Cancer-Associated Fibroblasts Confer Gemcitabine Resistance to Pancreatic Cancer Cells through PTEN-Targeting miRNAs in Exosomes.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related death in the United States. Even though the poor prognosis of PDAC is often attributed to late diagnosis, patients with an early diagnosis who undergo tumor resection and adjuvant chemotherapy still show tumor recurrence, highlighting a need to develop therapies which can overcome chemoresistance. Chemoresistance has been linked to the high expression of microRNAs (miRs), such as miR-21, within tumor cells. Tumor cells can collect miRs through the uptake of miR-containing lipid extracellular vesicles called exosomes. These exosomes are secreted in high numbers from cancer-associated fibroblasts (CAFs) within the tumor microenvironment during gemcitabine treatment and can contribute to cell proliferation and chemoresistance. Here, we show a novel mechanism in which CAF-derived exosomes may promote proliferation and chemoresistance, in part, through suppression of the tumor suppressor PTEN. We identified five microRNAs: miR-21, miR-181a, miR-221, miR-222, and miR-92a, that significantly increased in number within the CAF exosomes secreted during gemcitabine treatment which target PTEN. Furthermore, we found that CAF exosomes suppressed PTEN expression in vitro and that treatment with the exosome inhibitor GW4869 blocked PTEN suppression in vivo. Collectively, these findings highlight a mechanism through which the PTEN expression loss, often seen in PDAC, may be attained and lend support to investigations into the use of exosome inhibitors as potential therapeutics to improve the effectiveness of chemotherapy.

Surface Acoustic Wave Lysis and Ion-Exchange Membrane Quantification of Exosomal MicroRNA.

MicroRNA detection and quantification are commonly explored techniques for diagnostic and prognostic predictions. Typically, microRNAs are extracted and purified from a biological source, converted into complementary DNA (cDNA), and amplified using real time polymerase chain reaction (RT-PCR). The number of RT-PCR cycles required to reach the threshold of detection provides a relative quantification of the target microRNA when this data is normalized to the quantity of a control microRNA. This methodology has several drawbacks, including the need to artificially amplify the target microRNA for detection as well as quantification errors that can occur due to expression level differences of the control microRNAs for normalization in various sample sources. Here, we provide a technique to quantify actual concentrations of target microRNAs directly from any biological source without the requirement of these additional steps. In addition, we describe an alternative approach for obtaining exosomal microRNAs directly from biological samples without the use of harsh detergents and RNA isolation.