Urinary MicroRNA-based Early Cancer Detection Using Nanowire-based Devices

Sci-fi style computer graphicDetecting cancer at early stages when interventions are possible is important to improve survival. However, screening tests for many cancer types do not meet such needs due to the low sensitivity1 and specificity2, meaning that some patients will receive a false positive result in the screening even if they do not have cancer, while others will receive a false negative result despite having cancer. In addition, many cancer types even do not have any means for early detection.

Liquid biopsy is an alternative to conventional tissue biopsy by measuring biomarkers from body fluids such as blood and urine. It is less invasive, quick, and easily repeatable compared to conventional biopsy. To date, most liquid biopsy analyses are performed on blood samples and look for a wide range of circulating cancer biomarkers, from fragments of DNA and RNA shed from dying cells, to whole cancer cells and exosomes containing tumor material. While the golden standard of liquid biopsy is based on blood, urine-based liquid biopsy has some advantages compared to blood-based liquid biopsies, since urine is a completely non-invasive sample with huge advantages of easy and high-volume collection and low cost, making it extremely suitable for the mass screening of healthy individuals. Even though it has some advantages compared to blood-based biopsy, urine-based liquid biopsy has not been fully investigated because a satisfactory way to collect urinary biomarkers is lacking.

MicroRNAs (miRNAs) are short non-coding RNAs that regulate gene expression post-transcriptionally. They are released from cancer cells, thereby they can modulate gene expression in recipient cells in a tumor’s surrounding microenvironment, and they have been associated with the development and progression of the disease in various cancer types. They are recognized as promising biomarkers that could lead to clinical applications, since their profiles differ in healthy and cancer groups of people. Identification of tumor-specific microRNAs presents a powerful opportunity to potentially reduce cancer mortality through early detection. It has also been reported that there is a high degree of correlation between the expression patterns of microRNAs in tissues and those in blood and urine. Here, we have developed a mass-producible and sterilizable nanowire device that can extract urinary microRNAs easily and efficiently.

We designed an assembly-type microfluidic nanowire device for extracting urinary miRNAs and acquiring miRNA expression profiles. We fabricated the assembly-type microfluidic nanowire device by two processes: first, we grew zinc oxide (ZnO) nanowire scaffolds from a thermally oxidized chromium layer on a silicon (Si) substrate; and second, we assembled the ZnO nanowire scaffolds, cyclo-olefin polymer (COP) resin microfluidic substrate, a COP resin substrate, two stainless steel holders, and polyether ether ketone (PEEK) tubes into the device. The device was connected to PEEK tubes to introduce urine and lysis buffers and to collect flow-through urine and miRNA-containing solutions. Since no bonding process was required, each component of the assembly-type microfluidic nanowire device could be sterilized, such as by autoclave treatments, ethanol treatments, and dry-heat treatments, to prevent contamination by miRNAs in saliva and sweat of persons handling the device. Furthermore, the fact that each part is independent simplifies the fabricating processes, fabrication time was shortened, and the device could be mass-produced.

The device, which is equipped with 100 million ZnO nanowires, can extract a significantly greater variety and quantity of miRNAs from only a milliliter of urine compared to conventional methods. Using the device, we revealed that miRNAs in urine could be a promising biomarker to diagnose brain tumors. We analyzed microRNAs collected using the device from the urine of patients with brain tumors and non-cancer individuals and found that many microRNAs derived from brain tumors do exist in urine in a stable condition. Next, we examined whether urinary miRNAs could serve as a biomarker of brain tumors, using a diagnostic model developed based on the expression pattern of miRNAs in urine. The results showed that the model can distinguish the patients from non-cancer individuals at a sensitivity of 100% and a specificity of 97%, regardless of the malignancy and size of tumors. This means that the model correctly detected as positive all individuals with cancer no matter how early stage it is, and it labelled only 3% of healthy individuals as having the disease. We thus concluded that microRNAs in urine are a promising biomarker of brain tumors.

We immediately began working on the social implementation of this technology. Craif.Inc, a liquid biopsy startup, was established in 2018 to identify cancer-related microRNA ensembles in urine through a combination of nanowire devices and machine learning-based analysis.

Craif logoWith a nanowire-based device, Craif first enriches exosomes from urine and efficiently extract exosome-encapsulated miRNAs as well as cell-free miRNAs, then measure miRNA expression. Using this technology in combination with machine learning algorithms, Craif has succeeded to accurately distinguish cancer patients from healthy individuals, especially those at early stages. Initial data regarding early detection of urothelial cancer and ovarian cancer has been presented at AACR Annual Meeting 2021 and ESMO Congress 2021. A number of clinical studies are currently underway to demonstrate the usefulness of the test to detect many types of cancer. Regarding the development of a clinical test for brain tumors, a research project is underway aiming for regulatory approval with support from Japan Agency for Medical Research and Development.

Just by taking a urine sample at home, one can detect early signs of any disease, including cancer. This is not the stuff of science fiction. Such a future is about to become a reality.

  1. 1. Sensitivity = a test's ability to designate an individual with disease as positive. A highly sensitive test means that there are few false negative results (the test finds the individuals with the disease well).
  2. 2. Specificity = a test’s ability to designate an individual who does not have a disease as negative. A highly specific test means that there are few false positive results (the test does not erroneously indicate healthy individuals as having a disease).



Biography of the author

Takao Yasui is an Associate Professor of the Department of Biomolecular Engineering, Graduate School of Engineering at Nagoya University; he is also a PRESTO researcher at Japan Science and Technology Agency (JST) and a technical advisor at Craif Inc. (co-founder). In 2011, he received his Ph.D. in the Department of Applied Chemistry, Graduate School of Engineering from Nagoya University. He has received over 35 awards for his contribution to the field of nano-space science. His research interests are focused on designing, fabricating, and characterizing nano spaces for applications in healthcare, diagnosis, sensing biomolecules, and engineering biology.




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