Ultrasensitive quantification of tumor mRNAs in extracellular vesicles with an integrated microfluidic digital analysis chip

Abstract

Extracellular vesicles (EVs) present a promising liquid biopsy for cancer diagnosis. However, it remains a daunting challenge to quantitatively measure molecular contents of EVs including tumor-associated mRNAs. Herein, we report a configurable microwell-patterned microfluidic digital analysis platform combined with a dual-probe hybridization assay for PCR-free, single-molecule detection of specific mRNAs in EVs. The microwell array in our device is configurable between the flow-through assay mode for enhanced hybridization capture and tagging of mRNAs and the digital detection mode based on femtoliter-scale enzymatic signal amplification for single-molecule counting of surface-bound targets. Furthermore, a dual-probe hybridization assay has been developed to enhance the sensitivity of the digital single-molecule detection of EV mRNAs. Combining the merits of the chip design and the dual-probe digital mRNA hybridization assay, the integrated microfluidic system has been demonstrated to afford quantitative detection of synthetic GAPDH mRNA with a LOD as low as 20 aM. Using this technology, we quantified the level of GAPDH and EWS-FLI1 mRNAs in EVs derived from two cell lines of peripheral primitive neuroectodermal tumor (PNET), CHLA-9 and CHLA-258. Our measurements detected 64.6 and 43.5 copies of GAPDH mRNA and 6.5 and 0.277 copies of EWS-FLI1 fusion transcripts per 105 EVs derived from CHLA-9 and CHLA-258 cells, respectively. To our knowledge, this is the first demonstration of quantitative measurement of EWS-FLI1 mRNA copy numbers in Ewing Sarcoma (EWS)-derived EVs. These results highlight the ultralow frequency of tumor-specific mRNA markers in EVs and the necessity of developing highly sensitive methods for analysis of EV mRNAs. The microfluidic digital mRNA analysis platform presented here would provide a useful tool to facilitate quantitative analysis of tumor-associated EV mRNAs for liquid biopsy-based cancer diagnosis and monitoring.

Ewing sarcoma family of tumors-derived small extracellular vesicle proteomics identify potential clinical biomarkers

“…A limitation in pediatric studies such as this is in part due to the incidence of ESFT in children and young adults within the United States and the volume of blood ethically and safely obtainable, hence we have utilized 10 pediatric patients in this study and were able to detect EWS-ETS fusion transcripts in both metastatic and localized subset of patients utilizing only 250 µL of plasma. We have expanded and improved upon prior published accomplishments [52][53][54] by directly identifying ESFT sEV-associated protein biomarkers which enabled us to enrich for ESFT-specific sEVs (AUC = 0.92) and concordantly detecting the EWS-ETS fusion transcript (PPV = 1.00 and NPV = 0.67) from as little as 250 µl of archival plasma samples. The approach by Benini et al [55], Allegretti et al [56], and our own circumvent the requirement to sequence patient-specific breakpoints, obtain long tumor DNA fragments from fresh tumor, and design patient-specific primer sets.…”

Sensor-Integrated Microfluidic Approaches for Liquid Biopsies Applications in Early Detection of Cancer

“…Zhang et al [121] described an approach to effectively isolate and detect mRNA, which consisted of a microwell-based microfluidic platform that integrates two different steps; first, the target capture and tagging and then a PCR-free mRNA detection ( Figure 5A). A pneumatic control system was designed to optimize the workflow and to reliably monitor the flow rates and binding kinetics, which had an enormous impact on the reproducibility of the test [121,122].…”

Microfluidic systems for cancer diagnostics

“…Exosomes can be found in most body fluids [39], are more stable and abundant than ctDNA, and are present in circulation at early stages of cancers, features that have made them potential cancer biomarker candidates and garnered them considerable attention in recent years [41]. The exosome analysis workflow includes their isolation and quantitation followed by the characterization of their intra-and extra-vesicular contents, size, and morphology [40,42]. Current techniques for isolating exosomes (e.g.…”

“…Although highly sensitive, these biosensors still require a significant level of manual preparation either for serum isolation or exosome enrichment [44,45,47] and only a few of them have been integrated into microfluidic platforms [40,51]. Microfluidic methods for exosome capture and analysis are based mostly on immune-affinity [42,[52][53][54]] but there are reports of size-based separation [55,56]. The most popular approach is to coat a chip surface with specific antibodies against exosome surface markers, while the opposite surface is patterned with micro-or nano-structures to enhance capture efficiency [41, [57][58][59][60].…”

“…Some of these devices allow surface phenotyping of exosomes by staining them with different antibodies, which is important to find associations between tumor and exosome markers. Reports of on-chip analysis of exosome contents are emerging [42,50,53,62]; however, once a consensus panel of exosome generic markers is obtained, we expect integration of on-chip bioanalytical approaches such as PCR, digital PCR, biosensors, and digital ELISA for their analysis.…”