Carbon nanotubes enhance CpG uptake and potentiate antiglioma immunity - PubMed (original) (raw)

Carbon nanotubes enhance CpG uptake and potentiate antiglioma immunity

Dongchang Zhao et al. Clin Cancer Res. 2011.

Abstract

Purpose: Stimulation of toll-like receptor-9 (TLR9) by CpG oligodeoxynucleotides (CpG) has been shown to counteract the immunosuppressive microenvironment and to inhibit tumor growth in glioma models. Because TLR9 is located intracellularly, we hypothesized that methods that enhance its internalization may also potentiate its immunostimulatory response. The goal of this study was to evaluate carbon nanotubes (CNT) as a CpG delivery vehicle in brain tumor models.

Experimental design: Functionalized single-walled CNTs were conjugated with CpG (CNT-CpG) and evaluated in vitro and in mice bearing intracranial GL261 gliomas. Flow cytometry was used to assess CNT-CpG uptake and antiglioma immune response. Tumor growth was measured by bioluminescent imaging, histology, and animal survival.

Results: CNT-CpG was nontoxic and enhanced CpG uptake both in vitro and intracranial gliomas. CNT-mediated CpG delivery also potentiated proinflammatory cytokine production by primary monocytes. Interestingly, a single intracranial injection of low-dose CNT-CpG (but not free CpG or blank CNT) eradicated intracranial GL261 gliomas in half of tumor-bearing mice. Moreover, surviving animals exhibited durable tumor-free remission (>3 months), and were protected from intracranial tumor rechallenge, demonstrating induction of long-term antitumor immunity.

Conclusions: These findings suggest that CNTs can potentiate CpG immunopotency by enhancing its delivery into tumor-associated inflammatory cells.

©2010 AACR.

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Figures

Figure 1

Figure 1

CNTs enhance CpG uptake in vitro. Primary bone marrow-derived monocytes (BMM) from wt or CX3CR1GFP mice or GL261 gliomas were incubated with PEG-functionalized single-walled CNTs conjugated to Cy5.5-labeled sCpG (CNT-sCpG5.5, 2.5 µg CNT- 5µg sCpG5.5/ml) or free sCpG5.5 (5µg/ml) and sCpG5.5 uptake was visualized by fluorescent microscopy and quantified by flow cytometry. A, dot plot demonstrating CNT-sCpG uptake by BMM to be more efficient than free sCpG5.5. B, CpG5.5 signal was stronger in BMM.gfp cells incubated with CNT-sCpG and was mostly confined to cytoplasmic compartments. C, histogram and D, time course experiments demonstrating CNT’s to also enhance sCpG5.5 uptake by GL261 gliomas, but not as efficiently as in BMM. Data is representative of three separate experiments. (n=3 samples /point, ±SEM); *, P<0.05, _t_-test.

Figure 1

Figure 1

CNTs enhance CpG uptake in vitro. Primary bone marrow-derived monocytes (BMM) from wt or CX3CR1GFP mice or GL261 gliomas were incubated with PEG-functionalized single-walled CNTs conjugated to Cy5.5-labeled sCpG (CNT-sCpG5.5, 2.5 µg CNT- 5µg sCpG5.5/ml) or free sCpG5.5 (5µg/ml) and sCpG5.5 uptake was visualized by fluorescent microscopy and quantified by flow cytometry. A, dot plot demonstrating CNT-sCpG uptake by BMM to be more efficient than free sCpG5.5. B, CpG5.5 signal was stronger in BMM.gfp cells incubated with CNT-sCpG and was mostly confined to cytoplasmic compartments. C, histogram and D, time course experiments demonstrating CNT’s to also enhance sCpG5.5 uptake by GL261 gliomas, but not as efficiently as in BMM. Data is representative of three separate experiments. (n=3 samples /point, ±SEM); *, P<0.05, _t_-test.

