Event Title

Characterization of Exosomal microRNAs in Pancreatic Cancer

Location

Hager-Lubbers Exhibition Hall

Start Date

19-4-2016 3:30 PM

Description

PURPOSE: Early detection of pancreatic cancer (PaCa) is difficult as the organ is deep inside the body and patients usually have no symptoms until PaCa starts spreading. Exosomes, vesicles for cell-to-cell communications, which contain small molecule microRNAs, are proposed as a good source of biomarkers for early diagnosis. SUBJECTS: Frozen serum samples from PaCa patients and patient-derived-xenograft (PDX) models were used. PDX models allows sample consistency for assay development/ analysis and more accurate cancer growth by preserving key tumor characteristics to avoid bias observed in tissue culture models. METHODS AND MATERIALS: Exosomes were enriched using ExoQuick (SBI). Pulldown of exosomes derived from specific-cell-types was performed by using Pierce Protein-G magnetic beads (ThermoFisher) and anti-human CD63, EpCAM, FAP, and CD14. Western blot was performed to characterize the exosomes and RNA was isolated using SeraMir kit (SBI). Reverse-transcriptase and real-time PCR (qRT-PCR) (Qiagen) determined the presence of specific miRNAs. ANALYSES: No statistical tests were performed. RESULTS: Pulldowns were performed to enrich exosomes derived from epithelial cells (EpCAM), fibroblasts (FAP), and macrophages (CD14). Characterization of the exosomes suggested enrichment; however, qRT-PCR results were inconsistent. This brought some concerns about exosome integrity due to the use of frozen samples and protein contamination due to ExoQuick. CONCLUSIONS: The methodology suggests enrichment of specific-cell-type derived exosomes. However, it is necessary to improve exosome integrity and address protein contamination issues. Future directions include comparing fresh vs. frozen samples and how this pre-analytical variable affects detection of PaCa-associated microRNAs (miR-21, let7-a, miR-10b, and miR-155) in specific-cell-type derived exosomes.

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Apr 19th, 3:30 PM

Characterization of Exosomal microRNAs in Pancreatic Cancer

Hager-Lubbers Exhibition Hall

PURPOSE: Early detection of pancreatic cancer (PaCa) is difficult as the organ is deep inside the body and patients usually have no symptoms until PaCa starts spreading. Exosomes, vesicles for cell-to-cell communications, which contain small molecule microRNAs, are proposed as a good source of biomarkers for early diagnosis. SUBJECTS: Frozen serum samples from PaCa patients and patient-derived-xenograft (PDX) models were used. PDX models allows sample consistency for assay development/ analysis and more accurate cancer growth by preserving key tumor characteristics to avoid bias observed in tissue culture models. METHODS AND MATERIALS: Exosomes were enriched using ExoQuick (SBI). Pulldown of exosomes derived from specific-cell-types was performed by using Pierce Protein-G magnetic beads (ThermoFisher) and anti-human CD63, EpCAM, FAP, and CD14. Western blot was performed to characterize the exosomes and RNA was isolated using SeraMir kit (SBI). Reverse-transcriptase and real-time PCR (qRT-PCR) (Qiagen) determined the presence of specific miRNAs. ANALYSES: No statistical tests were performed. RESULTS: Pulldowns were performed to enrich exosomes derived from epithelial cells (EpCAM), fibroblasts (FAP), and macrophages (CD14). Characterization of the exosomes suggested enrichment; however, qRT-PCR results were inconsistent. This brought some concerns about exosome integrity due to the use of frozen samples and protein contamination due to ExoQuick. CONCLUSIONS: The methodology suggests enrichment of specific-cell-type derived exosomes. However, it is necessary to improve exosome integrity and address protein contamination issues. Future directions include comparing fresh vs. frozen samples and how this pre-analytical variable affects detection of PaCa-associated microRNAs (miR-21, let7-a, miR-10b, and miR-155) in specific-cell-type derived exosomes.