Intracerebral CpG immunotherapy with carbon nanotubes abrogates growth of subcutaneous melanomas in mice - PubMed (original) (raw)

Intracerebral CpG immunotherapy with carbon nanotubes abrogates growth of subcutaneous melanomas in mice

Haitao Fan et al. Clin Cancer Res. 2012.

Abstract

Purpose: Recently, we showed that intratumoral delivery of low-dose, immunostimulatory CpG oligodeoxynucleotides conjugated with carbon nanotubes (CNT-CpG) was more effective than free CpG and not only eradicated intracranial (i.c.) gliomas but also induced antitumor immunity that protected mice from subsequent i.c. or systemic tumor rechallenge. Here, we examined whether the same "intracerebral immunotherapy" strategy could be applied to the treatment of metastatic brain tumors.

Experimental design: Mice with both i.c. and s.c. melanomas were injected intratumorally with CNT-CpG into either location. Antitumor responses were assessed by flow cytometry, bioluminescent imaging, and animal survival.

Results: When given s.c., CNT-CpG response was mostly local, and it only modestly inhibited the growth of i.c. melanomas. However, i.c. CNT-CpG abrogated the growth of not only brain but also s.c. tumors. Furthermore, compared with s.c. injections, i.c. CNT-CpG elicited a stronger inflammatory response that resulted in more potent antitumor cytotoxicity and improved in vivo trafficking of effector cells into both i.c. and s.c. tumors. To investigate factors that accounted for these observations, CNT-CpG biodistribution and cellular inflammatory responses were examined in both tumor locations. Intracranial melanomas retained the CNT-CpG particles longer and were infiltrated by Toll-like receptor (TLR-9)-positive microglia. In contrast, myeloid-derived suppressive cells were more abundant in s.c. tumors. Although depletion of these cells before s.c. CNT-CpG therapy enhanced its cytotoxic responses, antitumor responses to brain melanomas were unchanged.

Conclusions: These findings suggest that intracerebral CNT-CpG immunotherapy is more effective than systemic therapy in generating antitumor responses that target both brain and systemic melanomas.

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Conflict of interest statement

Conflicts of Interest: None

Figures

Figure 1

CNT-CpG abrogates growth of melanomas following intracranial (i.c.) injection. Mice bearing both i.c. and subcutaneous (s.c.) B16.F10-luc melanomas were treated with i.c. intratumoral injections of either blank CNTs (2.5 μg) or CNT-CpG (2.5 μg CNT-5 μg CpG) 7 and 11 days (arrows) after initial tumor implantation. Tumor growth was evaluated by Xenogen (left graphs in A and B) and direct tumor size measurements with calipers (right graphs in A and B). Top panel C, Representative Xenogen images 7 and 13 days after tumor implantation; bottom panel C, brain and s.c. tumor cross sections 14 days after implantation illustrating the size of black-pigmented tumors. Data is representative of two separate experiments. n=5 mice/group, *: P< 0.05, **: P< 0.01, ***: P< 0.001.

Figure 2

Comparison of intracranial (i.c.) and subcutaneous (s.c.) CNT-CpG therapy in melanomas. Mice bearing both i.c. and s.c. B16.F10-luc melanomas were treated with either intratumoral i.c. PBS, i.c. CNT-CpG (2.5 μg CNT-5 μg CpG), or s.c. CNT-CpG (2.5 μg CNT-5 μg CpG) 5, 8, and 12 days after tumor implantation (arrows). Tumor growth was evaluated by Xenogen imaging. A and B, Intracranial CNT-CpG inhibited the growth of both i.c. and s.c tumors while s.c. CNT-CpG antitumor response was mostly local. Intracranial injection of CNT-CpG into mice bearing only s.c. tumors (open circles) has no systemic antitumor response (B). C, Representative Xenogen images of CNT-CpG-treated mice 17 days after tumor implantation. Arrows in C indicate site of CNT-CpG injections. Data is representative of two separate experiments. n=6 mice/group, *: P< 0.05, ***: P< 0.001.