Figure 2

Figure 2

CNT-mediated CpG delivery potentiates monocyte activation in vitro. BMM (5×104 cells/well in 24-well plates) were incubated with sCpG (5µg/well), CNT-sCpG (CNT 2.5µg-sCpG 5µg /well), blank CNT (2.5µg/well), or CNT/sCpG mixture (CNT + sCpG) and cytokine/chemokine expression evaluated by qRT-PCR and ELISA. CNTs-CpG significantly upregulated the expression of proinflammatory cytokines and chemokines, but not VEGF, by BMM. Data is representative of two separate experiments. (n=3 samples /point, ±SEM); *, P<0.05, **, P<0.001 for comparisons to control group (ANOVA).

Figure 2

Figure 2

CNT-mediated CpG delivery potentiates monocyte activation in vitro. BMM (5×104 cells/well in 24-well plates) were incubated with sCpG (5µg/well), CNT-sCpG (CNT 2.5µg-sCpG 5µg /well), blank CNT (2.5µg/well), or CNT/sCpG mixture (CNT + sCpG) and cytokine/chemokine expression evaluated by qRT-PCR and ELISA. CNTs-CpG significantly upregulated the expression of proinflammatory cytokines and chemokines, but not VEGF, by BMM. Data is representative of two separate experiments. (n=3 samples /point, ±SEM); *, P<0.05, **, P<0.001 for comparisons to control group (ANOVA).

Figure 3

Figure 3

CNTs promote CpG uptake by tumor-associated inflammatory cells. A, Four days after i.c. implantation of GL261 cells into CX3CR1GFP mice, tumors were injected with CNT-sCpG (2.5 µg CNT-5 µg sCpG/10 µl PBS) or sCpG, (5µg/10µl PBS) and tumor-associated GFP+ cells (mostly microglia and macrophages, MG/MP) examined for Cy5.5 uptake at various time-points (left). Most of the intracellular CNT-sCpG (arrows) was seen in GFP-expressing MG/MP (right). B, GL261.egfp cells were implanted i.c. into wt mice in order to measure sCpG uptake by tumor cells. Ten days later, tumors were injected with CNT-sCpG or sCpG (same concentrations as A), and GFP+ tumor cells examined for Cy5.5 uptake at various time-points (left). Most of the CNT-sCpG (arrows) was seen in non-GFP-expressing cells (right). C, Representative fluorescent micrographs of brains from A demonstrating persistence of sCpG5.5+ MG/MP at the injection sites (arrows) in animals treated with CNT-sCpG5.5. (n=3 mice/time-point ±SEM); *, P<0.05, _t_-test; T: tumor, ns: not significant.

Figure 3

Figure 3

CNTs promote CpG uptake by tumor-associated inflammatory cells. A, Four days after i.c. implantation of GL261 cells into CX3CR1GFP mice, tumors were injected with CNT-sCpG (2.5 µg CNT-5 µg sCpG/10 µl PBS) or sCpG, (5µg/10µl PBS) and tumor-associated GFP+ cells (mostly microglia and macrophages, MG/MP) examined for Cy5.5 uptake at various time-points (left). Most of the intracellular CNT-sCpG (arrows) was seen in GFP-expressing MG/MP (right). B, GL261.egfp cells were implanted i.c. into wt mice in order to measure sCpG uptake by tumor cells. Ten days later, tumors were injected with CNT-sCpG or sCpG (same concentrations as A), and GFP+ tumor cells examined for Cy5.5 uptake at various time-points (left). Most of the CNT-sCpG (arrows) was seen in non-GFP-expressing cells (right). C, Representative fluorescent micrographs of brains from A demonstrating persistence of sCpG5.5+ MG/MP at the injection sites (arrows) in animals treated with CNT-sCpG5.5. (n=3 mice/time-point ±SEM); *, P<0.05, _t_-test; T: tumor, ns: not significant.

Figure 4

Figure 4

CNT-sCpG uptake by glioma-associated inflammatory cells. GL261 tumors in wt mice were injected with PBS (control), CNT-sCpG5.5, free sCpG5.5 or unconjugated CNT/sCpG5.5 mixture at the same concentrations as Fig. 3 and phenotypes of Cy5.5+ inflammatory cells characterized by FACS at different time intervals. A, Dot plots of tumor-inflammatory cells illustrating stronger CNT-sCpG5.5 uptake by tumor-associated microglia and macrophages (MG/MP), NK, and dendritic cells (DC) as compared to free sCpG or CNT/sCpG. B, The proportion of CNT-sCpG5.5-positive MG/MP, NK and DC increased within 24 hr of CNT-sCpG injection. C, Injection of CNT-sCpG increased total and CNT-sCpG5.5-positive inflammatory cells into tumors. Representative data from two experiments is shown. (n=3 mice/time-point ±SEM); *, P<0.05, **, P<0.001 for comparisons to control group in B and to 24 hr time-point in C (ANOVA).

