Cancer associated fibroblasts (CAFs) comprise the majority of the tumor bulk of pancreatic adenocarcinomas (PDACs). Current efforts to eradicate these tumors focus predominantly on targeting the proliferation of rapidly growing cancer epithelial cells. We know that this is largely ineffective with resistance arising in most tumors following exposure to chemotherapy. Despite the long-standing recognition of the prominence of CAFs in PDAC, the effect of chemotherapy on CAFs and how they may contribute to drug resistance in neighboring cancer cells is not well characterized. Here we show that CAFs exposed to chemotherapy play an active role in regulating the survival and proliferation of cancer cells. We found that CAFs are intrinsically resistant to gemcitabine, the chemotherapeutic standard of care for PDAC. Further, CAFs exposed to gemcitabine significantly increase the release of extracellular vesicles called exosomes. These exosomes increased chemoresistance-inducing factor, Snail, in recipient epithelial cells and promote proliferation and drug resistance. Finally, treatment of gemcitabine-exposed CAFs with an inhibitor of exosome release, GW4869, significantly reduces survival in co-cultured epithelial cells, signifying an important role of CAF exosomes in chemotherapeutic drug resistance. Collectively, these findings show the potential for exosome inhibitors as treatment options alongside chemotherapy for overcoming PDAC chemoresistance.

There has been increasing evidence that micro and messenger RNA derived from exosomes play important roles in pancreatic and other cancers. In this work, a microfluidics-based approach to the analysis of exosomal RNA is presented based on surface acoustic wave (SAW) exosome lysis and ion-exchange nanomembrane RNA sensing performed in conjunction on two separate chips. Using microRNA hsa-miR-550 as a model target and raw cell media from pancreatic cancer cell lines as a biological sample, SAW-based exosome lysis is shown to have a lysis rate of 38%, and an ion-exchange nanomembrane sensor is shown to have a limit of detection of 2 pM, with two decades of linear dynamic range. A universal calibration curve was derived for the membrane sensor and used to detect the target at a concentration of 13 pM in a SAW-lysed sample, which translates to 14 target miRNA per exosome from the raw cell media. At a total analysis time of ~1.5 h, this approach is a significant improvement over existing methods that require two overnight steps and 13 h of processing time. The platform also requires much smaller sample volumes than existing technology (~100 μL as opposed to ~mL) and operates with minimal sample loss, a distinct advantage for studies involving mouse models or other situations where the working fluid is scarce.

Extracellular vesicles (EV) containing microRNAs (miRNAs) have tremendous potential as biomarkers for the early detection of disease. Here, we present a simple and rapid PCR-free integrated microfluidics platform capable of absolute quantification (<10% uncertainty) of both free-floating miRNAs and EV-miRNAs in plasma with 1 pM detection sensitivity. The assay time is only 30 minutes as opposed to 13 h and requires only ~20 μL of sample as oppose to 1 mL for conventional RT-qPCR techniques. The platform integrates a surface acoustic wave (SAW) EV lysing microfluidic chip with a concentration and sensing microfluidic chip incorporating an electrokinetic membrane sensor that is based on non-equilibrium ionic currents. Unlike conventional RT-qPCR methods, this technology does not require EV extraction, RNA purification, reverse transcription, or amplification. This platform can be easily extended for other RNA and DNA targets of interest, thus providing a viable screening tool for early disease diagnosis, prognosis, and monitoring of therapeutic response.

Exosomes carry microRNA biomarkers, occur in higher abundance in cancerous patients than in healthy ones, and because they are present in most biofluids, including blood and urine, these can be obtained noninvasively. Standard laboratory techniques to isolate exosomes are expensive, time consuming, provide poor purity, and recover on the order of 25% of the available exosomes. We present a new microfluidic technique to simultaneously isolate exosomes and preconcentrate them by electrophoresis using a high transverse local electric field generated by ion-depleting ion-selective membrane. We use pressure-driven flow to deliver an exosome sample to a microfluidic chip such that the transverse electric field forces them out of the cross flow and into an agarose gel which filters out unwanted cellular debris while the ion-selective membrane concentrates the exosomes through an enrichment effect. We efficiently isolated exosomes from 1× PBS buffer, cell culture media, and blood serum. Using flow rates from 150 to 200 μL/h and field strengths of 100 V/cm, we consistently captured between 60 and 80% of exosomes from buffer, cell culture media, and blood serum as confirmed by both fluorescence spectroscopy and nanoparticle tracking analysis. Our microfluidic chip maintained this recovery rate for more than 20 min with a concentration factor of 15 for 10 min of isolation.