Extracellular shed vesicles including exosomes and microvesicles are disseminated throughout the body and represent an important conduit of cell communication. populace and that extracellular shed vesicle size provides information about provenance and cargo. We have designed and implemented a novel microfluidic technology that separates microvesicles as a function of diameter from heterogeneous populations of cancer-cell-derived extracellular shed vesicles. We measured cargo carried by the microvesicle subpopulation processed through this microfluidic platform. Such analyses could enable future investigations to more Rabbit Polyclonal to STK17B. accurately and reliably determine provenance functional activity and mechanisms of transformation in cancer. and cancer-cell-derived to refer to ESVs contained within multivesicular bodies (MVBs) that are trafficked to SNS-032 (BMS-387032) the cell surface and released via fusion of MVBs with the cell membrane. Exosomes are thought to be generated by both normal and cancerous cells (Johnstone et al. 1987; Peinado et al. 2011). We use the term to refer to ESVs that bud from cancer cell surfaces (D’Souza-Schorey and Clancy 2012; Antonyak et al. 2011; Lee et al. 2011). ESVs represent an important conduit of cell communication (Keller et al. 2006; Peinado et al. 2011; van Niel et al. 2006) and have potential as a disease state biomarker (Burgess 2013; Skog et al. 2008; Wang et SNS-032 (BMS-387032) al. 2013; D’Souza-Schorey and Clancy 2012; Nilsson et al. 2009). ESVs contain membrane-associated cytosolic and nuclear molecules including specifically packaged signaling proteins enzymes miRNAs and RNA transcripts (Grange et al. 2011; Skog et al. 2008; Mathivanan and Simpson 2009; Cocucci et al. 2009; Antonyak et al. 2012; Lee et al. 2011; Li et al. 2012; Al-Nedawi et al. 2008; Al-Nedawi et al. 2009; Di Vizio et al. 2012). Recipient cells upon ESV uptake can experience a change in their behavior and function (Keller et al. 2006; Peinado et al. 2011; van Niel et al. 2006) due to cargoes in the ESVs. ESVs play a role in many systems including immune responses (Kim et al. 2006; van Niel et al. 2006; Valenti et al. 2007) reproduction (Mincheva-Nilsson and Baranov 2010; Dragovic et al. 2011) computer virus proliferation (Gy?rgy et al. 2011; van der Pol et al. 2012; Schorey and Bhatnagar 2008) and cancer progression (Muralidharan-Chari et al. 2010; D’Souza-Schorey and Clancy 2012; Peinado et al. 2011). Cancer-cell-derived ESVs represent a heterogenous populace that exhibits a large range of sizes with unique subpopulations (Antonyak et al. 2011; Muralidharan-Chari et al. 2010; van der Pol et al. 2010; Cocucci et al. 2009; Choi et al. 2007; Santana et al. 2014). We have recently exhibited that cancer-cell-derived ESVs exhibit a bimodal size distribution (Santana et al. 2014). It is likely that the two constituent cancer-cell-derived ESV subpopulations in this size distribution represent an exosome populace and a cancer-cell-specific microvesicle populace (Santana et al. 2014) and that size correlates with biological properties of interest (van der Pol et al. 2012; D’Souza-Schorey and Clancy 2012). Microvesicles are ubiquitous in populations shed by cancer cells and decorate the surface of these cells (Antonyak et al. 2011; Santana et al. 2014). ESV characterization is usually difficult because ESVs are small and exist in a complex biological milieu. The ability to discern chemical biological or physical differences among ESV subpopulations emanating from the same cell populace SNS-032 (BMS-387032) is extremely challenging. Current microvesicle harvesting approaches SNS-032 (BMS-387032) concentrate ESVs by means of ultracentrifugation (Choi et al. 2007; Jorgensen et al. 2013; Wubbolts et al. 2003) filtration (Antonyak et al. 2011; Simpson et al. 2009; Lawrie et al. 2009; Mathivanan et al. 2010) and immunoaffinity (Coren et al. 2008; Tauro et al. 2012; Mathivanan et al. 2010) or some combination thereof. Although centrifugation and immunoaffinity approaches enable measurements reflecting averaged properties of heterogeneous ESV populations they neither enable subpopulation cargo analysis nor efficiently isolate an intact ESV subpopulation for use in a biological assay. Centrifugation and filtration can concentrate ESVs within a sample but centrifugation does not individual subpopulations. Filtration can isolate a targeted size populace but to date the recovery efficiency and purity have not been quantified. Furthermore pressure drops across filters may damage the isolated ESV subpopulation. To address these limitions we have designed and implemented a novel microfluidic technology that separates microvesicles as a function of diameter from.