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.

Common variable immunodeficiency (CVID) patients have reduced gut microbial diversity compared to healthy controls. The reduced diversity is associated with gut leakage, increased systemic inflammation and ten “key” bacteria that capture the gut dysbiosis (dysbiosis index) in CVID. Rifaximin is a broad-spectrum non-absorbable antibiotic known to reduce gut leakage (lipopolysaccharides, LPS) in liver disease. In this study, we explored as a ‘proof of concept’ that altering gut microbial composition could reduce systemic inflammation, using CVID as a disease model. Forty adult CVID patients were randomized, (1:1) to twice-daily oral rifaximin 550 mg versus no treatment for 2 weeks in an open-label, single-centre study. Primary endpoints were reduction in plasma/serum levels of soluble (s) CD14, sCD25, sCD163, neopterin, CRP, TNF, LPS and selected cytokines measured at 0, 2 and 8 weeks. Secondary endpoint was changes in intra-individual bacterial diversity in stool samples. Rifaximin-use did not significantly change any of the inflammation or gut leakage markers, but decreased gut microbial diversity compared with no treatment (p = 0.002). Importantly, the gut bacteria in the CVID dysbiosis index were not changed by rifaximin. The results suggest that modulating gut microbiota by rifaximin is not the chosen intervention to affect systemic inflammation, at least not in CVID.

As many as 30% to 40% of locally advanced rectal cancer (LARC) patients experience metastatic progression of the disease. Recognizing the potential of the genetic cargo in tumor-derived exosomes, we hypothesized that plasma exosomal microRNA (miRNA) may reflect biological aggressiveness in LARC and provide new markers for rectal cancer aggressiveness and risk stratification. In a prospective LARC cohort ( NCT01816607 ), plasma samples were collected from 29 patients at the time of diagnosis, before neoadjuvant therapy and surgery. Exosomes, precipitated from plasma using a commercial kit, were verified by cryo-electron microscopy, nanoparticle tracking analysis, and western blotting. Expression of exosomal miRNAs was profiled using a miRCURY LNA miRNA microarray and validation of six miRNAs associated with pathological and clinical end-points was undertaken in plasma collected at the time of diagnosis from 64 patients in an independent prospective LARC cohort ( NCT00278694 ). In both cohorts, exosomal miR-141-3p and miR-375 were higher in patients with synchronous liver metastasis than in those without ( P = .010 and P = .017 respectively in the investigative cohort, and P < .001 for both in the validation cohort). Further , high exosomal miR-141-3p was associated with post-operative metastatic liver progression in the investigative cohort ( P = .034). Because both miRNAs are associated with tumor angiogenesis and immune modulation, we propose that these miRNAs in circulating exosomes may reflect rectal cancer aggressiveness and accordingly be candidate biomarkers for further investigations.