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Purity and yield of melanoma exosomes are dependent on isolation method

La Shu, Shin, Yang, Yunchen, Allen, Cheryl L., Hurley, Edward, Tung, Kaity H., Minderman, Hans, Wu, Yun, Ernstoff, Marc S.·2020·Journal of Extracellular Vesicles
DOI 10.1080/20013078.2019.1692401PMC PMC6882439
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Abstract

Both exosomes and soluble factors have been implicated in the generation of an immunosuppressive tumour microenvironment. Determining the contribution of each requires stringent control of purity of the isolated analytes. The present study compares several conventional exosome isolation methods for the presence of co‐enriched soluble factors while isolating exosomes from human melanoma‐derived cell lines. The resultant preparations were analysed by multiplex bead array analysis for cytokine profiles, and by electron microscopy and nanotracking analysis for exosome size distribution and concentration. It is demonstrated that the amount and repertoire of soluble factors in exosome preparations is dependent upon the isolation method used. A combination of ultrafiltration and size exclusion chromatography yielded up to 58‐fold more exosomes than ultracentrifugation, up to 836‐fold lower concentrations of co‐purified soluble factors when adjusted for exosome yield, and a greater than two‐fold increase in PD‐L1 expressing exosomes. Mechanistically, in context of the immunomodulatory effects of exosomes, the exosome isolation method should be carefully considered in order to limit any effects due instead to co‐eluted soluble factors.

