Harnessing the potential of small extracellular vesicle biomarkers for cancer diagnosis and prognosis with advanced analytical technologies

Predict outcomes and monitor progression

sEV biomarkers provide valuable insights into disease progression and survival rates in HCC. HCC patients with larger tumours or at later TNM stage have been found to exhibit lower blood sEV miRNA-638 levels, and those with these conditions additionally displayed poorer three- and five-year survival rates.^[36] This suggests the role of sEV miR-638 as a circulating cancer biomarker to predict poor prognosis for HCC patients. It has also been discovered that sEV PD-L1 contributes to survival prediction. Shorter progression-free survival and overall survival are independently predicted by a rise in sEV PD-L1.^[37] The progression of cancer can be monitored by tracking changes in sEV biomarker profiles overtime. SEV biomarkers can also be used to determine the likelihood of cancer recurring after initial treatment. For example, significant differences in miR-718 expression were observed in serum sEVs of HCC cases with recurrence following liver transplantation compared to those without recurrence.^[38] This facilitates the identification of patients who require liver transplantation and aids in predicting HCC recurrence after surgery. sEV lncRNAs display disease-specific expression patterns, making them useful biomarkers for evaluating recurrences. To differentiate patients with recurrent colorectal cancer from those without recurrence, a 5-sEV lncRNAs panel was created.^[39] Another study also utilized serum sEV lncRNAs in breast cancer for recurrence prediction.^[40] The execution of an aggressive follow-up approach for patients who are at high risk of recurrence can be justified by stratifying patients based on their risk of recurrence.

Classify stages and subtypes

sEVs contains biomarkers which contribute to the staging of cancer by providing information about the tumor’s size, invasiveness, and spread. This information aids in determining the appropriate treatment approach and prognosis for patients. Plasma sEV Sox2ot has been shown to correlated with TNM stage. Sox2ot is crucial for inducing EMT and stem cell-like characteristics, and sEVs with a high concentration of Sox2ot can facilitate tumour invasion and metastasis.^[41] Another biomarker, sEV TGF-B1, has shown a strong correlation with TNM. Patients with advanced gastric cancer, defined as TNM stages 2, 3, or 4, have demonstrated increased levels of sEV TGF-B1 compared to patients with stage 1 disease.^[42] This indicates that monitoring the expression level of sEV biomarkers can be a useful tool in classifying the TNM stage.

sEV biomarkers also play a crucial role in classifying cancer into different subtypes. In the context of breast cancer subtypes, patients with HER2− and HR+ tumours have better prognoses than those with more aggressive triple-negative (ER−PR−HER2−; TNBC) or HR−HER2+ malignancies.^[43] sEV miRNAs have been reported in these subtypes, with miR-335, miR-422a, and miR-628 showing significant differences between TNBC and HER2-positive individuals. The AUC values for miR-335, miR-422a, and miR-628 are of 0.737, 0.655, and 0.759 respectively. These sEV miRNAs have a sensitivity of 65% and 68% and a specificity of 84% and 81%, respectively, for differentiating between TNBC and HER2-positive individuals.^[44] The classification utilizing sEVs can assist in tailoring treatment strategies to specific cancer subtypes.

Assess resistance in cancer

Chemotherapy, radiation, immunotherapy, and surgical excision are commonly used treatment modalities. Nonetheless, it is not uncommon for cancer patients to develop primary or acquired medication resistance, and emerging evidence suggests that sEVs may play a role in the dissemination of drug resistance. It was discovered that cisplatin-resistant cells and stomach cancer cells have elevated levels of sEV circ-0063526. This Cir-0063526 has been shown to be packaged in sEVs and delivered to sensitive cells, thereby promoting resistance to cisplatin. In patients with gastric cancer, high expression of sEV circ-0063526 has been associated with a poor response to cisplatin treatment.^[45] Similarly, differential expression of several microRNAs (miR-425-3p, miR-1273h, miR-4755-5p, miR-9-5p, miR-146a-5p, and miR-215-5p) has been observed, with the highest fold change in platinum-resistant non-small cell lung cancer (NSCLC) patients when compared to platinum-sensitive NSCLC patients. In patients with NSCLC, high miR-425-3p has been identified as a powerful prognostic biomarker for low responsiveness to platinum-based chemotherapy.^[46]

