Therefore, the use of microfluidic-based technologies is a promising technique in research related to electric vehicles, with potential clinical applications. An official website of the United States government Official websites use. gov A. The gov website belongs to an official government organization of the United States.
In recent years, it has been discovered that nanoscale vesicles that originate in tumor cells and that can be found circulating in the blood (i.e., exosomes and microvesicles) contain a large amount of proteomic and genetic information to control cancer progression, metastasis and drug efficacy. However, the use of exosomes and microvesicles as biomarkers to improve patient care has been limited by their small size (30 nm—1 μm) and the extensive sample preparation required for isolation and measurement. In this critical review, we analyze the emerging use of microtechnology and nanotechnology to isolate and detect exosomes and microvesicles in clinical samples and the application of this technology to cancer monitoring and diagnosis. Cancer is usually located in parts of the body that are difficult to access, such as the brain, ovaries or pancreas, making measurements of molecular biomarkers in tumor cells impractical for routine clinical monitoring or disease diagnosis.1 In recent years, nanoscale exosomes (30 nm-100 nm) and microvesicles (100 nm to 1 μm), which originate in tumor cells and can be found circulating in the blood, have been found to contain a large amount of information proteomics and genetics for the diagnosis of diseases, as well as for the monitoring of cancer progression, metastasis and pharmacological efficacy, 2 Unfortunately, establishing the clinical utility of exosomes and microvesicles as biomarkers for improving patient care has been limited by the fundamental technical challenges that arise from their small size and the extensive sample preparation required before measurement. The scarcity of exosomes and microvesicles derived from tumors in relation to those that originate in healthy cells, their overlap in size with other nanoscale objects (for example, protein aggregates, cell debris) present in clinical samples and the short half-life of protein surface markers once removed from the body further complicate these measurements, 3 platforms that use microscale structures (for example, microfluidic), in which dimensions are designed to match those of cells, have been used with great success to selectively and sensitively classify 4 to 8 and detect 9 to 12 cells. However, it is difficult to translate these approaches to the optimal nanoscale sizes for the classification and detection of microvesicles and exosomes, due to the cost of nanolithography, the inherently low performance and the susceptibility to obstruction of nanoscale fluid channels, and the unfavorably strong scaling of many of the forces used to classify microfluidic objects as they move to the nanoscale.
In this manuscript, we will review the current state of the art in the use of microdevices and nanodevices to isolate and detect exosomes and microvesicles and share our vision of the future clinical and translational relevance of this rapidly growing field. Exosomes are vesicles of 30 to 100 nm that are released during the fusion of multivesicular endosomes (MVE) with plasma membrane 13 (fig. CD63, CD9, CD8), heat shock proteins (e.g.The biogenesis of exosomes and microvesicles from cells. A diagram showing the contents of exosomes and microvesicles.
A cartoon showing where circulating exosomes and microvesicles can be found in clinical samples that can be obtained in a non-invasive way. In addition to proteins and lipids, exosomes have also been found to contain nucleic acids such as miRNA and mRNA, 14 as well as double-stranded DNA, 15 Most RNAs found within exosomes are between 20 and 200 base pairs long, including full-length miRNA and tRNA, and fragments of mRNA and rRNA, 19 Different methods of RNA isolation have resulted in inconsistent reports as to the total amount of exosomal RNA and the relative amount of types of RNA and, as such, a detailed accounting of the charge of nucleic acids remain an open question 18. The few molecular markers that are released from tumor cells to the peripheral circulation have great potential for routine clinical monitoring of the molecular status of hard-to-reach tumor sites, 21 These markers, which include soluble proteins, cell-free DNA, circulating tumor cells (CTCs) and circulating microvesicles and exosomes (the table), have been shown to contain valuable information on the molecular state of a cancer, 22,23 Comparison of soluble proteins, cell-free DNA and tumor cells circulants (CTCs), and circulating exosomes and microvesicles for non-invasive cancer monitoring engineers have devised many ingenious strategies to isolate and measure both rare CTCs 4,5,8,9 and soluble proteins dispersed in the blood, 24-27 However, the fundamental limitations of these detection modalities preclude their clinical application. CTCs are limited by their extremely low blood concentrations (0 to 1000 or more of CTC in 7.5 ml of blood), causing long run times, excessive consumption of valuable clinical samples, and Poisson counting error.28 The detection of biomarkers based on soluble proteins, such as prostatic specific antigen (PSA) in prostate cancer, has been limited by issues of specificity. Because the biogenesis of the soluble protein found in the blood cannot be determined, diagnoses based on protein detection have high false positive rates, 29,30 The isolation of intact CTCs offers the promise of combining phenotypic characterization with molecular analysis of extracted nucleic acids. However, CTCs can be difficult to detect using existing platforms, especially in the case of minimal residual disease or early-stage cancer 31,32. The FDA-approved CellSearch device has had limited sensitivity to diseases such as melanoma, pancreatic and ovarian cancer31, 32, even in the context of advanced or metastatic disease.
