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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

Workflow for preparation of PBMC and CD61-depleted EV from blood (a), with DotScan profiling (b–d). The key (b) shows antibody locations, with shaded antibodies indicating cell capture. DotScan analyses are shown for 3×106 PBMC (c) and CD61-depleted EV from 10 ml of blood (d) from an 87-year-old female CLL patient (Patient 1) with a white blood cell count of 45.3 x109/L. EV were tested in the presence of heat inactivated human AB serum (2%). Detection of captured cells was by optical scanning (c). EV were detected by ECL using biotinylated CD19 antibody, with a 30 min exposure on ECL film (d). NanoSight analysis (e) compares the average size distributions (tested in triplicate) of EV in the plasma and purified CD61-depleted EV from the plasma. The results are shown as average number of particles per ml of plasma before and after enrichment for CD61-depleted EV; the numbers above the peaks represent mode sizes in nm. A Venn diagram (f) compares surface profiles of patient CLL cells and their EV.
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Figure 0003: Workflow for preparation of PBMC and CD61-depleted EV from blood (a), with DotScan profiling (b–d). The key (b) shows antibody locations, with shaded antibodies indicating cell capture. DotScan analyses are shown for 3×106 PBMC (c) and CD61-depleted EV from 10 ml of blood (d) from an 87-year-old female CLL patient (Patient 1) with a white blood cell count of 45.3 x109/L. EV were tested in the presence of heat inactivated human AB serum (2%). Detection of captured cells was by optical scanning (c). EV were detected by ECL using biotinylated CD19 antibody, with a 30 min exposure on ECL film (d). NanoSight analysis (e) compares the average size distributions (tested in triplicate) of EV in the plasma and purified CD61-depleted EV from the plasma. The results are shown as average number of particles per ml of plasma before and after enrichment for CD61-depleted EV; the numbers above the peaks represent mode sizes in nm. A Venn diagram (f) compares surface profiles of patient CLL cells and their EV.

Mentions: Figure 3a summarizes the protocol for preparation of PBMC and CD61-depleted EV from the blood of CLL patients and healthy controls. Enrichment for CLL-derived EV from plasma was necessary to achieve the required sensitivity for the assay. CLL-derived EV profiles were not clearly detected when plasma (300 µl) was tested directly on DotScan, probably due to interference from plasma proteins and platelet-derived EV. The removal of CD61+ EV by magnetic beads was confirmed by comparing DotScan profiles for CD61-depleted and non-depleted EV using biotinylated CD61 antibody for detection (results not shown); also the CD61+ EV captured on magnetic beads showed distinct platelet-like profiles on DotScan (CD41+, CD42a+, CD61+, CD62P+; unpublished data). Although citrate anti-coagulant has been recommended for proteomic studies because it induces fewer platelet-derived MV ex vivo (42,43), results from 3 independent experiments comparing blood samples collected into different anti-coagulants (citrate, heparin, EDTA) from healthy donors (unpublished data) demonstrated no significant differences (p>0.05 by two-tailed, paired student's t-test) in yield of CD61-depleted EV (determined by NanoSight analysis) or their normalized DotScan profiles (with CD45 detection). However, EV pellets from heparin were stickier than those from EDTA or citrate. The average yields of CD61-depleted EV were almost identical for heparin and citrate, but ~1.5-fold lower for EDTA. Despite this difference in average yield, the intensity of DotScan binding before normalization was similar for CD61-depleted EV from citrate and EDTA, but ~2-fold lower from heparin. Results were also more consistent between duplicate microarray panels for EDTA and citrate.


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)

Workflow for preparation of PBMC and CD61-depleted EV from blood (a), with DotScan profiling (b–d). The key (b) shows antibody locations, with shaded antibodies indicating cell capture. DotScan analyses are shown for 3×106 PBMC (c) and CD61-depleted EV from 10 ml of blood (d) from an 87-year-old female CLL patient (Patient 1) with a white blood cell count of 45.3 x109/L. EV were tested in the presence of heat inactivated human AB serum (2%). Detection of captured cells was by optical scanning (c). EV were detected by ECL using biotinylated CD19 antibody, with a 30 min exposure on ECL film (d). NanoSight analysis (e) compares the average size distributions (tested in triplicate) of EV in the plasma and purified CD61-depleted EV from the plasma. The results are shown as average number of particles per ml of plasma before and after enrichment for CD61-depleted EV; the numbers above the peaks represent mode sizes in nm. A Venn diagram (f) compares surface profiles of patient CLL cells and their EV.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 0003: Workflow for preparation of PBMC and CD61-depleted EV from blood (a), with DotScan profiling (b–d). The key (b) shows antibody locations, with shaded antibodies indicating cell capture. DotScan analyses are shown for 3×106 PBMC (c) and CD61-depleted EV from 10 ml of blood (d) from an 87-year-old female CLL patient (Patient 1) with a white blood cell count of 45.3 x109/L. EV were tested in the presence of heat inactivated human AB serum (2%). Detection of captured cells was by optical scanning (c). EV were detected by ECL using biotinylated CD19 antibody, with a 30 min exposure on ECL film (d). NanoSight analysis (e) compares the average size distributions (tested in triplicate) of EV in the plasma and purified CD61-depleted EV from the plasma. The results are shown as average number of particles per ml of plasma before and after enrichment for CD61-depleted EV; the numbers above the peaks represent mode sizes in nm. A Venn diagram (f) compares surface profiles of patient CLL cells and their EV.
Mentions: Figure 3a summarizes the protocol for preparation of PBMC and CD61-depleted EV from the blood of CLL patients and healthy controls. Enrichment for CLL-derived EV from plasma was necessary to achieve the required sensitivity for the assay. CLL-derived EV profiles were not clearly detected when plasma (300 µl) was tested directly on DotScan, probably due to interference from plasma proteins and platelet-derived EV. The removal of CD61+ EV by magnetic beads was confirmed by comparing DotScan profiles for CD61-depleted and non-depleted EV using biotinylated CD61 antibody for detection (results not shown); also the CD61+ EV captured on magnetic beads showed distinct platelet-like profiles on DotScan (CD41+, CD42a+, CD61+, CD62P+; unpublished data). Although citrate anti-coagulant has been recommended for proteomic studies because it induces fewer platelet-derived MV ex vivo (42,43), results from 3 independent experiments comparing blood samples collected into different anti-coagulants (citrate, heparin, EDTA) from healthy donors (unpublished data) demonstrated no significant differences (p>0.05 by two-tailed, paired student's t-test) in yield of CD61-depleted EV (determined by NanoSight analysis) or their normalized DotScan profiles (with CD45 detection). However, EV pellets from heparin were stickier than those from EDTA or citrate. The average yields of CD61-depleted EV were almost identical for heparin and citrate, but ~1.5-fold lower for EDTA. Despite this difference in average yield, the intensity of DotScan binding before normalization was similar for CD61-depleted EV from citrate and EDTA, but ~2-fold lower from heparin. Results were also more consistent between duplicate microarray panels for EDTA and citrate.

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