More than half of cancer patients use radiotherapy to treat localized cancer, relieve symptoms, or slow disease progression. Nonetheless, radioresistance continues to be the primary cause of radiation failure. A comparison between radioresistant and radiosensitive NSCLC patients revealed that sEV miR-96 was significantly overexpressed in the radioresistant group. An AUC value of 0.7496 was obtained for sEV miR-96’s radioresistance diagnostic capability, suggesting its potential as a useful biomarker for distinguishing patients with radioresistant NSCLC from those with radiosensitive NSCLC.^[47]

Recently, immunotherapy has gained popularity as a treatment for cancer, encompassing various strategies such as immune system modulators, cancer vaccines, and immune checkpoint inhibitors. sEVs have been discovered to be a useful biomarker for choosing the most suitable patients for immunotherapy. sEV PD-L1 expression has been shown to be higher in patients receiving immune checkpoint inhibitor treatment. Its expression can be used to predict an overall survival of NSCLC patients. Patients who demonstrated a fold change in sEV PD-L1 expression equivalent to or higher than 1.86 exhibited a higher progression-free survival rate.^[48] This suggests that sEV PD-L1 expression can help identifying NSCLC patients who are likely to benefit from immune checkpoint inhibitor therapy and have a more favourable prognosis. Moreover, a prognostic and diagnostic model using two sEV-derived genes, MYL6B and THOC2, has been developed. High expression of these genes was associated with greater expression patterns of immune checkpoint genes, such as PD-1, B7H, CTLA4, and TIM3, in patients with HCC.^[49] Patients whose immune checkpoint expression is higher may likely to benefit from immunotherapy as it can boost the immune response resulting in greater therapeutic benefits.

Size-based isolation

Deterministic lateral displacement (DLD) is a technique employed in microfluidics for the separation of particles within the nanometre to micrometre size range. It utilizes a series of bifurcations in the laminar flow pattern created by an array of regularly spaced pillars. This innovative method enables the efficient separation of particles from biological samples.^[55] Larger vesicles were laterally displaced across the array and collected at a side channel, while smaller vesicles flew out of the array in a zigzag pattern, thereby achieving the collection of sEVs. This resulted in the production of a nanoscale DL that can separate particles between 20 and 110 nm. This method demonstrates the size sorting of sEVs and enables fast colloid sorting in a continuous flow with single-particle resolution, paving the way for on-chip separation and diagnostics.^[56] The use of nanoscale DLD also has been proved to successfully isolate extracellular vesicles from serum and urine samples.^[57] Using double linked harmonic oscillations, EXODUS, an ultrafast isolation technology, combined two membrane filter configurations. Larger sEVs stayed in the central chamber while smaller particles and fluids were able to flow through the nonporous anodic aluminium oxide membrane due to periodic negative pressure oscillations caused by switching between periods of negative pressure and air pressure.^[58] The low yields and membrane pore blockage issues with the conventional approaches are resolved by these techniques.

Magnetic beads immunization

The utilization of magnetic bead-based immunoaffinity enrichment has gained significant interest due to its notable advantages in terms of convenience and high efficiency. This method involves the use of magnetic beads coated with specific antibodies that target surface markers of sEVs. Through this immunomagnetic approach, the process of capturing and enriching sEVs becomes more efficient and effective. Through the application of magnetic beads that specifically bound to the CD63 protein on sEVs derived from serum of mice having breast cancer, a nanodevice was able to separate target sEVs. The target sEVs could be eluted from magnetic beads by controlling light excitation while preserving their integrity.^[59] The problem of costly antibody applications on the beads is resolved by the aptamer approach. sEV-containing solutions can be selectively recognized and CD63 positive sEVs can be isolated using beads linked with CD63-1 aptamer.^[60] In another approach, magnetic beads conjugated with a synthetic peptide, Vn96, were used to isolate sEVs from MCF7 cell culture medium. This method achieved high efficiency in isolating sEVs without affecting their morphology.^[61] Numerous research endeavours have been dedicated to enhancing the functionality of magnetic beads. According to one study, immunomagnetic hedgehog particles (IMHPs) with nano-spikes can improve antibody-antigen based sEV binding and targeting by offering a greater surface area for immobilization of antibodies. These IMHPs demonstrated a capture efficiency of 91.7% in extracting sEVs from MCF-7 cells.^[62] These advancements in magnetic bead-based immunomagnetic techniques contribute to the effectiveness and functionality of sEV isolation.

