalized, low-tumourigenic, non-metastatic prostate epithelial cell collection,14 whereas highly tumourigenic and metastatic M12 is derived Rabbit Polyclonal to Histone H2A from P69 and mainly contains a deletion of 19q13.1– 19pter.15 We first used the xCELLigence RTCA-DP System real-time monitoring the migration curves of P69 and M12. The impedance increase correlates to increasing numbers of migrated cells.16, 17 P69 displayed a flat collection in cell index of migration; in contrast, M12 exhibited a strong migration curve tending to upward in 24?h (Physique 1a). This suggests that P69 has a very low motility capacity while M12 endows with the high motility ability. Open in a separate window Physique Angiotensin (1-7) 1 Morphological and metastatic differences between P69 and Angiotensin (1-7) M12. (a) Migration kinetics of P69 and M12, as shown by real-time monitoring of live cell migration (P69-reddish, M12-green). (b) Light microscopy images of P69 and M12 were taken from cultures produced in 3D culture matrix. Magnification, 20. (c) Immunofluorescence staining of P69 and M12 produced in 3D Culture Matrix (Vimentin-red, E-cadherin-green, DAPI-blue). (d) The expression levels of E-cadherin in P69 (green collection) and M12 (blue collection) were detected by circulation cytometry. IgG isotype antibody was used as a negative control Consistent Angiotensin (1-7) with above, 3D culture assays displayed morphologic changes that defined different tumourigenic and metastatic characteristics of these two cell lines. P69 produced acini morphology whereas M12 displayed a highly disorganized mass of cells and star-like morphology (Physique 1b). The loss of E-cadherin is usually a hallmark of epithelialCmesenchymal transition (EMT) and coincides with the transition from well-differentiated adenoma to invasive carcinoma.18 Thus, immunostaining for the mesenchymal marker Vimentin and the epithelial marker E-cadherin was conducted to observe the 3D culture morphologic structures. P69 displayed almost no expression of Vimentin but abundant E-cadherin; conversely, M12 showed high Vimentin but loss of E-cadherin (Physique 1c). This was confirmed by circulation cytometric analysis (Physique 1d). Collectively, these results indicate that these two cell lines are very different in metastatic potential and can be used for the following studies. P69 is usually more sensitive to expanded as explained previously.19, 20 We examined the expression levels of receptors on these NK cells showing a highly activated NK cell receptor expression pattern, which was characterized by high expressions of NKG2D and CD226 (DNAM-1), and moderate expressions of natural cytotoxicity receptors and low expressions of inhibitory receptors (Supplementary Figure S1). To verify whether there is different immune response between P69 and M12, we performed calcein acetyoxymethyl ester (calcein-AM) cytotoxicity assays to evaluate the activities of and CD56. The gated on CD56-posivtive cells were analyzed and the figures in boxes indicated percentage of CD107(IFN-(TNF-(a subunit of LFA-1) mAbs to block function of LFA-1 or Angiotensin (1-7) isotype-matched IgG as a control for 30?min and then mixed with P69 or M12 at an E/T ratio of 5C1 for calcein release assay. Data are the meanS.E.M. of three impartial experiments Binding of LFA-1 to ICAM-1 on target cell initiates activation signals for NK cell cytotoxicity, contributing to strong adhesion to target cell and polarization of cytolytic granules in NK cells.8, 22 To verify contribution of LFA-1/ICAM-1 to the differential NK cell cytotoxicities between P69 and M12, we knocked down ICAM-1 by shRNA-based lentiviral system in.