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Figure 1. HMEX size distribution, concentration, morphology and protein characteristics in 888-mel and 2183-Her4 when various exosome isolation methods are used. 15 mL of supernatant was isolated from 16.5 × 106 cells and was processed for exosome isolation using various methods. UC: Ultracentrifugation/REIUS: Rapid Exosome Isolation using Ultrafiltration and Size Exclusion Chromatography/EqU: ExoQuick Ultra. (a) HMEX size distribution in 888-mel and (b) 2183-Her4 is consistent regardless of exosome isolation method used. Concentrations of HMEX vary depending on the method of exosome isolation chosen. Exosome polydispersity index (NePdi) is the ratio of standard deviation over mean exosome size based on NTA (Zetaview). (c) Transmission Electron Microscopy images of isolated exosomes with negative staining by Uranyless. Circular morphology and the absence of internal staining indicate intact, compartmentalized vesicles. Size is consistent with results from Zetaview. (d) Protein characteristics of exosomes using western blotting technique. Exosomes isolated using different techniques were all concentrated to a 200 µl final volume, of which 50 µl was loaded for western blotting for comparison of yield. CD63 is an exosome-enriched marker and TSG-101 is an endosomal pathway marker specific for exosomes. β-tubulin and β-actin are cytoplasmic markers. The absence of β-tubulin and β-actin compared to cell lysate sample confirms the presence of purified exosomes with minimal cytoplasmic contaminants. CD63 forms a single fused band at higher protein concentrations but forms two distinct bands when loaded at lower protein concentrations in western blot (Supplementary Figure 7).
Figure 1. HMEX size distribution, concentration, morphology and protein characteristics in 888-mel and 2183-Her4 when various exosome isolation methods are used. 15 mL of supernatant was isolated from 16.5 × 106 cells and was processed for exosome isolation using various methods. UC: Ultracentrifugation/REIUS: Rapid Exosome Isolation using Ultrafiltration and Size Exclusion Chromatography/EqU: ExoQuick Ultra. (a) HMEX size distribution in 888-mel and (b) 2183-Her4 is consistent regardless of exosome isolation method used. Concentrations of HMEX vary depending on the method of exosome isolation chosen. Exosome polydispersity index (NePdi) is the ratio of standard deviation over mean exosome size based on NTA (Zetaview). (c) Transmission Electron Microscopy images of isolated exosomes with negative staining by Uranyless. Circular morphology and the absence of internal staining indicate intact, compartmentalized vesicles. Size is consistent with results from Zetaview. (d) Protein characteristics of exosomes using western blotting technique. Exosomes isolated using different techniques were all concentrated to a 200 µl final volume, of which 50 µl was loaded for western blotting for comparison of yield. CD63 is an exosome-enriched marker and TSG-101 is an endosomal pathway marker specific for exosomes. β-tubulin and β-actin are cytoplasmic markers. The absence of β-tubulin and β-actin compared to cell lysate sample confirms the presence of purified exosomes with minimal cytoplasmic contaminants. CD63 forms a single fused band at higher protein concentrations but forms two distinct bands when loaded at lower protein concentrations in western blot (Supplementary Figure 7).CC BY-NC · PubMed Central
Figure 2. Significant proportion of exosomes remain in supernatant after UC. 15 ml of UC supernatant was concentrated using 3kD MWCO ultrafiltration columns to examine for the presence of exosomes in supernatant. UC supernatant size distribution and TEM images from both (a) 888-mel and (b) 2183-Her4. Significant protein aggregation is observed. (c) Physical characteristics of exosomes. UC supernatant has exosomes consistent in size to the UC-isolated exosome fraction. REIUS FT was concentrated using 3kD MWCO ultrafiltration columns to examine for the presence of exosomes. REIUS FT has no observable particles based on NTA. 1: below detection limit. (d) Protein characteristics of exosomes using western blotting technique. CD63 is an exosome-enriched marker and TSG-101 is an endosomal pathway marker specific for exosomes. β-tubulin and β-actin are cytoplasmic markers. The absence of β-tubulin and β-actin compared to cell lysate sample confirms the presence of purified exosomes with minimal cytoplasmic contaminants. For consistency of comparison, 20 µg of sample was loaded per well for western blotting.
Figure 2. Significant proportion of exosomes remain in supernatant after UC. 15 ml of UC supernatant was concentrated using 3kD MWCO ultrafiltration columns to examine for the presence of exosomes in supernatant. UC supernatant size distribution and TEM images from both (a) 888-mel and (b) 2183-Her4. Significant protein aggregation is observed. (c) Physical characteristics of exosomes. UC supernatant has exosomes consistent in size to the UC-isolated exosome fraction. REIUS FT was concentrated using 3kD MWCO ultrafiltration columns to examine for the presence of exosomes. REIUS FT has no observable particles based on NTA. 1: below detection limit. (d) Protein characteristics of exosomes using western blotting technique. CD63 is an exosome-enriched marker and TSG-101 is an endosomal pathway marker specific for exosomes. β-tubulin and β-actin are cytoplasmic markers. The absence of β-tubulin and β-actin compared to cell lysate sample confirms the presence of purified exosomes with minimal cytoplasmic contaminants. For consistency of comparison, 20 µg of sample was loaded per well for western blotting.CC BY-NC · PubMed Central
Figure 3. Schematic representation comparing exosome isolation strategies using SEC method. In conventional SEC, melanoma cell culture supernatant is harvested and is directly applied to SEC columns. In some cases, when concentration is required, low (3 kD) MWCO ultrafiltration is used to reduce excess water and electrolytes to load the correct volume onto SEC (100 μL). It is assumed that soluble biologics (e.g. cytokines) that remain are an integral part of exosomes. For REIUS method, 100 kD MWCO ultrafiltration is used to reduce soluble factors that are under 100 kD, followed by SEC. This method is proposed to reduce soluble factors more efficiently than SEC alone. For both methods, presence of non-exosomal soluble factors was examined in detail. Portions of this figure were made using templates from Motifolio Scientific Illustration Toolkits (www.motifolio.com).