Figure 3

Role of tumor microenvironment on CNT-CpG therapy. Mice bearing both intracranial (i.c.) and s.c. subcutaneous (s.c.) tumors were injected with CNT-CpG (2.5 μg CNT-5 μg CpG,) in either location four days after implantation. A and B, Proportion of tumor inflammatory cells was quantified by flow cytometry in both brain and s.c. tumors 24 hours after CNT-CpG injection. n=4 mice/group; C, Ex vivo cytotoxicity. Mice bearing either i.c. or s.c. tumors (_n_=4/group) were injected with intratumoral CNT-CpG four days after initial tumor implantation and every 3 days thereafter. Splenocytes were harvested 48 hours after the final treatment and examined for killing of B16.F10 target cells. Non-tumor bearing naive mice were used as controls. n=4 mice/group, D, In vivo leukocyte trafficking. Mice bearing either i.c. or s.c. tumors (_n_=3/group) were injected with intratumoral CNT-CpG four days after initial implantation and every 3 days thereafter. Splenocytes were isolated 24 hours after the third CNT-CpG injection, labeled with CFSE and re-injected into recipient mice (1×105 cells/mouse) bearing 10 day-old untreated i.c. and s.c. melanomas (n=5/group). Tumors and blood were harvested 24 hours later and tested for the presence of CFSE-labeled NK and CD8 cells by flow cytometry. *: P< 0.05, **: P< 0.01, ***: P< 0.001, Bars: P< 0.05.

Figure 4

CNT-CpG and CpG clearance in intracranial (i.c.) and subcutaneous (s.c.) melanomas. Mice bearing four day-old i.c. and s.c. B16.F10-luc melanomas (green) were injected intratumorally (indicated by arrows in A) with Cy5.5-labeled (red) CpG (A, top panel) or CNT-CpG (A, bottom panel). Tumor growth and Cy5.5 signal were measured by Xenogen (B and C). A, Representative mouse from each group demonstrating CNT-CpG and CpG clearance in relation to tumor growth. B and C, CpG and CNT-CpG clearance from s.c. and brain tumors. Data is representative of two separate experiments. Dashed lines represent background signal. n=6 mice/group. D, CNT-CpG and CpG distribution in i.c. tumors. Mice bearing four day-old melanomas were injected intratumorally with Cy5.5-labeled (red) CpG or CNT-CpG. At different time intervals brains were harvested, sectioned and imaged with fluorescent microscopy. CpG diffused away from the injection site, while CNT-CpG appeared to disperse around the tumor.

Figure 5

Quantification of TLR9 expression in brain and subcutaneous (SC) melanomas. Seven days after tumor implantation, brain and SC tumors were assessed for leukocyte infiltration (A) and TLR9 expression (B) by flow cytometry. C, Representative dot plots of tumor-associated macrophages (MP, CD45high CD11b+) showing TLR9 expressing cells as red events. Microglia (MG: CD45low CD11b+) were the most common TLR9+ cells in brain tumors. Data is representative of two separate experiments. n=4 mice/group.

Figure 6

Role of myeloid-derived suppressive cells (MDSC) in CNT-CpG therapy. A and B, Quantification of MDSC. Mice bearing both intracranial (i.c.) and subcutaneous (s.c.) tumors were injected with i.c. PBS (baseline), i.c., or s.c. CNT-CpG four days after tumor implantation. Proportion of MDSC was quantified by flow cytometry in both brain (A) and s.c. (B) tumors 24 hours after CNT-CpG injection. Monocytic MDSC (M-MDSC; Ly6Chigh) and granulocytic MDSC (G-MDSC; Ly6Ghigh) are shown as red and green events, respectively, in representative dot plots. n=4 mice/group. C and D, Impact of Gr-1 depletion on CNT-CpG antitumor response. Mice were treated with anti-Gr-1 mAb or control IgG one day prior and every 3 days after tumor implantation. A, Mice (n=4) bearing four day-old i.c. and s.c. tumors were treated with intratumoral PBS or CNT-CpG (three injections as in Fig 3C), splenocytes were harvested 48 hours later and examined for killing of B16.F10 target cells. D, Antitumor response was measured in mice bearing both i.c. and s.c. tumors treated only with s.c. CNT-CpG four, seven and ten days (arrows) after tumor implantation. n=7 mice/group. Data is representative of two separate experiments, bars and *: P< 0.05, **: P< 0.01, ***: P< 0.001.

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Intracerebral CpG immunotherapy with carbon nanotubes abrogates growth of subcutaneous melanomas in mice - PubMed (original) (raw)