Figure 4

Figure 4

CNT-sCpG uptake by glioma-associated inflammatory cells. GL261 tumors in wt mice were injected with PBS (control), CNT-sCpG5.5, free sCpG5.5 or unconjugated CNT/sCpG5.5 mixture at the same concentrations as Fig. 3 and phenotypes of Cy5.5+ inflammatory cells characterized by FACS at different time intervals. A, Dot plots of tumor-inflammatory cells illustrating stronger CNT-sCpG5.5 uptake by tumor-associated microglia and macrophages (MG/MP), NK, and dendritic cells (DC) as compared to free sCpG or CNT/sCpG. B, The proportion of CNT-sCpG5.5-positive MG/MP, NK and DC increased within 24 hr of CNT-sCpG injection. C, Injection of CNT-sCpG increased total and CNT-sCpG5.5-positive inflammatory cells into tumors. Representative data from two experiments is shown. (n=3 mice/time-point ±SEM); *, P<0.05, **, P<0.001 for comparisons to control group in B and to 24 hr time-point in C (ANOVA).

Figure 5

Figure 5

CNT-sCpG eradicates established gliomas. Mice bearing four day-old i.c. GL261.luc gliomas (n=6) were given a single i.t. injection of PBS (control), free sCpG (5µg/10µl PBS), blank CNT (2.5 µg) mixed with free sCpG (CNT + sCpG; 5µg CpG/10µl PBS), or sCpG conjugated to CNT (CNT-sCpG; 2.5 µg CNT-5 µg sCpG/10 µl PBS). A, Intracranial tumor burden was assessed by biophotonic imaging. B, Kaplan-Meier analysis demonstrates improved survival of mice treated with a single injection of CNT-sCpG.

Figure 6

Figure 6

CNT-sCpG-treated mice develop immunity against tumor rechallenge. A, Naïve mice (n=6/group) were depleted of CD8 or NK cells by i.p. injection of relevant mAb (αCD8 and αNK) or control IgG (200 µg/injection) one day prior to both tumor implantation and CNT-sCpG (2.5 µg CNT/5 µg CpG/10 µl PBS) treatment. Intracranial tumor burden was assessed by biophotonic imaging at day 4, 7, 14, and 21 post tumor implantation (top panel). NK and CD8-depleted mice exhibited more rapid tumor growth and lower survival rates (bottom panel). B, Normal naïve mice and CNT-sCpG-treated GL261-bearing mice (n=5/group) that had survived for at least three months after the initial tumor inoculation were rechallenged with an i.c. injection of GL261 glioma (1×105). Intracranial tumor burden was assessed at 4 h, days 1, 4, and 7 after tumor implantation.

Figure 6

Figure 6

CNT-sCpG-treated mice develop immunity against tumor rechallenge. A, Naïve mice (n=6/group) were depleted of CD8 or NK cells by i.p. injection of relevant mAb (αCD8 and αNK) or control IgG (200 µg/injection) one day prior to both tumor implantation and CNT-sCpG (2.5 µg CNT/5 µg CpG/10 µl PBS) treatment. Intracranial tumor burden was assessed by biophotonic imaging at day 4, 7, 14, and 21 post tumor implantation (top panel). NK and CD8-depleted mice exhibited more rapid tumor growth and lower survival rates (bottom panel). B, Normal naïve mice and CNT-sCpG-treated GL261-bearing mice (n=5/group) that had survived for at least three months after the initial tumor inoculation were rechallenged with an i.c. injection of GL261 glioma (1×105). Intracranial tumor burden was assessed at 4 h, days 1, 4, and 7 after tumor implantation.

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