Electro-deposition

Electro-deposition is a technique that leverages electrostatic forces to isolate sEVs based on their electrical properties. These forces facilitate the movement of charged sEVs towards a conductive electrode, which can be modified to enhance their adhesion. By selectively depositing sEVs onto the conductive electrode, this method enables effective isolation and subsequent analysis and characterization of sEVs.^[63] For the purpose of isolating superparamagnetic nanobeads, a superparamagnetic track-etched membrane has been created. These beads possess a high capture capacity, short incubation time, and achieve capture rates of up to 99%, making them a viable option for isolating sEVs from physiological samples.^[64] Improved detection sensitivity was demonstrated by an electrode modified with chitosan composite, ionic liquid, and multi-walled carbon nanotubes. Breast cancer cell-derived sEVs containing HER-2 and EpCAM were found with high selectivity and sensitivity. Additionally, this approach presents the possibility of multiplex diagnosis detection of several sEV biomarkers.^[65] Electro-deposition enables the precise isolation of sEVs while maintaining their integrity, minimizing any potential damage or alterations that may occur during the deposition process.

Acoustic-based isolation

Acoustic forces can be utilized for the separation of sEVs from biological samples as well. By generating acoustic waves, spatial pressure nodes and antinodes are created, effectively causing sEVs to migrate towards specific regions for isolation. Due to their smaller size and lower density compared to cells and debris, sEVs can be selectively separated from larger and denser particles. This enables the efficient isolation of sEVs using acoustic forces.^[66] An example of this is the Acoustic Separation and Concentration of sEVs for Nucleotide Detection (ASCENDx) device, which utilizes a rotating microfluidic disc to enrich sEVs. In order to enable centrifugation and fluid actuation within the microfluidic channels on the disc surface, surface acoustic waves and the fluid layer on which the disc floats can be coupled to form the acoustofluidic disc rotation. With excellent selectivity and specificity of 95.8% and 100%, respectively, the enriched sEVs demonstrated diagnostic potential for identifying circulating colorectal cancer miRNA biomarkers from patient plasma samples.^[67] From undiluted blood samples, sEVs can also be directly isolated using a different acoustofluidic technology. The platform consists of two sequential surface acoustic wave microfluidic modules: one for isolating sEVs and the other for removing cells. The sEV-isolation module purifies the sEVs by eliminating other EV subgroups, while the cell-removal module eliminates microscale blood components. This approach effectively yields high purity and quantity of sEVs from undiluted blood samples.^[68]

Label-free optical methods

(SPR) is an optical sensing method that detects changes in the refractive index near a sensor surface caused by the binding of biomolecules, such as sEVs or their proteins.^[71] This technique has been used to precisely and sensitively detect HER2-positive sEVs. This technique offers a potential breast cancer diagnostic approach by differentiating patients with HER2-positive breast cancer from healthy individuals.^[72] This sensing approach can be expanded to accurately detect more sEV subtypes by simply altering the aptamer types. Utilizing sEV epidermal growth factor receptor and PD-L1 as biomarkers, SPR has also been used to diagnose lung cancer.^[73] Another analytical method for the identification and characterization of molecules is surface-enhanced Raman scattering (SERS). SERS uses chemical and electromagnetic processes to increase the Raman signal of tiny molecules affixed to the uneven metal surface.^[74] It has been used extensively in the search for sEV biomarkers. Using this method, a portable Raman sEV assay for sEV detection and protein profiling was developed. SERS can be used to diagnose breast cancer, and biomarkers for HER2 and EpCAM have been found to have diagnostic potential on sEVs in plasma from patients with HER2-positive breast cancer. According to these proof-of-concept investigations, this assay could accelerate research on sEVs and open the door for the creation of innovative liquid biopsies for cancer monitoring and detection.^[75] It is possible to precisely identify sEVs generated from cancer cells by combining statistical pattern analysis with SERS. Through principal component analysis of the entire SERS spectra of the sEVs, lung cancer cell-derived sEVs were identified with 95.3% sensitivity and 97.3% specificity from sEVs originated from normal cells.^[76]

