Dual‐Isolation and Profiling of Circulating Tumor Cells and Cancer Exosomes from Blood Samples with Melanoma Using Immunoaffinity‐Based Microfluidic Interfaces

Abstract

Melanoma is among the most aggressive cancers, and its rate of incidence continues to grow. Early detection of melanoma has been hampered due to the lack of promising markers for testing. Recent advances in liquid biopsy have proposed noninvasive alternatives for cancer diagnosis and monitoring. Circulating tumor cells (CTCs) and cancer-exosomes are gaining influence as promising biomarkers because of their cancer-associated molecular markers and signatures. However, technologies that offer the dual-isolation of CTCs and exosomes using a single sample have not been thoroughly developed. The dual-utilization OncoBean (DUO) device is conjugated with melanoma specific antibodies, MCAM and MCSP, enabling simultaneous CTC and exosome isolations. Using blood samples from patients, CTCs and exosomes are specifically isolated from a single sample and then undergo molecular profiling for comprehensive study. Melanoma patients have 0-17CTCs mL −1 and 299 µg exosomal protein mL −1 while healthy donors display fewer than 2CTCs and 75.6 µg of exosomes mL −1 , respectively. It is also demonstrated that both markers express melanoma-associated genes using multiplex qRT-PCR to test for expression pattern of a 96 gene panel. The dual isolation and molecular characterization will allow for further research into melanoma to identify viable markers for disease progression and treatment efficacy.

Isolation of Circulating Biomarkers for Liquid Biopsy using Immunoaffinity‐Based Stimuli‐Responsive Hybrid Hydrogel Beads

“…First, as we evaluated in a recent study, there was no significant relationship between number of CTCs and concentration of total exosomes. [32] However, we noted a direct correlation between the fraction of CTCs expressing mesenchymal markers and total exosome concentration; this is similar to what has been previously reported. [52] Second, results from one biomarker correlated to data on other biomarkers, demonstrating a major advantage of simultaneous detection of two biomarkers from the same patients.…”

“…biomarker collection steps (Figure S1). Previously, we devised a microfluidic dual-isolation platform that isolates CTCs and circulating exosomes in melanoma, [32] however, it still needs sophisticated chemical strategy to release isolated circulating markers from the device and is not easy to scale up to potential larger volumes due to the device saturation. The proposed concept has the advantage of being able to detect multi-level analytes, both CTCs and circulating exosomes (Figure 1B) and easily release them via stimuli-driven hydrogel release.…”

A Holistic Review of the State‐of‐the‐Art Microfluidics for Exosome Separation: An Overview of the Current Status, Existing Obstacles, and Future Outlook

“…Affinity-based isolation of exosomes on chips usually relies on capture probes (typically antibodies or aptamers) to identify generic membrane markers (e.g., CD9 and CD63) or specific exosome biomarkers, such as epithelial cell adhesion molecule (EpCAM), EGFR, melanoma cell adhesion molecule, and melanoma-associated chondroitin sulfate proteoglycan. [101][102][103][104][105][106] Compared to size-based methods, which can only retrieve total exosomes from bio-fluids, immunocapture on chip provides an opportunity for separating either total exosomes or sub-type exosomes. Generally, there are two main types of immunocapture-on-a-chip strategies.…”

Extracellular Vesicles for the Diagnosis of Cancers

“…concentrate melanoma EVs as well as circulating tumor cells from blood. [182] This device could enrich about 5.5 circulating tumor cells and 299 μg EV (protein content) per milliliter of blood. Afterward, the isolated EVs and circulating tumor cells were tested against a 96-gene panel.…”