Unmasking a hidden killer

18 March 2020:

Pancreatic cancer is one of the most lethal cancers, but difficult to diagnose: few sufferers have symptoms until the cancer has become large or already spread to other organs. Even then, symptoms can be vague and easily misconstrued as more common conditions.

This is why Dr Yuling Wang is so excited by results of a trial completed in late 2019, which — using plasmonic nanoparticles developed by the Centre for Nanoscale BioPhotonics (CNBP) — successfully detected signs of the cancer in blood of patients undergoing treatment. The paper was recently published in the premier journal, American Chemical Society — Sensors.

‘The test gave a very high signal in the blood for late-stage or very serious tumours, where other techniques cannot detect anything,’ said Dr Wang, an associate investigator at the Centre’s Macquarie University node in Sydney, in work led by Prof Nicolle Packer. ‘We need to test many more patient samples to validate the approach, but the strength of the signal was very encouraging.’

They did this by developing a method, using surface-enhanced Raman spectroscopy nanotags, that simultaneously detects three known pancreatic cancer biomarkers in blood. Known as extracellular vesicles, or EVs, they contain DNA and proteins for cell-to-cell communication and are shed from pancreatic cancer cells into surrounding body fluids. The CNBP method zeroes in on three: Glypican-1, epithelial cell adhesion molecules and CD44V6.

For the experiment, biopsies of healthy donors were provided alongside those of known sufferers of pancreatic cancer, in double-blind tests where the researchers did not know which was which. Nevertheless, the blood of sufferers was easily identified. The technique was so sensitive it could spot EVs as small as 113 nanometres in diameter — or less than 1% the width of a human hair — in every millilitre of blood.

Non-invasive screening of cancer biomarkers from blood with handheld Raman reader.

The pancreas is part of the digestive system, secreting insulin into the bloodstream to regulate the body’s sugar level as well as important enzymes and hormones into the small intestine to help break down food. Pancreatic cancer is the fifth biggest cancer killer in Australia and has a 5-year survival rate of 8.7%. More than 3000 Australians are diagnosed annually, and surgery to remove the cancer is a long and complex process, requiring long hospital stays.

Because existing blood tests for the protein biomarkers of pancreatic cancer are unreliable, imaging with endoscopic ultrasound or MRI scans is necessary. Even then, anomalies can only be confirmed with a biopsy of the organ, which is invasive and ultimately relies on a trained pathologist to recognise signs of the cancer under a microscope. As a result, there’s some subjectivity involved and cancer can be present but still be missed.

‘Our approach is non-invasive — we don’t need to take tissue from the patient, we just use a handheld device to test blood for targeted biomarkers, which gives a very quick result,’ Dr Wang said. It may also help provide earlier diagnosis of the cancer.

Dr Yuling Wang

While the work is a proof-of-concept, it was also able to detect colorectal and bladder cancer biomarkers — although not as clearly as those for pancreatic cancer. Nevertheless, the results are so encouraging that a commercial partner has committed funding to CNBP so it can develop a handheld spectrometer for cancer biomarkers in blood.

Journal: American Chemical Society — Sensors

Publication Title: Enabling sensitive phenotypic profiling of cancer-derived small extracellular vesicles using surface-enhanced Raman spectroscopy nanotags

Authors: Wei Zhang, Lianmei Jiang, Russell Diefenbach, Douglas H. Campbell, Bradley John Walsh, Nicolle H. Packer and Yuling Wang

Summary: Circulating cancer-derived small extracellular vesicles (EVs) are nanoscale membranous vesicles shed from cancer cells that are released into surrounding body fluids. Small EVs contain biomolecules associated with cancer such as DNA and proteins for cell-to-cell communication. Therefore, small EVs have been regarded as important cancer biomarkers for liquid biopsy-based cancer diagnosis and drug treatment monitoring. However, due to the high heterogeneity and low level of small EVs in body fluids, there is a high demand for sensitive detection and characterization of such vesicles at a molecular level. In this study, we have developed a sensitive and effective approach to simultaneously profile multiple protein biomarkers ex-pressed on cancer-derived small EVs using surface-enhanced Raman spectroscopy (SERS) nanotags in a single test, without complex isolation steps. Rapid and multiplexed phenotypic profiling of small EVs is achieved by mixing specific detection antibody-coated SERS nanotags, filtered conditioned EV suspended medium (conditioned EVs), and capture antibody (CD63)-conjugated magnetic beads to form a sandwich immunoassay. As a proof-of-concept demonstration, we applied this approach to characterize pancreatic cancer derived EVs by simultaneously detecting three specific EV surface receptors including Glypican-1, Epithelial cell adhesion molecule (EpCAM) and CD44 variant isoform 6 (CD44V6). The sensitivity of this method was measured down to 2.3×106 particles/mL, which is more sensitive and shows higher multiplexing capability than most other reported EV profiling techniques, such as Western blot, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Furthermore, phenotypic profiling of small EVs from colorectal cancer and bladder cancer cell lines (SW480 and C3) was conducted and compared to those derived from pancreatic cancer (Panc-1), highlighting the significant differ-ence in EV phenotypes for various cancer cell types suspended in both PBS and plasma. Thus, we believe this technology enables a comprehensive evaluation of small secreted EV heterogeneity with high sensitivity, offering strong potential for accurate non-invasive cancer diagnosis and monitoring of drug treatment. In addition, this assay provides point-of-care use because of the easy sample preparation and portable nature of the Raman spectrometer.

Link: https://pubs.acs.org/doi/10.1021/acssensors.9b02377