Conversely, exosomes have been easily detected in early and late stage pancreatic cancer33,34, melanoma,35, glioblastoma,36 and other types of cancer for which isolation of the CTC is currently difficult or impossible. The clinical value of tumor-derived exosomes and microvesicles can be considered on their own or in combination with other “liquid biopsy” assays, including CTC, cfDNA and soluble proteins. In making this consideration, it is worth noting that the relative importance of these liquid biopsies in the clinic is a topic of ongoing debate. 21,46 It is believed that the exosomal content, including nucleic acids and proteins, is representative of the cell of origin and can therefore be interrogated in order to guide therapy and monitor the response to treatment. An important aspect of personalized medicine is the detection of genetic variants that can be treated with targeted therapeutic agents, either as monotherapy or together with chemotherapeutic agents.
For example, driver mutations, such as BRAF V600E in melanoma, EGFR L858R in lung cancer, and HER2 amplification in breast cancer, can be treated with specific drugs.
Recently, Thakur and colleagues reported the specific detection of the BRAF V600E mutation in melanoma exosomes and the EGFR L858R and T790M mutations in lung cancer exosomes, while another group was able to detect gene amplification in exosomal DNA 47. Together, these experiments suggest the possibility of developing exosome-based genotyping to guide treatment selection when CTCs or cfDNA are undetectable
. Perhaps the most promising area for the clinical use of exosomes is the detection and monitoring of early-stage disease. It is believed that if cancers such as pancreatic and ovarian cancers could be detected before the advanced stage at which most patients are initially diagnosed, significant improvements could be achieved in the terrible survival rates of these diseases, 54-56. While current approaches for isolating CTC and detecting variants in cfDNA are quite sensitive in patients with advanced or metastatic disease, it is an open question in the field whether current trials can achieve the necessary sensitivity for early detection of or even the detection of cancer.On the contrary, several researchers have recently reported not only easily detectable tumor exosomes in pancreatic cancer and other early-stage cancers, but also the presence of protein markers associated with disease progression. In a study of patients with stage I pancreatic ductal adenoma (PDAC) cancer, it was found that higher levels of exosome-resident macrophage migration inhibitory factor (MIF) predict a greater risk of eventually developing liver metastasis.34 Strangely enough, this group also demonstrated, in a mouse model with PDAC, that blocking the MIF could prevent liver metastasis, suggesting that the content of the tumor exosome may be clinically relevant to both prognosis and development of new targeted therapies. Using mass spectrometry to assess the protein load of early-stage pancreatic cancer exosomes, Melo and colleagues recently identified glypican-1 (GPC) as a cell surface marker highly expressed in tumor-derived exosomes 33. By evaluating the serum exosomes of mice and humans with cancer, they were able to show that the number of GPC1+ exosomes correlated with tumor burden and survival. These results suggest that, just as CTC counts and the allelic fraction of variants detected in cell-free DNA have been shown to correlate with tumor volume, exosomes can also function as a substitute for tumor volume for real-time monitoring of response to treatment. Finally, and perhaps the most interesting result published by this team, was the finding that the measurement of GPC1+ exosomes could distinguish between patients with a precursor form of pancreatic cancer, an intraductal papillary mucinous neoplasm (IPMN), and healthy donors.33 The subsequent development of these tests could therefore lead to highly sensitive screening tests to identify one of the earliest and most treatable forms of one of the most lethal cancers. Density gradient separation offers an improvement in purity and recovery rate compared to differential centrifugation.
