Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma

Abstract

Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

Figure 1. The immunomagnetic exosomal RNA (iMER) platform.

(a) The iMER platform is designed to enable exosome enrichment, RNA extraction, reverse transcription and real-time analyses of distinct RNA targets in one small device. Cancer exosomes in serum are first captured onto magnetic microbeads containing affinity ligands (for example, anti-CD63 and anti-EGFR). The immuno-enriched exosomal population is then lysed and the lysate flows through a glass bead filter, where RNA efficiently adsorbs onto packed glass beads. Finally, the collected RNA is eluted and reverse-transcribed for real-time amplification and quantitation. (b) Scanning electron micrographs of magnetic microbeads after immunoaffinity capture. Microbeads (left, 3 μm) functionalized with antibodies against EGFRvIII, a cancer-specific deletion mutant, captured innumerable tumour vesicles from GLI36vIII conditioned medium. High-magnification micrographs (right) show that many of the captured vesicles exhibit cup-shaped characteristic of exosomes. Scale bars, 500 nm, 100 nm (inset). (c) Photograph of the microfluidic iMER prototype. The cartridge was developed to house all components of the iMER procedure, including (1) an immunomagnetic capture site, (2) a filter composed of densely packed glass beads for RNA extraction, (3) a main reservoir for reverse transcription and (4) qPCR chambers for multiplexed detection. Scale bar, 1 cm.

Figure 2. Enrichment and analysis.

(a) Exosome enrichment efficiency. Exosomes from SKMG3 cells were incubated with magnetic immunobeads (functionalized with either anti-EGFR, CD63 or IgG antibodies). Following magnetic separation, the achieved enrichment efficiency was determined. Note the high capture efficiency (>95%) for anti-EGFR and anti-CD63 microbeads as opposed to the IgG control (<2%). (b) Exosome enrichment specificity. Exosomes from EGFRvIII-positive GLI36vIII cells and negative GBM20/3 cells were mixed in 1:1 ratio (‘mixture') and subjected to EGFR-specific immunomagnetic isolation. As compared with equal amounts of GLI36vIII exosomes (‘positive control'), western blotting on the isolated exosomes (‘enriched') confirmed the enrichment of specific targets from a mixture of exosomes of different origins. (c) Comparison of RNA extraction. iMER-extracted RNA showed better structural integrity and quality profile to that extracted by a commercially available column (Qiagen). a.u.=arbitrary unit. (d) Correlation between iMER-extracted and column-extracted mRNA. The abundance of mRNA targets measured by iMER and a commercial system matched well (R²=0.986). All analyses were normalized against GAPDH. (e) iMER enrichment in serum. SKMG3 exosomes were spiked into normal human serum (‘serum mixture'). After targeted EGFR enrichment, captured exosomes were lysed and analysed on-chip (‘iMER filtered'). All measurements were performed in triplicate and the data are displayed as mean values.

Figure 3. Exosome mRNA analysis of 14 cell lines.

(a) mRNA drug resistance markers (GSTπ1, MGMT, APNG, ERCC1, ERCC2, MVP, ABCC3, CASP8 and IGFBP2), exosome marker (CD63) and representative diagnostic markers (EGFR, PDPN and EPHA2) were profiled in both parental cells (left, by conventional qRT-PCR) and corresponding exosomes (right, by iMER). mRNA quantities were normalized to intrinsic GAPDH mRNA. Note the excellent correlation between exosomal mRNA markers and those found in parental cells (R²=0.918). (b) Comparison of exosomal MGMT mRNA analysis with MGMT promoter DNA methylation in parental cells. The numbers on the x-axis (left) refer to the specific CpG islands in the MGMT DNA promoter sequence. There is an inverse correlation between promoter DNA methylation in the parental cells (left, determined via bisulfite sequencing for the extent of methylation at different CpG islands) and levels of exosomal MGMT mRNA (right). ND=non-detectable. All measurements were performed in triplicate and the data are shown as mean±s.d.

Figure 4. Effects of TMZ treatment.

(a) Exosomal MGMT and APNG mRNA levels correlate with in vitro TMZ sensitivity (ED₅₀). Cell lines were treated with varying doses of TMZ to determine their respective drug sensitivities (top panel). iMER analysis revealed that the levels of MGMT, APNG or both were elevated in resistant cell lines, whereas they were both low in sensitive ones (bottom panel). Row maximum and minimum refer, respectively, to the highest and lowest exosomal mRNA expression for a target marker across different GBM cell lines. (b) Higher average levels of exosomal MGMT/APNG were observed in resistant cell lines than in sensitive ones. However, there was overlap in exosomal mRNA levels between resistant and sensitive cell lines, demonstrating that a single marker was unable to distinguish drug resistance. Dotted line indicates the mean. (c) Serial analysis of exosomal MGMT and APNG mRNA levels following TMZ treatment. In resistant cell lines, exosomal MGMT/APNG mRNA levels increased within hours of drug treatment, while, in sensitive cell lines, the exosomal target levels decreased. GLI36vIII cell lines expressed non-detectable exosomal MGMT mRNA (* MGMT: ND). All experiments were performed in triplicate and the data are shown as mean±s.d.

Figure 5. Analysis of clinical samples.

(a) Measurement of mRNA for EPHA2, EGFR, PDPN in serum samples. GBM patients (n=17) generally showed higher levels of markers compared with healthy controls (n=15). (b) Correlation of exosomal MGMT mRNA against tumour tissue methylation status. Tissue methylation correlates inversely with exosomal MGMT copy number. The mRNA levels were significantly higher (*P<0.001; Tukey's multiple comparison test) in GBM patients with negative tissue MGMT methylation, than that of patients with positive methylation or healthy controls. The latter categories were non-distinguishable (NS) from each other. (c) Longitudinal exosomal MGMT and APNG mRNA analyses were performed in seven GBM patients; two representative examples are shown (see Supplementary Fig. 15 for other patients). Clinical assessments (NR, S, R) were based on radiological findings, clinical examination and lab values. All changes were plotted as mean±s.d. (d) Sequential exosomal mRNA changes between two time points were analysed in GBM patients (n=7) undergoing TMZ treatment. All changes were normalized to their initial values and plotted according to clinical evaluation at the end of the assessment period (the later time point). All changes were independent of initial tissue MGMT methylation status.