Figure 3. Schematic representation comparing exosome isolation strategies using SEC method. In conventional SEC, melanoma cell culture supernatant is harvested and is directly applied to SEC columns. In some cases, when concentration is required, low (3 kD) MWCO ultrafiltration is used to reduce excess water and electrolytes to load the correct volume onto SEC (100 μL). It is assumed that soluble biologics (e.g. cytokines) that remain are an integral part of exosomes. For REIUS method, 100 kD MWCO ultrafiltration is used to reduce soluble factors that are under 100 kD, followed by SEC. This method is proposed to reduce soluble factors more efficiently than SEC alone. For both methods, presence of non-exosomal soluble factors was examined in detail. Portions of this figure were made using templates from Motifolio Scientific Illustration Toolkits (www.motifolio.com).CC BY-NC · PubMed Central
Figure 4. 100 kD MWCO ultrafiltration elimininates substantial cytokine contamination from cell culture supernatant. Circular cytokine plot overview of the 13-plex luminex assay result displaying cytokines that were reduced through 100 kD MWCO ultrafiltration (flow-through) and cytokines present with exosomes after REIUS method. Soluble factors detected in (a) 888-mel and (b) 2183-Her 4 supernatants from 100 kD MWCO UF flow-through that contains non-exosome associated soluble factors. Soluble factors detected in HMEX isolated using REIUS method from (c) 888-mel and (d) 2183-Her4 cell line. Individual data points used to generate the plot is included in supplementary data (supplementary table 2) (n = at least 6).
Figure 4. 100 kD MWCO ultrafiltration elimininates substantial cytokine contamination from cell culture supernatant. Circular cytokine plot overview of the 13-plex luminex assay result displaying cytokines that were reduced through 100 kD MWCO ultrafiltration (flow-through) and cytokines present with exosomes after REIUS method. Soluble factors detected in (a) 888-mel and (b) 2183-Her 4 supernatants from 100 kD MWCO UF flow-through that contains non-exosome associated soluble factors. Soluble factors detected in HMEX isolated using REIUS method from (c) 888-mel and (d) 2183-Her4 cell line. Individual data points used to generate the plot is included in supplementary data (supplementary table 2) (n = at least 6).CC BY-NC · PubMed Central
Figure 5. Determining efficiency of REIUS method in removing cytokines. (a,b) comparison between REIUS and REIUS flow-through (REIUS FT). (c,d) direct comparison between SEC alone and REIUS method and the presence of representative soluble factors (IFN-γ, IL-7, IL-8 and IL-10) in either 888-mel or 2183-Her4 supernatant isolates. REIUS method is capable of removing more soluble factors than SEC alone (n = at least 6).
Figure 5. Determining efficiency of REIUS method in removing cytokines. (a,b) comparison between REIUS and REIUS flow-through (REIUS FT). (c,d) direct comparison between SEC alone and REIUS method and the presence of representative soluble factors (IFN-γ, IL-7, IL-8 and IL-10) in either 888-mel or 2183-Her4 supernatant isolates. REIUS method is capable of removing more soluble factors than SEC alone (n = at least 6).CC BY-NC · PubMed Central
Figure 6. Ratio of soluble cytokine concentration to the number of exosomes isolated. When controlled for number of exosomes, cytokine profile per 1012 exosomes were up to 836 fold lower in REIUS compared to UC. A decrease was observed in both (a) 888-mel HMEX and (b) 2183-Her4 HMEX (n = at least 6).
Figure 6. Ratio of soluble cytokine concentration to the number of exosomes isolated. When controlled for number of exosomes, cytokine profile per 1012 exosomes were up to 836 fold lower in REIUS compared to UC. A decrease was observed in both (a) 888-mel HMEX and (b) 2183-Her4 HMEX (n = at least 6).CC BY-NC · PubMed Central
Figure 7. 15 ml of supernatant was isolated from 16.5 × 106 cells and was processed for exosome isolation using various methods. Real-time assay measurement results for Compact Surface Plasmon Resonance Biosensor (SPR) detection of PD-L1. (a) The clone used, MIH1, is as effective as 28–8 for use in SPR assay when normalized by number of exosomes (1 × 1010). (b) Linear regression analysis of PD-L1 positive exosomes using MIH1 clone (R2 = 0.9818). When normalized by number of exosomes (1 × 1010), PD-L1 expression of exosomes in (c) 888-mel HMEX and (d) 2183-Her4 HMEX is as shown (n = at least 6).
Figure 7. 15 ml of supernatant was isolated from 16.5 × 106 cells and was processed for exosome isolation using various methods. Real-time assay measurement results for Compact Surface Plasmon Resonance Biosensor (SPR) detection of PD-L1. (a) The clone used, MIH1, is as effective as 28–8 for use in SPR assay when normalized by number of exosomes (1 × 1010). (b) Linear regression analysis of PD-L1 positive exosomes using MIH1 clone (R2 = 0.9818). When normalized by number of exosomes (1 × 1010), PD-L1 expression of exosomes in (c) 888-mel HMEX and (d) 2183-Her4 HMEX is as shown (n = at least 6).CC BY-NC · PubMed Central

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Metadata

Authors
La Shu, S., Yang, Y., Allen, C. L., Hurley, E., Tung, K. H., Minderman, H., Wu, Y., & Ernstoff, M. S.
Venue
Journal of Extracellular Vesicles
Publisher
Wiley
Published
2019-11-20
Volume
9
Issue
1
ISSN
2001-3078
DOI
10.1080/20013078.2019.1692401
PMC
PMC6882439
Cited by
141
References
1
Language
en
Licence
Funders
  • National Cancer Institute (5R33CA19124, S10OD018048)
  • National Institutes of Health (1R50CA211108)
  • Roswell Park Comprehensive Cancer Center (P30CA016056)
  • Roswell Park Cancer Institute (P30CA016056)

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