The team designed the nanoparticles to shed short sequences of DNA that are excreted in the urine when they encounter a tumor, analysis of the resulting DNA barcodes can reveal an assortment of distinguishing features of a particular tumor. Additionally, the test was designed to be performed using a strip of paper, similar to at-home testing kits, which is hoped to keep the diagnostic simple, affordable, and accessible to as many people as possible.
“We are trying to innovate in a context of making technology available to low- and middle-resource settings. Putting this diagnostic on paper is part of our goal of democratizing diagnostics and creating inexpensive technologies that can give you a fast answer at the point of care,” says senior author Sangeeta Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Technology and of Electrical Engineering and Computer Science at MIT and a member of MIT’s Koch Institute for Integrative Cancer Research and Institute for Medical Engineering and Science.
Testing has demonstrated that the sensors could detect the activity of five different enzymes that are expressed by tumors and that the approach could be scaled up to distinguish at least 46 different DNA barcodes in a single sample by using a microfluid device to analyze the samples.
Bhatia has been working on developing synthetic biomarkers that could be used in the diagnosis of cancer, building on the concept of detecting biomarkers like proteins or circulating tumor cells in blood samples. Naturally occurring biomarkers are rare and nearly impossible to find at early stages, but synthetic biomarkers could be used to amplify smaller-scale changes that occur within small tumors.
Previously Bhatia and his team created nanoparticles to detect the activity of protease enzymes that help cancer cells escape their original locations and/or settle into new locations by cutting through extracellular matrix proteins. The nanoparticles were designed to be coated with peptides that are cut by different proteases that once released into the bloodstream can be concentrated and more easier to detect in urine samples. These peptide biomarkers were also designed to be detected based on small variations in their masses by using a mass spectrometer.
However, mass spectrometers may not be available in all resource settings so the team sought to develop other sensors that could be analyzed more affordably using DNA barcodes that can be detected/read using CRISPR technology. For the new approach to work, phosphorothioate chemical modification was used to protect the circulating DNA reporter barcodes from being broken down in the blood. Similar modification techniques have already been used to improve the stability of modern RNA vaccines, allowing them to survive longer within the body.
The DNA barcodes are attached to a nanoparticle by a linker that is cut with a specific protease, when that protease is present the DNA molecule is released making it free to circulate and end up in urine. Two different types of nanoparticles were used in this study: one made of FDA-approved polymers and the other is a nanobody antibody fragment that can be designed to accumulate at tumor sites.
Once secreted in urine samples the sensors can be analyzed using paper strips that recognize a reporter that is activated using Cas12a CRISPR enzymes. The Cas12a enzymes amplify the signals of particular DNA barcodes when they are present in samples so that they are seen as dark strips on the paper test. The nanoparticles can be designed to carry different DNA barcodes that detect different types of protease activity allowing for multiplexed sensing. Additionally, using a greater number of sensors provides a boost in specificity and sensitivity so that the test can distinguish between tumor types more easily.
“Our goal here is to build up disease signatures and to see whether we can use these barcoded panels not only read out a disease but also to classify a disease or distinguish different cancer types,” Hao says.
The team showed that a panel of five DNA barcodes accurately distinguished between tumors first occurring in the lungs from tumors that were formed by colorectal cancer cells that metastasized to the lungs. The team expects to need more than 5 barcodes because there is much variety between tumors from person to person. As such they worked with researchers from the Broad Institute of MIT and Harvard led by Harvard University Professor Pardis Sabeti, resulting in the development of a microfluidic chip that can be used to read up to 46 different DNA barcodes from one sample.
The team believes that this testing could also be used for measuring how a tumor is responding to treatment and whether the tumor has recurred after treatment. The team is currently working on further development of the particles with the goal of testing them on humans. Earlier versions of the urinary diagnostic particles were investigated in phase 1 clinical trials which found them to be safe in patients.
In addition to Bhatia, Hao, and Sabeti, the study’s co-authors include Renee T. Zhao, Nicole L. Welch, Edward Kah Wei Tan, Qian Zhong, Nour Saida Harzallah, Chayanon Ngambenjawong, Henry Ko, and Heather E. Fleming.
The research was funded by the Koch Institute Support (core) Grant from the National Cancer Institute, a Core Center Grant from the National Institute of Environmental Health Sciences, the Marble Center for Cancer Nanomedicine at the Koch Institute, the Koch Institute Frontier Research Program, the Virginia and D.K. Ludwig Fund for Cancer Research, and a Pathway to Independence Award from the National Cancer Institute.
