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MA04 - Models and Biomarkers (ID 122)
- Event: WCLC 2019
- Type: Mini Oral Session
- Track: Biology
- Presentations: 1
- Now Available
- Moderators:Montse Sanchez-Cespedes, Kwok-kin Wong
- Coordinates: 9/08/2019, 13:30 - 15:00, Interlaken (1988)
MA04.09 - Study of Exosomes in NSCLC for Biomarkers Searching (Now Available) (ID 2417)
13:30 - 15:00 | Author(s): Andrea Moreno
Exosomes are small membranous vesicles secreted by a most type of cells (especially in tumoral processes), around 40-130 nm of size, that carry relevant information to distant tissues and being able to modulate its physiology. Exosomes have been detected in different clinical samples and may play a key role in NSCLC, participating in several processes such as horizontal transfer of RNA from tumor to microenvironmental cells, angiogenesis, pre-metastatic niche formation, immunosuppression; and could also be key elements in stem cell differentiation (from different origins).
The principal objective of this study was to analyze the exosomes cargo from NSCLC cell lines and primary cultures with diverse characteristics under different growth conditions: suspension cultures with cancer stem cells (CSCs) features and monolayer cultures.Method
Primary cultures from resected NSCLC patients and NSCLC cell lines were successfully established. Differentiated tumor cells were cultured under adherent conditions (2D) whereas CSCs were established in suspension cultures (3D tumorspheres). Exosomes isolation was performed by ultracentrifugation. Exosomes characterization was carried out through nanovesicles tracking analysis (NTA) and electron microscopy; and the determination of surface markers through immunoblot and flow cytometry. Exosomal DNA was extracted in order to determine the mutational status of the EGFR and RAS genes by BEAMing Digital PCR (Sysmex). Transcriptomic analysis has been carried out from exosomal RNA through whole genome gene expression microarrays, (Affymetrix). The data was normalized by Robust Multi-Array Average (RMA) and analyzed using Transcriptome Analysis Console (TAC), MultiExperiment Viewer (MeV) software and Partek Genomics Suite. Statistical significance was established at (p ≤ 0.01).Result
In reference to the characterization, NTA and electron microscopy showed that exosomes were obtained free of cellular debris and their size ranges from 108-125 nm, according to the size of tumor-derived microvesicles. Exosomal surface markers analyzed by immunoblot and flow cytometry were detected in samples, confirming proper isolation. Mutational analysis of EGFR and RAS genes performed on exosomal DNA shown the same pattern displayed by the origin cells. Transcriptomic analysis of the exosomal content showed that the expression of mRNAs, miRNAs and precursors were significantly different between 3D and 2D-derived exosomes. Finally, a pathway enrichment analysis was carried out to know in which biological processes (cancer-related) are involved.
Significant differential expressions were also found between mRNAs, miRNAs and pre-miRs present in exosomes from adenocarcinoma (ADC) vs. squamous cell carcinoma (SCC). Interestingly, 7 miRNAs differentially expressed in exosomes (miR-200c; miR-29a; miR-339; miR-224; miR-31; miR-21; miR-33a) had already been identified as overexpressed in tumor tissue from NSCLC patients by our group. Moreover, miR-339 y miR-21 were related to prognosis (p < 0.05) in ADC group.Conclusion
Differences in exosomal mRNA, miRNAs and pre-miRs expression have been observed between: i) lung-tumorspheres vs. more differentiated tumor cells and ii) ADC vs. SCC cultures. In addition, the same mutational pattern was detected in exosomes as compared with their parental cultures. Therefore, exosomes can be a useful source for biomarkers analysis in NSCLC.
Supported by grant GV/2018/026, PI18/00266, & Asociación Española Contra el Cáncer (AECC Valencia).
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