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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

DotScan analysis of LIM1215 cells (b) and their EV (c). The key (a) shows antibody locations, with shaded antibodies indicating cell capture. Duplicate antibody arrays (outlined) are surrounded by a frame of alignment dots consisting of a mixture of CD44/CD29 antibodies. Detection of captured cells was by optical scanning (b). EV (7.8×108 particles derived from 335 µl of LIM1215-conditioned medium) were detected by ECL using biotinylated EpCAM (CD326) antibody, with a 10 min exposure on ECL film (c). NanoSight analysis shows the size distribution of LIM1215 EV (d). The number above the peak represents mode size in nm. A Venn diagram compares surface profiles of LIM1215 cells with their EV (e). TCR, T-cell receptor; κ, λ, immunoglobulin light chains kappa, lambda; sIg, surface immunoglobulin; DCC, deleted in colorectal cancer protein; EGFR, epidermal growth factor receptor; FAP, fibroblast activation protein; HLA-ABC, HLA-DR, human leukocyte antigens A, B, C and DR, respectively; MICA, MHC class I chain-related protein A; MMP-14, matrix metallopeptidase 14; PIGR, polymeric immunoglobulin receptor; TSP-1, thrombospondin-1; Mabthera, chimeric mouse/human anti-CD20. G1, G2a, G2b and M are murine isotype control antibodies IgG1, IgG2a, IgG2b and IgM, respectively. The numbers 500, 200 and 50 refer to isotype control antibody concentrations in µg/ml. NB means no BSA in the antibody solution; these antibodies were at 500 µg/ml. Antibody details are listed in Supplementary Table 1 of Supplementary Material.
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Figure 0001: DotScan analysis of LIM1215 cells (b) and their EV (c). The key (a) shows antibody locations, with shaded antibodies indicating cell capture. Duplicate antibody arrays (outlined) are surrounded by a frame of alignment dots consisting of a mixture of CD44/CD29 antibodies. Detection of captured cells was by optical scanning (b). EV (7.8×108 particles derived from 335 µl of LIM1215-conditioned medium) were detected by ECL using biotinylated EpCAM (CD326) antibody, with a 10 min exposure on ECL film (c). NanoSight analysis shows the size distribution of LIM1215 EV (d). The number above the peak represents mode size in nm. A Venn diagram compares surface profiles of LIM1215 cells with their EV (e). TCR, T-cell receptor; κ, λ, immunoglobulin light chains kappa, lambda; sIg, surface immunoglobulin; DCC, deleted in colorectal cancer protein; EGFR, epidermal growth factor receptor; FAP, fibroblast activation protein; HLA-ABC, HLA-DR, human leukocyte antigens A, B, C and DR, respectively; MICA, MHC class I chain-related protein A; MMP-14, matrix metallopeptidase 14; PIGR, polymeric immunoglobulin receptor; TSP-1, thrombospondin-1; Mabthera, chimeric mouse/human anti-CD20. G1, G2a, G2b and M are murine isotype control antibodies IgG1, IgG2a, IgG2b and IgM, respectively. The numbers 500, 200 and 50 refer to isotype control antibody concentrations in µg/ml. NB means no BSA in the antibody solution; these antibodies were at 500 µg/ml. Antibody details are listed in Supplementary Table 1 of Supplementary Material.

Mentions: Figure 1 shows surface profiles of live LIM1215 cells acquired using optical detection with a DotScan scanner (Fig. 1b) and those of LIM1215-derived EV (Fig. 1c) using ECL detection. These EV particles, enriched for exosomes by differential centrifugation, ranged in size from 30 to 400 nm by NanoSight analysis (Fig. 1d), with a mode size of 101 nm (the value of the highest point of the peak, i.e. the most frequently occurring size). The Venn diagram (Fig. 1e) shows the antigens co-expressed on cells and EV, and those detected on cells or EV alone. Of the 34 antigens detected on the LIM1215 cells, 24 (70.6%) were also detected on their EV. TSP-1 was detected on the LIM1215 EV, but not the cells.