Antibody-based methods

The unique binding of antibodies to sEV surface indicators is the basis for antibody-based techniques such as fluorescence detection and lateral flow assays, which allows the detection and study of these markers in sEVs. The presence of target analyte is determined by antibody-antigen interactions in lateral flow tests. This technique has been used to detect isolated sEVs from a malignant melanoma cell line, with a detection limit of 8.54x10^5 sEVs/μL.^[77] Additionally, it has been used to create a point-of-care platform for the detection and tracking of colorectal cancer. Together with lateral flow experiments, CD147-containing sEVs were employed as a biomarker to identify and monitor colorectal cancer. This point-of-care tool was used to quantify the CD147 antigen embedded in sEVs that were isolated from plasma.^[78] By using fluorescent labelling, sEVs can be visualized using the fluorescent detection method. This method has been utilized to detect sEVs from biological samples. The estimated limit of detection for sEVs using this method was 1.29 × 10³ particles/μL. Additionally, the difference in sEV concentration between sera of healthy individuals and cancer patients was evaluated.^[79] This technique could potentially be developed into a platform for the precise and specific identification of sEVs in biological samples for the diagnosis of cancer. A study showed that the fluorescence approach may reliably identify plasma sEVs, with an AUC of 0.85 for cancer diagnosis, to differentiate lung cancer patients from healthy persons.^[80] By employing other tumour-related sEV proteins as recognition targets, it is possible to isolate and identify sEVs from specific subpopulations derived from tumour cells, thereby improving the sensitivity and specificity of tumour diagnosis.

Nanoparticle-based methods

Colorimetric assays can be employed to detect sEVs using nanozymes, which are nanomaterials possessing inherent catalytic activity similar to those of enzymes. Through surface modification with certain ligands, the nanozyme can selectively bind to sEV surface indicators. In a study, the CD63 aptamer was utilized to identify sEVs. The hybrid nanozyme’s peroxidase activity was increased and a colorimetric signal was produced with the aid of the CD63 aptamer-bound sEVs. As a result, sEVs with a detection limit of 3.37 × 10³ particles/μL could be found.^[81] Similarly, EpCAM aptamer was utilized in the nanozyme-based colorimetric assay to provide specific detection, hence enabling the differentiation of breast cancer patients from healthy individuals.^[82] Quantum dots (QDs), one of the several varieties of nanoparticle-based optical labels, are advantageous for sEV detection because of their small diameter (2–10 nm), which enables effective sEV labelling and detection in a smaller size range.^[83] Different surface protein markers on sEVs from various breast cancer cell lines were specifically and quantitatively detected using the QD-based technique, and cancer-associated surface protein indicators can be used to distinguish sEVs produced from cancer cells from normal sEVs. By employing QDs to analyse HER2 expression on plasma sEVs, HER2-positive breast cancer was identified. Patients with the disease had HER2 expression that was around five times higher than that of healthy donors, with an AUC value of 0.96875.^[84]

Electrochemical methods

Electrochemical Impedance Spectroscopy (EIS) is a method that assesses the impedance response of a system when subjected to an alternating current signal across various frequencies. This technique proves valuable in detecting impedance changes arising from the interactions between sEVs and electrode surfaces. Considering that sEVs possess distinct compositions, particularly in terms of their membrane and cytosolic charge-dependent contents, variations in their opacity can serve as distinguishing factors. Consequently, EIS enables the differentiation of sEVs derived from different cellular origins based on their unique characteristics.^[85] Electrochemical biosensors, on the other hand, utilize electrochemical reactions taking place at the electrode surface, which can be influenced by the presence of sEVs. In a recent study, tumour cell-derived sEVs were successfully detected using a combination of cyclic nicking enzyme cleavage and a hybridization chain reaction for dual-signal amplification. For this assay, a hairpin aptamer probe (HAP) containing an aptamer was designed. The aptamer specifically binds to PTK7, a protein found on the surface of sEVs, causing a conformational change in the HAP. This conformational change enables hybridization between the HAP and the linker DNA, initiating cyclic cleavage of the nicking endonuclease on the linker DNA. Consequently, sEV detection is transformed into DNA detection. By incorporating this approach with HCR signal amplification, the study achieved highly sensitive electrochemical detection of CCRF-CEM sEVs, with a limit of detection as low as 1.1 × 10⁴ particles/mL.^[86]

