(27aq) Quantifying Plasma Exosomes for Early Cancer Detection Using Rotational Brownian Motion of Janus Particles

Cancer is the second leading cause of death in the United States. 90% of these cancer deaths are due to metastasis because the cancer was not treated earlier. An article published by Etzioni et al extrapolates that effective early cancer screening for colorectal, breast, and prostate cancer would have >90% 5-year survival rate. Advancing diagnostic technology will have profound impact on how society identifies and treats the burden of cancer.

Cancer cells overexpress specific markers on their surface that aid in tumorigenesis, angiogenesis, and eventually metastasis. An example is glypican 1 (GPC1) in pancreatic ductal adenocarcinoma (PDAC). Screening these disease-specific biomarkers remains a promising non-invasive early screening technique. Because of the complex nature of cancers, studies have shown integration into the body is multifaceted, comprising numerous surface proteins and signaling pathways for survival. Observing complementary membrane markers in tandem is crucial to pinpoint the cancer progression and location in a non-invasive diagnostic application. Exosomes, once considered a disposal mechanism of cells, are now known to possess the biomarkers of their diseased parent cells. Exosomal proteins provide an avenue for detecting cancer without a priori knowledge of its location in the body. Studies show that exosomes are responsible for intercellular communication, reshaping the tumor microenvironment, suppressing immune cell response, and contributing to tumor proliferation. The circulation in bodily fluids of blood, urine, and saliva at concentrations up to 1E11 particles/mL makes exosome extraction and isolation straightforward.

Our lab has developed a technique for quantifying plasma exosomes that employs rotational Brownian motion. The technology utilizes Janus Particles (JPs), one-micron fluorescent beads coated with a thin layer of gold on a singular hemisphere. Fundamental rotational Brownian motion produces a spin on the spheres with a frequency inversely proportional to the cubed particle diameter. The JPs appear to blink on account of the dissimilar halves, and any increase in the effective size, i.e. via exosome coupling, will slow the rotation with great sensitivity. These beads provide rapid (<60 minute) detection of exosomes due to a small ~100 um average separation of the JPs and exosomes in solution. Additionally, with the use of monoclonal antibodies, the desired exosome-bound proteins are identified, absent of interference from non-targeted exosomes or soluble biomarkers. These benefits allow for the facile detection of cancer exosomes in patient plasma with simple filtration, incubation, and imaging steps. Anti-GPC1 functionalized Janus Particles could identify pancreatic cancer based on the rotational shift of JP-exosome conjugates.

The binding of whole exosomes means that the integrity of the exosomal membrane remains intact. Whole exosomes enable co-localization, a technique incompatible with individual protein detection methods. Co-localization more accurately pinpoints cancer’s origin, as multiple types of cancers can upregulate the same biomarker. The process works for instance, by capturing GPC1+ exosomes to form a JP-exosome conjugate. A quantum dot (QD) functionalized with a glypican 2 (GP2) antibody would then be added to solution, which forms a JP-exosome-QD sandwich scheme. Due to disparate excitation spectra, the rotation of JPs can be quantified separately from QDs with varying wavelengths of light. The blinking of GP2 functionalized quantum dots at the same frequency as the JPs would be indicative of pancreatic cancer. Multiplexed detection provides the benefit of detecting more than one organ/cancer marker in a single experiment. In conjunction with JPs, QDs allow for multiplexing with up to four different antibodies that are representative of different regions of the body, both increasing throughput and sensitivity of the Janus Particle platform.

John Alexander Sinclair and Sonu Kumar contributed equally to this work