This work presents chemically stable and biodegradable hydrogel beads for the isolation of circulating tumor cells (CTCs) and circulating exosomes in liquid biopsy. The liquid biopsy hydrogel beads (LBbeads) consisting of alginate and poly(vinyl alcohol) hydrogels show both chemical stability and stimuli‐degradable characteristics. Unlike single‐component hydrogels, this hybrid form is not easily degraded by buffers or cell culture media while its degradable characteristic remains; thus, it is useful in bio‐applications requiring multi‐step processes with various reagents and lengthy incubation periods. We applied our platform to clinical samples for isolating two promising circulating biomarkers for a liquid biopsy, CTCs and exosomes, by conjugating the hydrogel surface with anti‐EpCAM and anti‐CD63 antibodies, respectively, thus achieving 37.4 CTCs and comparable amount of exosome recovery per 1 milliliter of blood. The results show easy device‐free isolation and retrieval of CTCs and exosomes, with recovered circulating biomarkers successfully analyzed by western blot analysis and fluorescence microscopy. We believe that this simple and versatile platform enables us to isolate prominent circulating biomarkers for clinical use in cancer diagnosis.
Traumatic brain injury (TBI) is a global cause of morbidity and mortality. Initial management and risk stratification of patients with TBI is made difficult by the relative insensitivity of screening radiographic studies as well as by the absence of a widely available, noninvasive diagnostic biomarker. In particular, a blood-based biomarker assay could provide a quick and minimally invasive process to stratify risk and guide early management strategies in patients with mild TBI (mTBI). Analysis of circulating exosomes allows the potential for rapid and specific identification of tissue injury. By applying acoustofluidic exosome separation—which uses a combination of microfluidics and acoustics to separate bioparticles based on differences in size and acoustic properties—we successfully isolated exosomes from plasma samples obtained from mice after TBI. Acoustofluidic isolation eliminated interference from other blood components, making it possible to detect exosomal biomarkers for TBI via flow cytometry. Flow cytometry analysis indicated that exosomal biomarkers for TBI increase in the first 24 h following head trauma, indicating the potential of using circulating exosomes for the rapid diagnosis of TBI. Elevated levels of TBI biomarkers were only detected in the samples separated via acoustofluidics; no changes were observed in the analysis of the raw plasma sample. This finding demonstrated the necessity of sample purification prior to exosomal biomarker analysis. Since acoustofluidic exosome separation can easily be integrated with downstream analysis methods, it shows great potential for improving early diagnosis and treatment decisions associated with TBI.
more » « less- Award ID(s):
- 1807601
- NSF-PAR ID:
- 10216242
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Microsystems & Nanoengineering
- Volume:
- 7
- Issue:
- 1
- ISSN:
- 2055-7434
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Introduction Abnormal angiogenesis is central to vascular disease and cancer, and noninvasive biomarkers of vascular origin are needed to evaluate patients and therapies. Vascular endothelial growth factor receptors (VEGFRs) are often dysregulated in these diseases, making them promising biomarkers, but the need for an invasive biopsy has limited biomarker research on VEGFRs. Here, we pioneer a blood biopsy approach to quantify VEGFR plasma membrane localization on two circulating vascular proxies: circulating endothelial cells (cECs) and circulating progenitor cells (cPCs).
Methods Using quantitative flow cytometry, we examined VEGFR expression on cECs and cPCs in four age-sex groups: peri/premenopausal females (aged < 50 years), menopausal/postmenopausal females (≥ 50 years), and younger and older males with the same age cut-off (50 years).
Results cECs in peri/premenopausal females consisted of two VEGFR populations: VEGFR-low (~ 55% of population: population medians ~ 3000 VEGFR1 and 3000 VEGFR2/cell) and VEGFR-high (~ 45%: 138,000 VEGFR1 and 39,000–236,000 VEGFR2/cell), while the menopausal/postmenopausal group only possessed the VEGFR-low cEC population; and 27% of cECs in males exhibited high plasma membrane VEGFR expression (206,000 VEGFR1 and 155,000 VEGFR2/cell). The absence of VEGFR-high cEC subpopulations in menopausal/postmenopausal females suggests that their high-VEGFR cECs are associated with menstruation and could be noninvasive proxies for studying the intersection of age-sex in angiogenesis. VEGFR1 plasma membrane localization in cPCs was detected only in menopausal/postmenopausal females, suggesting a menopause-specific regenerative mechanism.
Conclusions Overall, our quantitative, noninvasive approach targeting cECs and cPCs has provided the first insights into how sex and age influence VEGFR plasma membrane localization in vascular cells.
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There is great interest in advancing methodologies for the isolation and characterization of exosomes (30–150 nm, extracellular vesicles (EVs)) for fundamental biochemical research and liquid biopsy applications. This is due to the accessibility of exosomal surface biomarkers, providing relevant biochemical information from their cells of origin. Exosome-based techniques hold potential for diagnostic applications through less invasive sampling ( versus the physical extraction methods of pathology). This study demonstrates a simple spin-down tip methodology for generic exosome capture, followed by immunoaffinity-based fluorescent labeling to classify EVs captured on a polyester capillary-channeled polymer (C-CP) fiber stationary phase. An antibody to the generic EV tetraspanin protein (CD81) is employed to confirm the presence of biologically active EVs on the fiber surface. An antibody to the CA125 protein, upregulated in the case of ovarian cell stress, is included as a cancer marker protein. Scanning electron microscopy and confocal fluorescence microscopy were performed directly on the capture fibers to visualize the morphology and assess the bioactivity/identity of captured vesicles. This report provides a proof-of-concept for an efficient means of isolating, purifying, immunolabeling, and fluorescent imaging for the biomarker assessment of extracellular vesicles on a single platform . Herein lies the novelty of the overall approach. The ability to affect the entire isolation, immunolabeling, and imaging process in <5 hours is demonstrated. The C-CP fiber spin-down tip is an efficient exosome isolation methodology for microliter samples from diverse media (human urine and cell culture media here) towards diverse means of characterization and identification.more » « less
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Abstract Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca2+chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. ThenewExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.
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This article is categorized under:
Diagnostic Tools > Biosensing
