3.2.8.2 – Lysate of exosomes with NP-40 lysis buffer Reagents:
6 Future perspectives
In this thesis we have established a protocol for exosome isolation from plasma using size-exclusion chromatography. With a combination of mass spectrometric analysis and electron microscopy, we were able to define fractions 7-8 as containing exosomes with least plasma protein contamination. These fractions also contained relative homogenously sized vesicles within the size-range of exosomes, and selective expression of two acknowledged exosomal markers. In this thesis we did not detect the exosomal marker CD63 by western blotting, an option might be to use LGALS3BP and CD5L instead. Also, the size of the isolated exosomes should be quantified by the use of the nanosight technology.
Further studies should include MS analysis of more control samples, as only exosomes from 2 healthy controls were included in this study. Also, exosomes from AML and T-ALL patients should be investigated by MS and compared to the results from pre-B ALL patients.
Moreover, another attempt at isolating blast-specific exosomes should be made, as we never attempted their separation from the SEC fractions. MS analysis of blast-enriched exosomes could yield detection of low abundance proteins, e.g. ligands for activating receptors, which could be below threshold for detection in a heterogeneous population as investigated here.
Also a protocol to perform immunophenotyping of the exosomes by flow cytometry could be developed. There exists protocols to capture exosomes on latex beads, which allows staining exosomes with antibodies for flow cytometric analysis. The advantage with this approach is the high sensitivity of antibodies that could lead to identification of low abundance proteins not detected by MS.
It would be interesting to test whether exosomes derived from patients have suppressive effects on NK cells. This could be achieved by culturing PBMC with exosomes, and analyze functional activity and expression of activating NK cell receptors by flow cytometry.
We isolated exosomes for future miRNA/RNA analysis, and a future directive could be to investigate whether the exosomes contain RNA for activating ligands or miRNA species that may influence gene expression of activating receptors in NK cells.
In terms of analysis of expression of activating ligands on leukemic cells, more samples should be included. In a follow-up study, also B7-H6, the other ligand for NKp30, and CD112 for DNAM-1 should be included. Also, it could be desirable to include flow
cytometric analysis of the ligands, as mRNA expression levels not necessarily predict protein expression.
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