An experimental strategy unveiling exosomal microRNAs 486‐5p, 181a‐5p and 30d‐5p from hypoxic tumour cells as circulating indicators of high‐risk rectal cancer

Abstract

Tumour hypoxia contributes to poor treatment outcome in locally advanced rectal cancer (LARC) and circulating extracellular vesicles (EVs) as potential biomarkers of tumour hypoxia and adverse prognosis have not been fully explored. We examined EV miRNAs from hypoxic colorectal cancer cell lines as template for relevant miRNAs in LARC patients participating in a prospective biomarker study (NCT01816607). Five cell lines were cultured under normoxia (21% O2) or hypoxia (0.2% O2) for 24 h, and exosomes were isolated by differential ultracentrifugation. Using a commercial kit, exosomes were precipitated from 24 patient plasma samples collected at the time of diagnosis. Exosome size distribution and protein cargo were determined by cryo-electron microscopy, nanoparticle tracking analysis, immunoblotting and flow cytometry. The vesicles harboured strong cell line-specific miRNA profiles with 35 unique miRNAs differentially expressed between hypoxic and normoxic cells. Six of these miRNAs were considered candidate-circulating markers of tumour hypoxia in the patients based on the frequency or magnitude of variance in hypoxic versus normoxic cell line experiments and prevalence in patient plasma. Of these, low plasma levels of exosomal miR-486-5p and miR-181a-5p were associated with organ-invasive primary tumour (p = 0.029) and lymph node metastases (p = 0.024), respectively, both attributes of adverse LARC prognosis. In line with this, the plasma level of exosomal miR-30d-5p was elevated in patients who experienced metastatic progression (p = 0.036). Our strategy confirmed that EVs from colorectal cancer cell lines were exosomes containing the oxygen-sensitive miRNAs 486-5p, 181a-5p and 30d-5p, which were retrieved as circulating markers of high-risk LARC.

How hypoxia regulate exosomes in ischemic diseases and cancer microenvironment?

“…Nanoparticle tracking analysis, cryo‐electron microscopy, flow cytometry, and immunoblotting assays were used to determine the differential expression of miRNAs in exosomes isolated from colorectal cancer (CRC) cell lines under hypoxia and normoxia. The results exhibited differential expression of 35 unique miRNAs between hypoxic and normoxic cells (see Table from). Six of these 35 miRNAs were subjected to candidate‐circulating markers in plasma from locally advanced rectal cancer patients.…”

“…Six of these 35 miRNAs were subjected to candidate‐circulating markers in plasma from locally advanced rectal cancer patients. Exosomal miRNA‐181a‐5p and miRNA‐486‐5p were related to lymph node metastasis and tumor invasion, respectively …”

Non-coding RNAs shuttled via exosomes reshape the hypoxic tumor microenvironment

“…In locally advanced rectal cancer (LARC), Tonje et al filtered a series of exosomal miRNAs from five hypoxic cell lines and validated in patient plasma samples. Among these, downregulated miR-486-5p and miR-181a-5p were correlated with T4 and lymph node metastasispositive disease, respectively, while upregulated exosomal miR-30d-5p was associated with the metastatic progression [123].…”

MicroRNA: Another Pharmacological Avenue for Colorectal Cancer?

“…miR-17, miR-20a, miR-18b, miR-193a-3p, and miR-31, are significantly reduced in pretreatment plasma of rectal cancer patients. Extracellular vesicles from colorectal cancer are exosomes containing the oxygen-sensitive miR 486-5p, 181a-5p and 30d-5p, which are retrieved as circulating markers of high-risk locally advanced rectal cancer ( Bjornetro et al, 2019 ). miR-155, miR-34a and miR-29a are downregulated in patients determined with colorectal by qRT-PCR assay.…”