Edited by Alex Toker The triple-negative phenotype is the most prevalent form of human breast cancer worldwide and is characterized by poor survival, high aggressiveness, and recurrence. Microvesicles (MV) are shredded plasma membrane components and critically mediate cell-cell communication, but can also induce cancer proliferation and metastasis. Previous studies have revealed that protease-activated receptor 2 (PAR2) contributes significantly to human triple-negative breast cancer (TNBC) progression by releasing nano-size MV and promoting cell proliferation, migration, and invasion. MV isolated from highly aggressive human TNBC cells impart metastatic potential to nonmetastatic cells. Over-expression of microRNA221 (miR221) has also been reported to enhance the metastatic potential of human TNBC, but miR221's relationship to PAR2-induced MV is unclear. Here, using isolated MV, immunoblotting, quantitative RT-PCR, FACS analysis, and enzymatic assays, we show that miR221 is translocated via human TNBC-derived MV, which upon fusion with recipient cells, enhance their proliferation, survival, and metastasis both in vitro and in vivo by inducing the epithelial-to-mesenchymal transition (EMT). Administration of anti-miR221 significantly impaired MV-induced expression of the mesenchymal markers Snail, Slug, N-cadherin, and vimentin in the recipient cells, whereas restoring expression of the epithelial marker E-cadherin. We also demonstrate that MV-associated miR221 targets phosphatase and tensin homolog (PTEN) in the recipient cells, followed by AKT Ser/Thr kinase (AKT)/NF-B activation, which promotes EMT. Moreover, elevated miR221 levels in MV derived from human TNBC patients' blood could induce cell proliferation and metastasis in recipient cells. In summary, miR221 transfer from TNBC cells via PAR2-derived MV induces EMT and enhances the malignant potential of recipient cells. This work was supported in part by Department of Science and Technology, Government of India DST SR/SO/BB-0125/2012 for project grants and a fellowship from the Council of Scientific and Industrial Research, India. The authors declare that they have no conflicts of interest with the contents of this article. This article contains Figs. S1-S14 and Tables S1-S3.

Apart from blood coagulation, coagulation proteases are involved inextricably in cancer progression/propagation via intra/inter-cellular signaling, mediated predominantly by protease-activated receptors (PARs). Microvesicles (MVs), a plasma membrane shredded component, has recently been identified as an important contributor to human breast cancer metastasis. However, the role of PAR2 in promoting MVs generation from breast cancer cells remains largely unexplored. The objective of this study is to investigate whether coagulation protease-mediated human breast cancer propagation commences via MVs and also to decipher the underlying signaling mechanism. Here, we elicited that coagulation factor-FVIIa and Trypsin activates PAR2, which governs MVs shedding from MDAMB231 cells by altering actomyosin dynamics. Treatment of cells with PAR2 activators facilitate MVs generation by activating three independent (MAPK, P38, and Rho) signaling cascades. MAPK, signals through activating MLCK followed by MLC phosphorylation to alter myosin organization whereas, P38 reorganizes actin dynamics by the sequential activation of MK2 and HSP27. RhoA-dependent ROCK-II activation again contributes to remodeling myosin II activity. Further, both our in vitro and in vivo analyses showed that these MVs potentiate invasive and migratory property to the recipient cells. Breast cancer patients blood show an elevation of TF-bearing, pro-metastatic MVs than normal. These findings give an insight into the detailed signaling mechanism involved in the production of MVs with transforming ability from PAR2-activated human breast cancer cells. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in MVs-associated human breast cancer metastasis.

Perylene bisimides (PBIs) with high quantum yields and high chemical and photophysical stabilities are usually suffering from poor solubility in various solvents, including water, which restricts their use in biomedical and other applications. Thus, in this study, as-synthesized hydrophilic poly(1-vinylimidazole) (PVim) is introduced at both the imide positions of the hydrophobic PBI unit by using L-cysteine (Cys) bearing two orthogonally reactive groups to produce a water-and organicsoluble PBI-based polymer conjugate. To do so, first, L-cysteine is used for thiol-mediated radical polymerization of 1-vinylimidazole (Vim). In the next step, the cysteine-end-capped poly(1-vinylimidazole) (Cys−PVim) with free −NH 2 is coupled with perylene-3,4,9,10-tetracarboxylic dianhydride (PDA) by employing a onestep microwave-assisted reaction to produce PBI−(Cys−PVim) 2 conjugate. The solution optical properties of this conjugate are thoroughly investigated to ascertain the extent of aggregation among PBIs units in both aqueous and organic media. The aqueous PBI−(Cys−PVim) 2 solution emits characteristic green fluorescence of PBI under UV irradiation of 365 nm wavelength. Owing to the presence of a protonable imidazole moiety, the PBI−(Cys−PVim) 2 conjugate shows pH-dependent optical properties. The amphiphilic PBI−(Cys−PVim) 2 molecules undergo self-assembly into vesicular nanostructures in water as confirmed from cryo-and high-resolution-transmission electron microscopy. The conjugate binds with ctDNA and plasmid DNA in water to form polyplexes. The fluorescent PBI−(Cys−PVim) 2 conjugate with low cytotoxicity and high quantum yield is efficiently used for the imaging of HeLa cells. The cellular uptake of the conjugate is studied at different time intervals and at different pHs using fluorescence microscopy.

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