In density gradient separation, a sample is rotated in a tube containing a density gradient of a viscous material, so that the objects are separated according to their isopycnic point, 57 While this technique can achieve a higher purity and recovery rate than conventional differential centrifugation,60, it cannot separate exosomes from viruses or microvesicles due to their similar floating density, 57 The execution times of density gradient separation are similar to those of ultracentrifugation conventional, and requires the same ultracentrifugation equipment, making it impractical for many clinical applications, 3,57 Unlike strategies that isolate exosomes or microvesicles based on their size, immunoaffinity-based approaches classify them by the expression of specific proteins on their surface. This approach has the advantage, compared to size-based purification, of reducing copurification with cellular debris and protein aggregates, as well as the ability to isolate specific subpopulations of exosomes or microvesicles based on the expression of a specific surface marker, 57,65—67 A non-microfluidic example of immunoaffinity-based classification is the use of conventional magnetic-activated cell sorting columns (MACS), 43 These columns are designed to separate cells directly from biological samples, taking advantage of the strong magnetic forces on the magnetic field of cells labeled with nanoparticles (MNP) and the lack of magnetism in biological samples. The Taylor group reused MACS to isolate exosomes from serum samples from normal controls, patients with benign diseases, and early-stage ovarian cancer 43. In this technique, exosomes that expressed the epithelial cell adhesion molecule (EpCAM) were incubated with anti-EpCAM magnetic microbeads and trapped with a separator Conventional MACS. After passing the unlabeled material and discarding it, the exosomes were released and collected.
Using the enriched exosomes, they profiled exosomal miRNAs to see which ones were elevated in exosomes compared to cells. Tumor-derived exosome levels were found to increase with the progression of ovarian cancer. By isolating exosomes by immunoaffinity on a microfluidic chip, the recovery rate can be improved and smaller sample volumes can be processed 61. The Toner group presented an immunoaffinity-based microfluidic device that rapidly and specifically isolates exosomes from cell culture media. or from serum samples.
The surface of the microfluidic channel was modified to coat the surface with biotinylated anti-CD63, a panexosome marker. The exosomes were captured from the serum by binding to the antibody. Since the isolation is carried out in a portable microfluidic device, the technique can be used as a tool at the point of care if combined with a subsequent analysis technique. Overview of exosome and microvesicle detection technologies Size measurement accuracy depends on size homogeneity of particles.
Dynamic range is a function of nanopore size. In DLS, the diameter of microvesicles and exosomes is determined by measuring the dynamic changes produced by the dispersion of coherent light (that is, that coming from a laser) through the suspension of exosomes and microvesicles, 45,73 These changes are due to Brownian motion, which causes the distance between the vesicles that scatter the light to fluctuate. By quantifying the time scale of the decay of this autocorrelation, the diffusion rate of the particles and, therefore, the size of the particles can be calculated. 73 The DLS is a commonly used laboratory tool that, in addition to microvesicles and exosomes, is used to characterize the size and concentration of a variety of particles, including nanoparticles, polymers and proteins, 74 For exosomes and microvesicles, the DLS is specified to measure particles in the size range of 0.3 nm to 10 μm (Malvern).The accuracy of DLS can be distorted by the presence of only a small number of large particles (e.g., platelets) and, as such, DLS measurements in exosomes require careful sample preparation, 45,74 DLS is not susceptible to molecular labeling and, therefore, cannot be used to profile specific molecular information on exosomes or microvesicles.
In flow cytometry, the most effective technique for high-performance cell analysis, each vesicle or exosome is individually passed through a laser spot and the fluorescent and scattered light emitted is measured 75. Because of the 100-fold smaller size of exosomes and microvesicles relative to cells, the challenge of applying flow cytometry to exosomes and microvesicles is the difficulty of recovering such weak signals. Therefore, the disadvantage is that only particles larger than 300 nm can be resolved, 45 The advantage of flow cytometry is that it allows individual exosomes to be resolved and allows multiple surface markers to be measured per exosome. 76 flow cytometry machines that are now entering the market are raising the detection limit to 100 nm or more (e.g. e.g.
A50-Micro-Plus, Apogee) could expand the utility of flow cytometers for analyzing exosomes. Because the diameter of exosomes is smaller than the optical wavelengths, they cannot be resolved with a conventional optical microscope. By using the short wavelength of electrons, TEM can resolve individual exosomes 45. In addition to the techniques described above, which can provide the size, concentration and analysis of surface markers, TEM images allow us to resolve the morphology and heterogeneity of exosomes. Extensive sample processing is required, including dehydration, fastening and metallization.
In resistive pulse detection (RPS), individual nanoparticles are measured as they are propelled, one by one, through a nanopore. 80 As the particle flows through the pore, a transient change in ionic current flow is created. The change in current is proportional to the size of the particle, allowing for the quantitative sizing of the particles with appropriate calibration. Adjustable resistive pulse detection (TRP) is an adaptation of resistive pulse detection, in which the pore size can be elastically stretched to the size of the particle to improve sensitivity.
