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Page 38 Extracell Vesicles Circ Nucleic Acids 2020;1:20-56 I http://dx.doi.org/10.20517/evcna.2020.10

vesicle pathway and differentially regulated by IMP1 expression. We found that IMP1 increases extracellular
vesicle secretion in HT-29 (4483 +/- 403 vs. 1934+/- 414, P = 0.006) and SW480 (3604+/- 399 vs. 2293+/-
464, P = 0.049) CRC cells vs. null controls. IMP1 modulates translational efficiency or protein levels of
early endosome proteins, including: EEA1, SNX15, PLA2G4B, and F8A2. IMP1 decreases expression of
autophagy proteins LAMP1 and LC3-II. By contrast, Imp1 overexpression in non-transformed enteroids
does not enhance vesicle secretion, but may alter expression of endocytic proteins.
Conclusions: Our novel findings suggest that IMP1 plays an important role in vesicle production and
secretion in the context of colon cancer through increased early endosomal pathway activity and reduced
autophagy-mediated degradation. Importantly, the effects of IMP1 expression differ between non-
transformed and cancer cells. Our findings have implications for the development of novel early detection
and therapeutic approaches in CRC where IMP1 is overexpressed.
Funding: AGA Research Scholar Award, Prevent Cancer Foundation Fellowship Award, Lustgarten
Family Colon Cancer Research Fund, NIH F32DK107052, T32CA1152999, R01DK056645, P30CA13696,
R01CA243445.

23. Simple workflow for isolation and Western blot detection of MISEV-recommended EV
protein-markers

1
3
3
1
1
2
Authors: Lisa Meyer , Daniel Enderle , Carsten Lück , Anne Bickel , Yasef Khan , Chris Heger , Martin
4
5
4
Schlumpberger , Markus Sprenger-Haussels , Johan Skog , Mikkel Noerholm 1
E-mail: lisa.meyer@bio-techne.com
Affiliations:
1 Exosome Diagnostics GmbH, Martinsried, Germany.
2 Bio-Techne, Analytical Solutions Division, Abingdon, IN, USA.
3 Bio-Techne, Analytical Solutions Division, San Jose, CA, USA.
4 QIAGEN GmbH, Hilden, Germany.
5 Exosome Diagnostics, Inc., Waltham, MA, USA.
Abstracts:
Introduction: Characterization of isolated extracellular vesicles (EV) is often challenging, due to the
limited amount of material and the variety in approaches for experimental EV preparations. The “Minimal
Information for Studies of Extracellular Vesicles” (MISEV) guidelines advice to characterize isolated EVs by
their protein composition. Nevertheless, following these recommendations can result in a significant effort
due to work-intensive and variable EV isolation procedures and time-consuming standard western blot
protocols. Here we present a complete workflow for isolating intact EVs via membrane-affinity columns
and detection of MISEV-recommended protein markers using Simple Western Blotting.
Methods: Intact EVs were isolated using a bind-wash elute procedure from 0.25-8 mL pre-filtered plasma
or 10 mL of pre-filtered urine (exoEasy Kit, QIAGEN). EVs were eluted in 250 µL elution buffer for
analysis and compared to the EV-depleted fraction in the column flow-through and to the neat biofluid
sample. Multiple EV and non-EV protein markers were analyzed by subjecting 4 μL sample directly to
Simple Western system, which entails automated capillary electrophoresis-based protein separation and
chemiluminescence-immunodetection (Jess platform, ProteinSimple, Bio-Techne).
Results: A total of ten primary antibodies were identified and optimized for the workflow, including
multiple targets in each of the three categories suggested by the MISEV-guidelines. Input volume titrations
were used to define the dynamic range for the complete workflow and replicate isolations were used to
demonstrate its repeatability. EV markers are generally low abundant in urine and plasma but were readily
detected after enrichment of EVs by isolation. The EV fractions showed a strong enrichment of EV-
proteins, whereas non-EV proteins were significantly reduced - and increasing the number of wash steps in
the EV isolation lead to a substantially improved signal-to-noise ratio.