Detection of circulating microRNAs with Ago2 complexes to monitor the tumor dynamics of colorectal cancer patients during chemotherapy

Abstract

Because of the different forms of circulating miRNAs in plasma, Argonaute2 (Ago2)-miRNAs and extracellular vesicles (EV-miRNAs), we examined the two forms of extracellular miRNAs in vitro and developed a unique methodology to detect circulating Ago2-miRNAs in small volumes of plasma. We demonstrated that Ago2-miR-21 could be released into the extracellular fluid by active export from viable cancer cells and cytolysis in vitro. As miR-21 and miR-200c were abundantly expressed in both metastatic liver sites and primary lesions, we evaluated Ago2-miR-21 as a candidate biomarker of both active export and cytolysis while Ago2-miR-200c as a biomarker of cytolysis in plasma obtained from colorectal cancer (CRC) patients before treatment and in a series of plasma obtained from CRC patients with liver metastasis who received systemic chemotherapy. The measurement of Ago2-miR-21 allowed us to distinguish CRC patients from subjects without CRC. The trend in ΔCt values for Ago2-miR-21 and -200c during chemotherapy could predict tumor response to ongoing treatment. Thus, capturing circulating Ago2-miRNAs from active export can screen patients with tumor burdens, while capturing them from passive release by cytolysis can monitor tumor dynamics during chemotherapy treatment.

Keywords: Ago2; chemotherapy; circulating miRNA; colorectal cancer; liquid biopsy.

Figure 1

Assessment of the release mechanism of extracellular miRNAs by a cytotoxic agent (5‐FU). Cells from a CRC cell line, HT29, were treated with 5‐FU solution 48 h after seeding. The supernatants were collected at 24, 48, 72, and 96 h after seeding. (a) Cell proliferation and cell biological activities measured using a WST assay were plotted. (b) Cytotoxicity measured using an LDH assay were plotted. (c) The relative amount of miR‐21, miR‐31 and miR‐200c in the culture medium of the HT29 cell line was estimated by quantitative reverse transcription PCR. The expression ratio was represented by a fold change based on the change in threshold cycle value (ΔCt) obtained at 24 h after seeding. “Control” and “5‐FU” denote the control group and the 5‐FU treatment group, respectively. Each bar represents the mean and SD of five independent experiments. The yellow shaded areas represent the period of treatment with 5‐FU. Abbreviations: 5‐FU, 5‐fluorouracil; Ago2, Argonaute2 protein; CRC, colorectal cancer; EVs, extracellular vesicles; miRNA, micro RNA; PCR, polymerase chain reaction; *p < 0.05 (vs. the base line at 24 h after seeding). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Global miRNA profiling of the primary tumors and liver metastases. The association of the global miRNA expression status of the primary tumor and the corresponding liver metastasis by miRNA microarray. CT1, 2 and LT1, 2 denote CRC and liver metastasis specimens from patients 1 and 2, respectively. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Development of a procedure for detecting circulating Ago2‐miRNAs. (a) Ago2‐miR‐451 was recovered from Ago2‐IP by quantitative reverse transcription PCR. Added ce‐miR‐39 was used as a normalization control for all samples. Data are presented as the relative fold changes in miRNA levels. (b) The expression levels of miR‐16, miR‐21, and miR‐451 recovered from the first Ago2‐IP and from a second Ago2‐IP obtained from the flow‐through. Anti‐β‐actin antibody was used as a negative control. Each bar represents the mean ± SD of three independent experiments. Data are presented as the relative fold changes in miRNA levels. (c) Scatter plot and correlation coefficient for miR‐451 recovered from Ago2‐IP and the plasma volume. Three independent experiments were performed for each volume. Data are presented as the relative fold changes in miRNA levels. (d) Scatter diagram of Ct values of each miRNA obtained from the Ago2‐IP and EV isolation procedures using 30 μL of plasma. Each plot represents the mean of three independent experiments. Abbreviations: Ago2, Argonaute2 protein; Ct, threshold cycle value; EVs, extracellular vesicles; IP, immunoprecipitation; miRNA, micro RNA; PCR, polymerase chain reaction. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Ago2‐miR‐451, −21 and ‐200c levels in plasma samples. (a) Ct values of Ago2‐miR‐451, −21 and ‐200c in blood plasma obtained from control subjects (CS, n = 20) and CRC patients (CRC, n = 39). (b) ΔCt (miR‐451) values of Ago2‐miR‐21 in blood plasma obtained from CS, CRC, and stages I, II, III and IV. (c) ΔCt (miR‐451) values for Ago2‐miR‐21 in plasma obtained from 11 patients with CRC before surgery (Pre) and 1 year after the surgical removal of primary tumors (Post). (d) ΔCt (miR‐451) values of Ago2‐miR‐200c in blood plasma obtained from CS, CRC, and stages I, II, III and IV. (e) ΔCt (miR‐451) values for Ago2‐miR‐200c in plasma obtained from 11 patients with CRC before surgery (Pre) and 1 year after surgical removal of primary tumors (Post). The y‐axes in (b), (c), (d), and (e) represent the ΔCt values of Ago2‐miRNAs normalized to Ago2‐miR‐451. Boxes represent the interquartile range, and the horizontal line across each box indicates the median value. Statistically significant differences were determined using the Wilcoxson test. NS, not statistically significant. Dotted lines represent the cut‐off values for each Ago2‐miRNA. ΔCt (miR‐451) values below the dotted line (beyond the dotted line on both Ago2‐miRNAs) are considered abnormal values. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Examples of the expression levels of Ago2‐miR‐21 and ‐200c during the clinical course of four patients receiving systemic chemotherapy. The numbers in circles represent the chemotherapy course number. Ago2‐miR‐21 and ‐200c on the y‐axes represent the ΔCt values of Ago2‐miRNAs normalized to ΔCt values of Ago2‐miR‐451. Red arrows denote the time points at chemotherapy with blood collection for analyzing Ago2‐miRNAs. Blue arrows denote chemotherapy alone and dashed arrow denote blood collection alone. Blue, yellow, and red shading represent the shrinkage phase, stable phase, and progressive phase, respectively. Dotted lines represent the cut‐off values for each Ago2‐miRNA. Black arrows denote the time point of image assessment for liver metastasis, and computed tomography or magnetic resonance imaging represents major lesions. Abbreviations: Ago2, Argonaute2 protein; Beva, bevacizumab; CRC, colorectal cancer; Ct, threshold cycle value; FOLFOX6, modified FOLFOX6 regimen; miRNA, micro RNA; SOX, TS1 with oxaliplatin regimen. [Color figure can be viewed at wileyonlinelibrary.com]