Microfluidics for detection of exosomes and microRNAs in cancer: State of the art

Abstract

Exosomes are small extracellular vesicles with sizes ranging from 30-150 nanometers that contain proteins, lipids, mRNAs, microRNAs, and double-stranded DNA derived from the cells of origin. Exosomes can be taken up by target cells, acting as a means of cell-to-cell communication. The discovery of these vesicles in body fluids and their participation in cell communication has led to major breakthroughs in diagnosis, prognosis, and treatment of several conditions (e.g., cancer). However, conventional isolation and evaluation of exosomes and their microRNA content suffers from high cost, lengthy processes, difficult standardization, low purity, and poor yield. The emergence of microfluidics devices with increased efficiency in sieving, trapping, and immunological separation of small volumes could provide improved detection and monitoring of exosomes involved in cancer. Microfluidics techniques hold promise for advances in development of diagnostic and prognostic devices. This review covers ongoing research on microfluidics devices for detection of microRNAs and exosomes as biomarkers and their translation to point-of-care and clinical applications.

Keywords: biomarkers; cancer; exosomes; microRNA; microfluidics.

Graphical abstract

Figure 1

Microfluidics technology in cancer studies (A) Circular tumor cell (CTC) isolation by immunomagnetic-based, immunoaffinity-based, and size-based techniques. (B) Molecular diagnosis: droplet-based PCR for identifying rare mutations, on-chip single-cell qRT-PCR conducted in every reaction chamber, and droplet-scale estrogen assay for quantifying small amounts of tissues. (C) Tumor biology: migration of cancer cells in a micro-capillary array under mechanical confinement conditions, cell migration platform to explore the co-culture environmental effect, and generation of 3D co-culture spheroids for investigating the PCa metastatic microenvironment. (D) Programmable cell culture array for drug screening. High-throughput screening: an integrated blood barcode chip to identify plasma proteins and a single-cell array consisting of micromechanical traps for screening anti-cancer drugs that resulted in apoptosis. This figure was adapted from other studies., , , , , , , , ,

Figure 2

A schematic of biogenesis of exosomes and their cargos

Figure 3

Summary of tumor-derived exosome-mediated functions Released exosomes from tumor cells modulate autocrine/paracrine induction of tumors and can induce angiogenesis, regulation of the immune system, re-education of stromal cells, organotropic metastasis, and remodeling the extracellular matrix. This figure was adapted from Tai et al.

Figure 4

Novel and conventional techniques for exosome isolation Conventional techniques of EV isolation are as follows: differential ultracentrifugation (dUC) and size-exclusion chromatography (SEC). SEC uses a porous stationary phase against biofluids as a mobile phase to elute the molecules differentially with an opposite speed relation to their size. That is, at first, larger particles will elute, continued by smaller vesicles. Because smaller vesicles will pass into and flow via the pores, it results in a longer route and time of elution. dUC is based on EV subpopulation separation using slowly increasing acceleration rates. Novel exosomal methods are as follows. Polyethylene glycol (PEG)-based precipitation applies a solution to promote polymer-entrapped vesicle aggregation in a more significant number. The immunoaffinity (IA) capture method involves antibodies targeted for exosomal surface proteins to isolate specific vesicle populations. Chips with specific antibody-mediated binding are applied by microfluidics (MF) technology to efficiently capture the exosomes. Ultrafiltration (UF) relies on a filter with a particular pore size that specifically produces a vesicle-rich filtrate to the desired size. This figure was adapted from Sidhom et al.

Figure 5

Illustration of an MF device for exosome analysis Plasma or serum has flows through an antibodiy-containing chamber that detects exosome surface proteins. Exosomes are captured in this chamber, and waste is piled up in an outlet. Retained exosomes are stained with various antibodies for profiling of surface protein. The exosomes can then be transferred to another chamber for lysis and deliver their cargos into various chambers. Proteins can be recognized by sandwich immunoassays, whereas RNA and DNA can be examined by DNA microarrays or PCR. Exosome cargo can be study off-chip for more molecular profiling. This figure was adapted from Garcia-Cordero et al.

Figure 6

Experimental strategy for exosome immobilization and characterization using ExoChip (A) Schematic of the exosome capture and analysis procedure using ExoChip. The blood is collected for serum extraction from healthy or diseased individuals, and then exosomes are captured by flowing serum through a CD63 antibody-coated ExoChip. To visualize the captured exosomes, the ExoChip is processed for membrane-specific dye (DiO) staining. (B) The ExoChip is designed to measure the levels of fluorescently stained exosomes through fluorescence intensity measurements using microplate readers and allows molecular characterization of exosome contents through a variety of standard assays, including protein analysis (western blot) and mRNA/miRNA analysis (RT-PCR/miRNA open array). This figure was adapted from other studies.^,

Figure 7

Down- or up-regulation of miRNAs contributes to the cancer-driving steps Often one miRNA affects more than one hallmark with one prevailing tissue-dependent mechanism. This figure was adapted from Detassis et al.

Figure 8

chip-based approaches (A) The PF MF device in which PDMS absorbs air in the outlet chamber, making it a self-stand pumping device. Probe DNA is immobilized on the glass surface, microchannels convey the sample to the probe, and miRNA hybridization and detection take place. (B) An enlarged view of a laminar flow in the microchannel. The laminar flow conveys fluorescein isothiocyanate (FITC)-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA). Sandwich hybridization and dendritic amplification take place at the intersection between the probe DNA-patterned surface and the interface of the laminar flow. This figure was adapted from other studies.^,,