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Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples.

Belov L, Matic KJ, Hallal S, Best OG, Mulligan SP, Christopherson RI - J Extracell Vesicles (2016)

Bottom Line: Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence.These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63.None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

View Article: PubMed Central - PubMed

Affiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia; larissa.belov@sydney.edu.au.

ABSTRACT
Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

No MeSH data available.


Related in: MedlinePlus

Comparison of DotScan surface profiles of PBMC (3×106 cells; a) and CD19+, CD61-depleted EV (b) from the blood of CLL patients (n=4). DotScan analysis was carried out as for Figure 3. After background and isotype control subtraction and median centred normalization, averaged duplicate binding intensities (expressed in arbitrary units, Au) are shown for 38 antigens, each of which was detected on the cells of at least 2 of 4 CLL samples.
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Figure 0004: Comparison of DotScan surface profiles of PBMC (3×106 cells; a) and CD19+, CD61-depleted EV (b) from the blood of CLL patients (n=4). DotScan analysis was carried out as for Figure 3. After background and isotype control subtraction and median centred normalization, averaged duplicate binding intensities (expressed in arbitrary units, Au) are shown for 38 antigens, each of which was detected on the cells of at least 2 of 4 CLL samples.

Mentions: Averaged normalized DotScan data from 4 CLL patients (Supplementary Table 2) are shown in Fig. 4. Of the 38 antigens detected on the cells of 2 or more patients (Fig. 4a), ~33% were also detected on the corresponding CD19+ EV (Fig. 4b), with strong expression of CD19, CD31, CD44, CD55 and CD62L; moderate levels of CD5, CD82, HLA-A,B,C and HLA-DR; and very low levels of CD21, CD49c and CD63. CD5 was not detected on CD19+ EV from Patient 4, whose CLL cells were only weakly positive for CD5 by DotScan. Flow cytometry confirmed that only 19% of this patient's CD19+ leukaemia cells co-expressed CD5 (clinical data). CD19 levels were similar for all 4 EV samples after normalization, confirming that CD19-biotin antibody was suitable for the detection of CLL-derived EV. By contrast, the tetraspanins CD9, CD63 and CD151 were very low or below the limit of detection, while CD62L (L-selectin) was high on EV compared to cells. Compared to CLL cells, EV showed low CD5, CD21, CD82, HLA-ABC and HLA-DR (relative to CD19), and the B-cell antigen CD20 was not detected by either of the CD20 antibodies.


Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples.

Belov L, Matic KJ, Hallal S, Best OG, Mulligan SP, Christopherson RI - J Extracell Vesicles (2016)

Comparison of DotScan surface profiles of PBMC (3×106 cells; a) and CD19+, CD61-depleted EV (b) from the blood of CLL patients (n=4). DotScan analysis was carried out as for Figure 3. After background and isotype control subtraction and median centred normalization, averaged duplicate binding intensities (expressed in arbitrary units, Au) are shown for 38 antigens, each of which was detected on the cells of at least 2 of 4 CLL samples.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834364&req=5

Figure 0004: Comparison of DotScan surface profiles of PBMC (3×106 cells; a) and CD19+, CD61-depleted EV (b) from the blood of CLL patients (n=4). DotScan analysis was carried out as for Figure 3. After background and isotype control subtraction and median centred normalization, averaged duplicate binding intensities (expressed in arbitrary units, Au) are shown for 38 antigens, each of which was detected on the cells of at least 2 of 4 CLL samples.
Mentions: Averaged normalized DotScan data from 4 CLL patients (Supplementary Table 2) are shown in Fig. 4. Of the 38 antigens detected on the cells of 2 or more patients (Fig. 4a), ~33% were also detected on the corresponding CD19+ EV (Fig. 4b), with strong expression of CD19, CD31, CD44, CD55 and CD62L; moderate levels of CD5, CD82, HLA-A,B,C and HLA-DR; and very low levels of CD21, CD49c and CD63. CD5 was not detected on CD19+ EV from Patient 4, whose CLL cells were only weakly positive for CD5 by DotScan. Flow cytometry confirmed that only 19% of this patient's CD19+ leukaemia cells co-expressed CD5 (clinical data). CD19 levels were similar for all 4 EV samples after normalization, confirming that CD19-biotin antibody was suitable for the detection of CLL-derived EV. By contrast, the tetraspanins CD9, CD63 and CD151 were very low or below the limit of detection, while CD62L (L-selectin) was high on EV compared to cells. Compared to CLL cells, EV showed low CD5, CD21, CD82, HLA-ABC and HLA-DR (relative to CD19), and the B-cell antigen CD20 was not detected by either of the CD20 antibodies.

Bottom Line: Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence.These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63.None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

View Article: PubMed Central - PubMed

Affiliation: School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, Australia; larissa.belov@sydney.edu.au.

ABSTRACT
Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

No MeSH data available.


Related in: MedlinePlus