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Complex evaluation of human monocyte-derived dendritic cells for cancer immunotherapy.

Vopenkova K, Mollova K, Buresova I, Michalek J - J. Cell. Mol. Med. (2012)

Bottom Line: Different maturation strategies were previously tested to obtain DC capable of anti-cancer responses in vitro, usually with limited clinical benefit.Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application.DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens.

View Article: PubMed Central - PubMed

Affiliation: Advanced Cell Immunotherapy Unit, Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. katka.skalova@gmail.com

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DC adherence to plastic tested in a ‘scratch assay’. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Scratch assay was performed after 24 hrs of maturation. The contrast of all pictures has been increased artificially and equally to facilitate the observation of the differences among DC types. (A) Scratch assay, 0 hr: a scratch without cells created in a DC monolayer by a pipette tip. The scratch is marked with an arrow on its both ends. (B) Scratch assay, after 6 hrs: adherent cells do not fill in the scratch, whereas migrating cells do. Each scratch is marked with an arrow. Pictures represent results obtained from four different donors, magnification of 400.
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fig03: DC adherence to plastic tested in a ‘scratch assay’. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Scratch assay was performed after 24 hrs of maturation. The contrast of all pictures has been increased artificially and equally to facilitate the observation of the differences among DC types. (A) Scratch assay, 0 hr: a scratch without cells created in a DC monolayer by a pipette tip. The scratch is marked with an arrow on its both ends. (B) Scratch assay, after 6 hrs: adherent cells do not fill in the scratch, whereas migrating cells do. Each scratch is marked with an arrow. Pictures represent results obtained from four different donors, magnification of 400.

Mentions: We observed differences in DC adherence to plastic and migratory capacity. We documented high adherence and minimal migration for DC4 and DC5 in a scratch assay. In these DC types, the scratch was almost empty even after 24 hrs (not shown). On the other hand, DC1, DC2 and DC6 exhibited higher migratory ability, as they filled in the scratch in less than 6 hrs (Fig. 3). As the scratch assay provides only semi-quantitative results, we proceeded with a transwell migration assay to establish the DC migratory ability more precisely. Spontaneous as well as CCL21 chemokine-induced migration was assessed in the transwell migration assay. DC3 exhibited the highest migratory capacity in both spontaneous (median 6719 events) and CCL21-induced migration (4175 events, P < 0.05) compared with all other alternatives. Higher migratory potential was also observed in DC2 and DC6 in both spontaneous and CCL21-induced migration. The lowest migration ability was documented in DC4 and DC5 (medians below 600 events) in both spontaneous and CCL21-induced migration (Fig. 4A and B). Interestingly, we did not observe a positive effect of CCL21 on DC migration in most cases (Fig. 4C). This finding corresponds to relatively low expression of CCR7 on all types of DC (Fig. 4D).


Complex evaluation of human monocyte-derived dendritic cells for cancer immunotherapy.

Vopenkova K, Mollova K, Buresova I, Michalek J - J. Cell. Mol. Med. (2012)

DC adherence to plastic tested in a ‘scratch assay’. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Scratch assay was performed after 24 hrs of maturation. The contrast of all pictures has been increased artificially and equally to facilitate the observation of the differences among DC types. (A) Scratch assay, 0 hr: a scratch without cells created in a DC monolayer by a pipette tip. The scratch is marked with an arrow on its both ends. (B) Scratch assay, after 6 hrs: adherent cells do not fill in the scratch, whereas migrating cells do. Each scratch is marked with an arrow. Pictures represent results obtained from four different donors, magnification of 400.
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Related In: Results  -  Collection

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fig03: DC adherence to plastic tested in a ‘scratch assay’. DC were matured with TNF-α (DC1); TNF-α, IL-1α, IL-6, PGE2 (DC2); TNF-α, IL-1β, IFN-γ, PGE2, R848 (DC3); IFN-γ, LPS (DC4); IFN-γ, R848 (DC5) or were cultured without maturation (DC6). Scratch assay was performed after 24 hrs of maturation. The contrast of all pictures has been increased artificially and equally to facilitate the observation of the differences among DC types. (A) Scratch assay, 0 hr: a scratch without cells created in a DC monolayer by a pipette tip. The scratch is marked with an arrow on its both ends. (B) Scratch assay, after 6 hrs: adherent cells do not fill in the scratch, whereas migrating cells do. Each scratch is marked with an arrow. Pictures represent results obtained from four different donors, magnification of 400.
Mentions: We observed differences in DC adherence to plastic and migratory capacity. We documented high adherence and minimal migration for DC4 and DC5 in a scratch assay. In these DC types, the scratch was almost empty even after 24 hrs (not shown). On the other hand, DC1, DC2 and DC6 exhibited higher migratory ability, as they filled in the scratch in less than 6 hrs (Fig. 3). As the scratch assay provides only semi-quantitative results, we proceeded with a transwell migration assay to establish the DC migratory ability more precisely. Spontaneous as well as CCL21 chemokine-induced migration was assessed in the transwell migration assay. DC3 exhibited the highest migratory capacity in both spontaneous (median 6719 events) and CCL21-induced migration (4175 events, P < 0.05) compared with all other alternatives. Higher migratory potential was also observed in DC2 and DC6 in both spontaneous and CCL21-induced migration. The lowest migration ability was documented in DC4 and DC5 (medians below 600 events) in both spontaneous and CCL21-induced migration (Fig. 4A and B). Interestingly, we did not observe a positive effect of CCL21 on DC migration in most cases (Fig. 4C). This finding corresponds to relatively low expression of CCR7 on all types of DC (Fig. 4D).

Bottom Line: Different maturation strategies were previously tested to obtain DC capable of anti-cancer responses in vitro, usually with limited clinical benefit.Mutual comparison of currently used maturation strategies and subsequent complex evaluation of DC functions and their stimulatory capacity on T cells was performed in this study to optimize the DC vaccination strategy for further clinical application.DC were characterized with regard to their surface marker expression, cytokine profiles, migratory capacity, allogeneic and autologous T cell stimulatory capacity as well as their specific cytotoxicity against tumour antigens.

View Article: PubMed Central - PubMed

Affiliation: Advanced Cell Immunotherapy Unit, Department of Pharmacology, Faculty of Medicine, Masaryk University, Brno, Czech Republic. katka.skalova@gmail.com

Show MeSH
Related in: MedlinePlus