Abstract
BACKGROUND
Cancer cells produce a heterogeneous mixture of vesicular, organelle-like structures (microvesicles or MVs) into their surroundings including blood and body fluid. In particular exosomes are biological nanovescicles (40-100 nm) that are formed by the inward budding of multivescicular bodies (MVB), as a component of the endocytic pathway. They are released from different cell types under both normal and pathological conditions. Exosomal content is composed by proteins, DNA, mRNA and microRNA (miRNA) that are transferred to distant site and mediate inter-cellular communication.
PURPOSE OF THE STUDY
The aim of this pilot study is to investigate whether exosomes isolation from clinical samples (plasma from NSCLC patients) is feasible. Furthermore we have analyzed a panel of 7 miRNA related to NSCLC.
EXPERIMENTAL DESIGN AND METHODS
The study population was selected among lung cancer patients diagnosed with NSCLC, histotype adenocarcinoma, in the Oncology department of Antwerp University Hospital. A total of 12 patients participated in the project. Six blood samples from healthy volunteers were also collected. After obtaining the informed consent, blood samples (10 ml) were stored in the UZA-tumor biobank. Clinical data were also collected from patients’ medical records.
Exosomes were isolated by means of both Density Gradient (DG) centrifugation and the Total Exosome Isolation kit (from plasma) (Invitrogen) according to manufacturers’ instructions. The Total exosome RNA and protein isolation kit (Invitrogen) was used for proteins recovery from exosome. Extracted proteins are used for western blot analysis aimed at the identification of specific exosome protein markers: CD63, ALIX and TSG101. Nanoparticle Tracking Analysis (NTA) was performed by means of the NanoSight NS500 instrument. In order to visualize the isolated exosomes a TEM (Transmission Electron Microscopy) analysis was performed.
The total exosome RNA and protein isolation kit (Invitrogen) was used to extract small-RNA from exosomal samples. The analysis of 7 miRNAs (miR-30b, -30c, -103, -122, -195, -221, -222) was performed on the LightCycler 480 (Roche) and the fold change was calculated according to the formula 2ααCq. miR-1228 was used has endogenous control to normalize the reaction.
RESULTS
• ALIX protein is a reliable protein marker for exosome identification
• TEM analysis have shown that exosomes can be isolated in NSCLC plasma samples
• 7 selected miRNAs are strongly deregulated in our clinical samples and seem related to staging
• miR30b and -30c might be related to squamous cell carcinoma histotype
CONCLUSION
The analysis of exosomes in NSCLC could represent a noninvasive test for patients’ management. With this pilot study we have demonstrated that exosome analysis and exosomal miRNA profiling is feasible. Due to the small sample size we cannot make significant statistic conclusion. Further analyses in a larger series are needed.
Citation Format: Christian D. Rolfo, Marta Castiglia, Marco Giallombardo, Jorge Chacartegui, Nele Van Der Steen, Inge Mertens, Marc Peeters, Antonio Russo, Patrick Pauwels. Exosomes analysis in non-small cell lung cancer: looking for a clinical application. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3382. doi:10.1158/1538-7445.AM2015-3382
- ©2015 American Association for Cancer Research.
Volume 75, Issue 15 Supplement