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)

DotScan analysis of LIM1215 cells (b) and their EV (c). The key (a) shows antibody locations, with shaded antibodies indicating cell capture. Duplicate antibody arrays (outlined) are surrounded by a frame of alignment dots consisting of a mixture of CD44/CD29 antibodies. Detection of captured cells was by optical scanning (b). EV (7.8×108 particles derived from 335 µl of LIM1215-conditioned medium) were detected by ECL using biotinylated EpCAM (CD326) antibody, with a 10 min exposure on ECL film (c). NanoSight analysis shows the size distribution of LIM1215 EV (d). The number above the peak represents mode size in nm. A Venn diagram compares surface profiles of LIM1215 cells with their EV (e). TCR, T-cell receptor; κ, λ, immunoglobulin light chains kappa, lambda; sIg, surface immunoglobulin; DCC, deleted in colorectal cancer protein; EGFR, epidermal growth factor receptor; FAP, fibroblast activation protein; HLA-ABC, HLA-DR, human leukocyte antigens A, B, C and DR, respectively; MICA, MHC class I chain-related protein A; MMP-14, matrix metallopeptidase 14; PIGR, polymeric immunoglobulin receptor; TSP-1, thrombospondin-1; Mabthera, chimeric mouse/human anti-CD20. G1, G2a, G2b and M are murine isotype control antibodies IgG1, IgG2a, IgG2b and IgM, respectively. The numbers 500, 200 and 50 refer to isotype control antibody concentrations in µg/ml. NB means no BSA in the antibody solution; these antibodies were at 500 µg/ml. Antibody details are listed in Supplementary Table 1 of Supplementary Material.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 0001: DotScan analysis of LIM1215 cells (b) and their EV (c). The key (a) shows antibody locations, with shaded antibodies indicating cell capture. Duplicate antibody arrays (outlined) are surrounded by a frame of alignment dots consisting of a mixture of CD44/CD29 antibodies. Detection of captured cells was by optical scanning (b). EV (7.8×108 particles derived from 335 µl of LIM1215-conditioned medium) were detected by ECL using biotinylated EpCAM (CD326) antibody, with a 10 min exposure on ECL film (c). NanoSight analysis shows the size distribution of LIM1215 EV (d). The number above the peak represents mode size in nm. A Venn diagram compares surface profiles of LIM1215 cells with their EV (e). TCR, T-cell receptor; κ, λ, immunoglobulin light chains kappa, lambda; sIg, surface immunoglobulin; DCC, deleted in colorectal cancer protein; EGFR, epidermal growth factor receptor; FAP, fibroblast activation protein; HLA-ABC, HLA-DR, human leukocyte antigens A, B, C and DR, respectively; MICA, MHC class I chain-related protein A; MMP-14, matrix metallopeptidase 14; PIGR, polymeric immunoglobulin receptor; TSP-1, thrombospondin-1; Mabthera, chimeric mouse/human anti-CD20. G1, G2a, G2b and M are murine isotype control antibodies IgG1, IgG2a, IgG2b and IgM, respectively. The numbers 500, 200 and 50 refer to isotype control antibody concentrations in µg/ml. NB means no BSA in the antibody solution; these antibodies were at 500 µg/ml. Antibody details are listed in Supplementary Table 1 of Supplementary Material.
Mentions: Figure 1 shows surface profiles of live LIM1215 cells acquired using optical detection with a DotScan scanner (Fig. 1b) and those of LIM1215-derived EV (Fig. 1c) using ECL detection. These EV particles, enriched for exosomes by differential centrifugation, ranged in size from 30 to 400 nm by NanoSight analysis (Fig. 1d), with a mode size of 101 nm (the value of the highest point of the peak, i.e. the most frequently occurring size). The Venn diagram (Fig. 1e) shows the antigens co-expressed on cells and EV, and those detected on cells or EV alone. Of the 34 antigens detected on the LIM1215 cells, 24 (70.6%) were also detected on their EV. TSP-1 was detected on the LIM1215 EV, but not the cells.

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