Molecular beacons

Molecular beacons are hairpin-shaped nucleic acid probes that detect specific target sequences. They form a stem-loop structure with a fluorophore and quencher in close proximity. When the target binds to the probes, the stem opens, resulting in the activation of fluorescence.^[90] It has been shown that miRNA-targeting molecular beacons can detect several miRNAs simultaneously in sEVs. In sEVs generated from MCF-7, molecular beacons hybridized with numerous miRNAs despite the high concentration of human serum.^[91] Even in the presence of human urine, molecular beacons were able to identify the markers miRNA-375 and miRNA-574-3p, which are present in sEVs produced by prostate cancer cells. This implies that they can be used to do liquid biopsies for prostate cancer using human urine.^[92]

Solid-state nanopore sensing

Solid-state nanopore sensing involves applying a voltage across a solid-state material that contains a nanopore. When nucleic acid passes through the nanopore, it temporarily blocks the flow of ions or electrons, causing a detectable change in the electrical current. This change in current can be measured and analyzed.^[93] This technique has been utilized to detect the only two cysteine-containing peptides from LRG-1, an emerging protein biomarker, that are uniquely present in the urine of ovarian cancer patients. The technique provided improved selectivity for detecting biomarkers in ovarian cancer.^[94] Moreover, solid-state nanopores have been explored as single-molecule counters for future digital diagnostic technologies, as evidenced by this technology’s capacity to quantify more than six distinct microRNA concentrations.^[95]

Droplet digital PCR

In digital droplet PCR (ddPCR), the nucleic acid sample of interest is divided into numerous separate reaction droplets, each containing a few target molecules or none at all. These droplets undergo PCR amplification within a thermal cycler, and upon completion, fluorescence-based detection methods are employed to analyze the results. Each individual droplet is examined to determine whether it exhibits a fluorescence signal or not. By quantifying the number of positive and negative droplets, the absolute quantity of target molecules in the initial sample can be accurately calculated using Poisson statistics.^[96] A study used the ddPCR technology with sEV DNA to develop a sensitive and accurate approach for diagnosing tuberculosis. sEV and total DNA that was isolated from respiratory samples were targeted to the IS6110 region. The use of sEV DNA in ddPCR resulted in greater sensitivity and specificity of 98.0% and 76.9%, respectively, similar to whole DNA.^[97] This suggests that the combination of ddPCR platform with sEV DNA has the potential to provide a sensitive and accurate methodology for diagnosis of cancer. Another study evaluated the efficacy of various methods for detecting EGFR mutation in pleural fluid and plasma samples; the results showed that ddPCR in conjunction with sEV DNA had the highest sensitivity. With NSCLC patients, this method may be able to detect genetic alterations linked to resistance to EGFR inhibitor treatment.^[98]

DNA tetrahedron

DNA tetrahedron is a versatile DNA nanostructure that offers precise control over its architecture. Through chemical modifications and DNA self-assembly, it can be engineered to provide a wide range of amplified signal tags.^[99] A study used DNA tetrahedron nanoprobe (DTNP) based on fluorescence resonance energy transfer (FRET) to establish a sensitive detection approach for has-miR-146b-5p, a tumour-related microRNA. The target miRNA was intended to cause a structural alteration in the DTNP, leading to a significant enhancement in the FRET signal. This method facilitated the assessment of miRNA expression levels in various cell lines and demonstrated a low limit of detection.^[100]

Total internal reflection fluorescence

Total internal reflection fluorescence (TIRF) imaging assay relies on the principle of total internal reflection, where a laser beam is directed at a specific angle onto a glass or prism surface, creating an evanescent wave that penetrated only a short distance into the sample.^[101] With the use of this method, single sEVs and their miRNA contents in serum microsamples can be directly visualized and measured. Serum sEV miR-21 is a commonly used cancer biomarker. The TIRF imaging test was used to analyse miR-21 and demonstrated a better performance than traditional real-time PCR assays in differentiating cancer patients from healthy individuals.^[102]