The ion occlusion detection of 81 nanopores has several key advantages compared to conventional techniques. Compared to techniques, such as DLS, which measures the average of the set of many exosomes and microvesicles, much smaller sample volumes (~100 times) can be interrogated. In addition, much lower concentrations of exosomes and microvesicles can be resolved, with the maximum detection limit being a single exosome or microvesicle. Compared to electron microscopy, which can also resolve individual exosomes and microvesicles, nanopore-based measurements are much faster, less expensive and higher-performing. External voltage polarizing electrodes (H, L) and sensing electrode (S); integrated nanoscale (F) filters; fluid resistance (FR); nanoconstriction (NC); pressure-regulated fluid ports (P1—P).
Expanded to view nanoparticle detection components. The nanoparticles flow in the direction of the arrows and change the electrical potential of the fluid adjacent to the nanoconstriction, which are detected by the sensor electrode S. Circuit model of the nanopore device. As a function of time, as a single nanoparticle crosses the nanopore, 85 Non-invasive diagnosis and monitoring of cancer continue to make inroads into the clinic, which translates into greater personalization of therapy, more sensitive monitoring of the disease and less discomfort for patients due to invasive procedures. Tumor-derived exosomes offer new clinical opportunities for minimally invasive diagnosis and disease monitoring, which complement existing circulating biomarkers.
To further advance the development of exosome-based clinical trials, validation studies are needed to establish the sensitivity, reproducibility and specificity of such tests in large cohorts of patients and for multiple cancers of interest. In addition, it will be essential for the field to establish the relative sensitivity and clinical utility of exosomes against CTCs and cell-free DNA, and to use more tests with patient samples to find the best type of test or tests with the clinical question being posed. Finally, our understanding of the biology of tumor-derived exosomes has only just begun. As the role of exosomes in promoting tumor growth and the seeding of metastases continues to be discovered, there is no doubt that this will drive the development of new therapeutic strategies and stronger and more sensitive tests to help take clinical decisions.
While the recent work highlighted in this review shows the promise of exosomes as cancer biomarkers, the extensive processing required to isolate and detect them has limited their clinical use.3 The use of micro and nanotechnologies designed to overcome these technical challenges is ongoing. By allowing for rapid sample preparation and molecular analysis from small volumes of samples, these platforms can help to fully exploit the clinical potential of these circulating biomarkers in cancer and other settings. Jina is a graduate student in Bioengineering at the University of Pennsylvania. His research focuses on the early detection of lethal diseases, such as pancreatic cancer and traumatic brain injury, using circulating cells and rare exosomes.
She is interested in developing point-of-care platforms that are low-cost, small, fast, and highly accessible. His doctoral work is carried out in close collaboration with the University of Pennsylvania's medical school, allowing him to quickly translate his research into clinical trials and their use. Low EXO concentrations are difficult to detect because of their inherently low refractive index and weak Raman scattering signal. Consequently, even a small alteration in the reflection index of the detection medium will prevent the appearance of SPR, allowing the detection of analytes.
Cholesterol, for example, is used as a reagent to detect the binding of a target to aptamers or surface proteins, such as the CD63-sensitive detection of exosomal proteins using a compact surface plasmon resonance biosensor for the diagnosis of cancer. In addition, although SERS is known for its extraordinary sensitivity, it is still difficult to reliably obtain low EXO detection limits. It is essential to achieve significant technical advances and to validate novel concepts about various detection methods before EXO-based liquid biopsy moves towards its definitive clinical application and disease monitoring. Clinical diagnosis is based on the analysis of cancer-related biomarkers, such as circulating tumor cells (CTC), circulating tumor DNA (ctDNA), microRNA and exogenous genes, which provide vital information for the detection of tumor metastases, the surveillance of postoperative recurrence and the early diagnosis of cancer.
However, nanodevices could offer an alternative, as they provide a sensitive and affordable real-time method for detecting circulating tumor EXOs. When developing a biosensor to detect cancer cells, the ability to detect poor concentrations of biomarkers (and sometimes even just a few cells) is one of the most important factors. A visible and colorimetric aptasensor based on single-walled carbon nanotubes coated with DNA for the detection of exosomes. Currently, the detection of exogenous, cell-free DNAs (cfDNA) and circulating tumor cells (CTCs) is considered to be one of the areas most important of the analysis of liquid biopsies.
