A microfluidic technology for integrated exosome analysis allows for specific immunoisolation, subpopulation profiling, and targeted proteomic analysis of circulating exosomes, with minimally invasive sample consumption and markedly improved assay speed and sensitivity.

During tumor development, constant interplay occurs between tumor cells and surrounding stromal cells. We report evidence that gastrointestinal stromal tumor (GIST) cells invade the interstitial stroma through the release of the oncogenic protein tyrosine kinase (KIT)-containing exosomes, which triggers the phenotypic conversion of progenitor smooth muscle cells to tumor-promoting cells. These recipient cells display morphologic changes and acquire tumor-associated phenotypes, including enhanced adhesion to extracellular matrix proteins, activation of intracellular pathways downstream of KIT, expression of Interstitial Cell of Cajal-like markers, and release of various matrix metalloproteinases (MMPs), particularly MMP1. This report shows stimulation of MMP1 production by stromal cells via uptake of tumorderived exosomes, which leads to tumor cell invasion. Exosomes derived from GIST patients but not healthy donors show enhanced MMP1 secretion by smooth muscle cells and tumor cell invasion, whereas selective blocking of exosome-mediated MMP1 secretion decreases tumor invasiveness. Our study indicates that exosome release and subsequent MMP1 induction creates a positive feedback mechanism established between tumor and stromal cells that drives GIST development and offers unique insights for potential therapeutic strategies to block GIST progression and metastatic spread.ICC | crosstalk | microvesicles | tumor microenvironment

Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells.

Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring.