By Deborah Borfitz
November 8, 2019 | Massachusetts General Hospital (MGH) has developed liquid biopsy technologies to do high-throughput exosome protein profiling and point-of-care (POC) exosome analysis. The research aim is to develop multiplexed assay systems to streamline the analysis of exosomes and evaluate their clinical utility for cancer management, both as diagnostic tools and to predict disease recurrence, says Hyungsoon Im, assistant professor of radiology at MGH and a speaker at the 2019 Next Generation Dx Summit.
Im focused his discussion on the nano-plasmonic exosome (nPLEX) sensor, in routine use at MGH, which detects exosome protein levels based on “spectral shifts” (intensity changes) of light through thousands of optimally spaced 200-nanometer (nm) holes, says Im. Antibodies against cancer biomarkers get immobilized on the nanopores for capture.
Nanoholes on the sensor are gold and exosomes are labeled by gold nanoparticles for signal amplification, says Im. Gold has proven to have the best chemical stability for this type of assay. The device is being manufactured on a glass-based substrate, as silicon wafers proved to be too fragile.
The iMEX (integrated magnetic-electrochemical exosome) device, meanwhile, integrates vesicle isolation and detection in a single platform, he says. Target-specific exosomes first get enriched through immunomagnetic selection, for high detection sensitivity. The sensors can be miniaturized and expanded for parallel measurements.
In studies, nPLEX has demonstrated good accuracy and 100 times greater sensitivity than the commonly used enzyme-linked immunosorbent assay (ELISA) and able to detect as few as 3,000 exosomes, Im says.
Exosome counts had been considered a poor diagnostic marker for ovarian cancer, since both cancer and non-cancer groups shed exosomes into their circulation, says Im. But as was shown in a 2014 study, the two groups can readily be distinguished by combining EpCAM (epithelial cell adhesion molecule) and cancer immunotherapy target CD24 as biomarkers. The proteins have good expression in both cells and exosomes, he notes.
MGH researchers have also come up with a protein signature for pancreatic ductal adenocarcinoma (PDAC) that is powerful enough for diagnostic purposes—a combination of EGFR (epidermal growth factor receptor), EpCAM, MUC1, GPC1 and WNT2. As demonstrated in a prospective study involving more than 100 patients and age-matched controls, “as tumors shrink the biomarker combination gets lower and lower,” Im says.
This offers higher sensitivity, specificity, and accuracy than the existing serum marker (CA 19-9) or analysis of a single circulating tumor-derived extracellular vesicle. The next step is a multi-institutional validation study of the signature with 600 patients, says Im.
More recently, MGH researchers used nPLEX technology to show how exosome protein expression of cancer cells changes with drug treatment. Discovery of these unique, drug-dependent protein signatures suggests the exosome screening assay will be a potentially powerful molecular screening tool.
The screening assay could be incorporated into POC instruments for “more rigorous testing of different drugs in different settings,” says Im, to see how well results reflect changes at the tumor site. In fact, Im and his MGH colleagues are now working with Exosome Diagnostics’ Shakky System—the world’s first instrument specifically for exosomal protein analysis—to improve their prototype assay for the early detection of pancreatic cancer.
Finding a foundry to manufacturer the POC device may be one of the bigger challenges, says Im, adding that fabrication could possibly happen in Korea. The nPLEX chip sits on the bottom plate of the device and has 100 sensing sites to automate reagent spotting and measurements.
In 2016, Nature Biotechnology selected nPLEX technology as one of the “greatest hits” in its 20-year publishing history, Im says. Current challenges are to push profiling down to the single exosome level and to determine if exosomes can be used therapeutically in cancer.