- Liquid biopsies currently focus on the number of circulating tumor cells (CTCs) in the blood to detect the spread of a tumor
- Cancer cells secrete proteases that are linked with metastasis; however, the success of protease inhibitors has been inconsistent because of tumor cell heterogeneity
- A new microfluidics device that isolates CTCs can analyze the proteases they secrete to gain insight into their function and heterogeneity
- Using the technology, researchers found that matrix metalloproteases secreted by CTCs indicate active malignant processes
Australian pathologist Thomas Ashworth first described “cells identical with those of the cancer itself” in the blood in 1869 (1). Today, the presence of such circulating tumor cells (CTCs) is associated with the aggressive spread of a tumor, which is thought to occur when CTCs secrete proteolytic enzymes that facilitate invasion. These matrix metalloproteases (MMPs) are synthesized as inactive preproenzymes, but become activated by pro-matrixins once secreted and then degrade extracellular matrix barriers.
Previous efforts to quantify the number of CTCs in patients, with the goal of predicting treatment effectiveness, have yielded mixed results. This is partly because of CTC phenotype heterogeneity; not all cells have a phenotype optimized for extravasation. Similarly, clinical trials of MMP inhibitors have not been overwhelmingly successful thus far, because CTCs vary in their secretion of MMPs. What if we could establish the level of MMP secretion by patients’ CTCs – rather than simply measuring the number of CTCs? And would such a tool allow us to identify patients who would benefit from MMP inhibitors?
A new technique developed by Dino Di Carlo and his team at the University of California, Los Angeles, uses liquid biopsy and a microfluidics device to isolate and analyze CTCs in the blood. “Solid tumors generally produce between one and 100 CTCs per milliliter of blood – a volume that contains around five billion red blood cells and 10 million white blood cells. That rarity is a critical challenge when attempting to pick out these cells for analysis,” Di Carlo says. “Existing technologies to isolate these cells are numerous, but don’t go any further – and, once they are isolated, there is a lot of downstream work using traditional techniques, such as staining. Unfortunately, most of the CTCs are lost in this process, leading to poor performance and the inability to quantify the properties of these cells.”
Di Carlo’s team combined the isolation and analysis of CTCs into a seamless integrated system. Their microfluidic technique captures CTCs from blood, exchanges the fluid around them to eliminate contaminants, adds an MMP substrate, and encapsulates them into droplets on the nanoliter scale. The process reduces cell loss, resulting in the ability to analyze individual cells to detect the secretion and activity of particular enzymes, such as MMPs. “Instead of looking at genetic information or protein levels in their non-functional form, we are able to study the activity of these CTCs in terms of proteases that they are secreting or expressing on their surfaces. The key breakthrough is that this device tells us how active the proteases are – because they can be secreted in an active or inactive form – which provides crucial information on the actual function of MMPs.”
The integrative aspect of the technology means that the process – from whole blood sample to isolated CTCs – takes just 15 minutes to complete. In addition, the sensitivity of the device enables as few as seven protease molecules to be counted per droplet, allowing for high levels of precision. The isolation technique, based on fluid dynamics, was discovered by Di Carlo serendipitously. “I began developing new types of microfluidic tools based on fluid inertia when I was a postdoctoral researcher. Such an approach was unheard of at the time, because everyone thought that small amounts of fluid were characterized by smooth, constant motions known as low Reynolds number flows. We challenged that way of thinking and found that, in rapid-moving inertial flows, randomly distributed cells will migrate across fluid streams, ordering themselves into preferred locations.”
The same inertial flows form the basis of the microfluidic device, in which rapid streams produce a jetting flow that stems off the main channel upon exposure to sudden expansions of small reservoirs. Micro-whirlpools form in these reservoirs; small cells (such as red and white blood cells) can enter the whirlpools and exit downstream, whereas larger cells (such as CTCs) become trapped inside by fluid dynamic lift forces. “The nice thing here is that, once they’re trapped and circulating, you can lower the flow in the channel and then the whirlpools dissipate, so all of the larger cancer cells are released into very small, highly concentrated volumes.”
Using the microfluidic system in their research, Di Carlo’s team applied the assay to analyze MMP secretion by cells in seven metastatic prostate cancer patients. Along with CTCs, other circulating cells that are known to secrete MMPs in prostate cancer patients include leukocytes, which do so at the sites of tissue inflammation and tumors. MMP activity from CTCs was found to be 2.6 ± 1.5 times higher than that from leukocytes in the same patients (2). The results of this study indicate that the relative increase in MMP secretion by CTCs compared with a leukocyte baseline could signal the presence of active malignant processes, helping to inform the prognosis of metastatic prostate cancer.
Future applications of the technique could include its use in studying subcategories of cancer-specific proteases, facilitating a better understanding of the proteolytic pathways associated with patient-specific disease. “It would make sense that there is variation in the mix of enzymes secreted by different cancer types, and there could be differences in the activity of these cells,” Di Carlo says.
MMPs are also involved in helping cancer cells evade the immune system – another interesting application of the new device. MMPs secreted in this instance cleave stress proteins expressed on the cell surface, preventing natural killer cells from identifying the tumor and eradicating it. “We’re seeing the development of more and more drugs that block the cleavage of these stress proteins, so that the immune system can start to re-attack those cancer cells. By characterizing the activity of MMPs, we will gain a better understanding of these processes and get one step closer to identifying patients who will benefit from certain treatments.”
“As an engineer, I’m particularly excited by fact that we are able to identify CTCs that are ‘one in a billion’ in the blood – and then measure a few molecules from each of those single cells. My hope now is that we can get this device into laboratories to help pathologists and oncologists make treatment decisions,” Di Carlo says. The vortex trapping technology has been licensed and is currently being developed into a complete assay that Di Carlo hopes will be on the market within the next three years. “The technology is getting ever closer to that stage, and we’re currently developing more downstream assays. Our ultimate aspiration is to help select the most effective drugs and improve the lifetime of patients, such as those who are helping us with our research studies.”
The trapping technology is a class I FDA-registered device currently being sold as a research instrument in the US. Alongside the analysis of proteases secreted by CTCs, the format of the technique – confining cells within droplets – is well-suited to nucleic acid level measurements, single cell sequencing, and other single cell-based assays. No wonder Di Carlo is optimistic for the future. “In the field of precision medicine, one of the key goals is obtaining samples that are as informative – but not as invasive – as traditional methods. And that’s where our device fits in. Now that we can receive functional information from individual cancer cells, I think it’s going to be an exciting new area with plenty to explore.”
Dino Di Carlo reports the following relevant disclosures: Board Member of Vortex Biosciences, the company that has taken the trapping technology to market in the US.
Dino Di Carlo is a Professor in the department of Bioengineering and Director of the Cancer Nanotechnology Program of the Jonsson Comprehensive Cancer Centre at the University of California, Los Angeles.
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References
- TR Ashworth, “A case of cancer in which cells similar to those in the tumors were seen in the blood after death”, Aust Med J, 14, 146–149 (1869). M Dhar et al., “Functional profiling of circulating tumor cells with an integrated vortex capture and single-cell protease activity assay”, PNAS, 40, 9986–9991 (2018). PMID: 30224472.
