Plasma extracellular vesicles detected by Single Molecule array technology as a liquid biopsy for colorectal cancer

Abstract

Circulating extracellular vesicles (EVs) were recognized as a promising source of diagnostic biomarker. However, there are limited studies published in this area, partly due to the limited number of detection platforms capable of detecting extracellular vesicles. In this study, extracellular vesicle immunoassays were developed using the Single Molecule array technology (SiMoa) and their clinical applications to cancer diagnosis were evaluated. Two extracellular vesicle detection assays, CD9-CD63 and Epcam-CD63, were designed to detect universal extracellular vesicles and tumour-derived extracellular vesicles, respectively. Our results show that CD9-CD63 and Epcam-CD63 SiMoa assays specifically detect extracellular vesicles but not free proteins with high sensitivities. The Epcam-CD63 levels detected in cancer cell culture media were consistent with levels of Epcam-expressing EVs isolated from the same cancer cell lines and detected by Western blot. Furthermore, the assays distinguish cancerous from non-cancerous plasma samples. The highest CD9-CD63 and Epcam-CD63 signals were observed in colorectal cancer patients comparing to healthy and benign controls. Both assays showed superior diagnostic performance for colorectal cancer. In addition, our results show that CD9-CD63 detection is an independent prognosis factor for both progression free survival and overall survival, while Epcam-CD63 detectionis an independent prognosis factor for OS.

PD-L1 detection on circulating tumor-derived extracellular vesicles (T-EVs) from patients with lung cancer

“…In order to screen the best antibody pair for Simoa prototype, three monoclonal antibodies of PD-L1 were purchased from Abcam and Origene (Table S1). An antibody for Epcam (MAB9601, R&D Systems) confirmed in a previously study (33) was taken as the capture antibody. Exosomes were collected from cell culture supernatant of HCT-116 for antibody pair testing due to its positive expression of Epcam and PD-L1 (35,36).…”

“…Recently, using a single molecule array (Simoa), a highly sensitive immunoassay technology (29)(30)(31)(32), we developed automatic EV/T-EV detection assays, enabling the direct profiling of EVs/T-EVs from the plasma of patients with cancer (33). In the current study, by capturing T-EVs with the same marker, epithelial cell adhesion molecule (Epcam), an important surface marker of epithelial tumor cell used as a biomarker of circulating tumor cells (CTCs) and T-EVs (34), we aim to develop a Simoa immunoassay prototype to detect PD-L1 + T-EVs from blood samples of patients with lung cancer and to compare PD-L1 expression in T-EVs and tumor tissues.…”

An insight into small extracellular vesicles: Their roles in colorectal cancer progression and potential clinical applications

“…Some work has tried to give solutions to this issue, and they build their own quantification platforms based on antibodies, immunoassay, and imaging flow cytometry, which are able to selectively detect tumor-derived EVs. [151][152][153] And in their work, compared to healthy donors, cancerous samples are able to release more EVs, and gene knockdown of organ-tropic membrane proteins could decrease organ-tropic EVs to specific organ. 154 This might indicate that increasing of EVs secretion promotes cancer progression.…”

“…Though there are many methods to quantify EVs such as quantification of particle number by nanoparticle tracking analysis, or measurement of particulate components such as proteins, lipids, and nucleic acids are relative suitable proxy for EVs quantification, it is still imperfect and need further improvement. Some work has tried to give solutions to this issue, and they build their own quantification platforms based on antibodies, immunoassay, and imaging flow cytometry, which are able to selectively detect tumor‐derived EVs 151–153 . And in their work, compared to healthy donors, cancerous samples are able to release more EVs, and gene knockdown of organ‐tropic membrane proteins could decrease organ‐tropic EVs to specific organ 154 .…”

CD9, a tetraspanin target for cancer therapy?

“…CD9 is found not only at the cell surface, but also at the membrane of EVs [85][86][87][88][89] (Figure 2(a)). EVs are classified according to their biogenesis as exosomes when derived from multivesicular bodies or as ectosomes/microvesicles when originating directly from the plasma membrane.…”