Microfluidic Electrophoresis Platform for Analysis of Pancreatic Cancer Exosomes
Kalluri, Raghu; Kono, Junichiro
Master of Science
Pancreatic cancer (PaCa) has a dismal prognosis, due to extremely late diagnosis, aggressive local metastasis, and limited treatment options. Thus, there is an urgent need for early detection methods for PaCa to increase the likelihood of treatment success. Liquid biopsy (marker detection in biofluids) is the only feasible approach for early diagnosis, as the biopsy of pancreatic tissue is not practical in patients lacking overt symptoms, and only minimally invasive methods can be used for sample collection. Recent studies demonstrate the use of exosomes as a means to improve the sensitivity of liquid biopsies. Most of the ongoing work utilizes stand-alone immunoaffinity-based capture of disease-associated exosomes in body fluids. The drawback of this approach is the heterogeneity of the exosome population, as the protein marker(s) of interest are typically expressed only by specific tumor subtypes, and the use of a single marker may result in false-negative or false-positive results or limit the enrichment capacity. In this work, we develop the novel principle of exosome fractionation based on their charge. The feasibility of the approach is based on our findings, which show a strong correlation between the negative charge of the exosomes and PaCa aggressiveness. Further analysis identified the enhanced incorporation of phosphatidylserine and DNA fragments in the outer leaflet of exosomal membranes, as the molecular cause of the highly anionic nature of cancer-derived exosomes. The use of a label-free separation approach allows us to collect intact exosomes and enables a broader range of downstream molecular analyses, unavailable in the permanent modifications by the tagging molecules on exosomes incurred by immunoaffinity techniques. Hence allowing for a multiplexed detection of biophysical and biomolecular properties of exosomes. We have designed and developed a prototype microfluidic platform that uses electrophoresis principles to isolate highly anionic exosomes, which correspond to the cancer-derived exosome population in patient serum. Further development of such a device for the detection of highly anionic exosomes in patient sera, would yield a much-needed tool for early PaCa diagnosis.
Pancreatic cancer, Exosomes, Early detection, Microfluidics, Electrophoresis