As potent stimulators of both innate and adaptive immune systems, CpG ODNs are currently being examined for cancer immunotherapy (5
). Anti-tumor function of CpG has also been studied in brain tumors. Although initial reports by Carpentier et al. demonstrated an 88% cure rate and long-term immunity in rats with i.c. gliomas (27
), others have shown variable CpG anti-glioma responses ranging from no efficacy (29
), partial response (4
), and complete tumor eradication (30
). One explanation for these contrasting reports may be differences in animal models and CpG constructs. Nevertheless, results from early-stage human clinical trials utilizing convection-enhanced CpG therapy in patients with recurrent malignant gliomas have been disappointing with only modest anti-tumor response in a few patients (2
). Although increasing the CpG dose may further enhance its pro-inflammatory effect, high i.c. CpG could exacerbate brain edema and result in CNS toxicity by activating TLR9-independent pathways (31
). Alternatively, pro-inflammatory CpG response can be augmented with repeated injections of low-dose CpG or nanoparticles delivery systems (21
). Both lipid and nonlipid-based nanoparticles have been shown to promote CpG uptake into TLR9-rich endosomes to enhance its immunostimulatory activity in vitro
and in vivo
). Here we demonstrate that functionalized CNTs may also be used to enhance CpG uptake by tumor-associated phagocytic cells as an immunotherapy approach. Although the use of CNTs for CpG delivery has been examined in vitro
), this is the first report demonstrating the anti-tumor efficacy of CNT-CpG complexes in vivo
. Our data indicate that improved delivery of CpG into endosomes by CNTs and “slow-clearance” of CNT-sCpG complexes in the CNS both played a role in augmenting CpG immunotherapy in this model.
Located in the endosomal compartment, TLR9 was initially shown to specifically respond to unmethylated CpG motifs, such as those present in bacterial DNA. As a result, synthetic CpGs were designed to contain repeat unmethylated CpG motifs to enhance TLR9 engagement, and phosphorothioate linkages to prevent breakdown (40
). Recent work, however, suggests that DNA recognition by TLR9 depends on the 2’ deoxyribose phosphate backbone and that neither phosphorothioate linkage nor specific sequences (i.e. CpG motifs) are necessary to induce the immune response (41
). These findings have challenged the dogma that unmethylated CpG motifs constitute the “foreign signature” that triggers TLR9. Instead, phosphorothioate linkages and CpG motifs appear to increase CpG stability, aggregation, and uptake into cells. Thus, any approach that stabilizes and improves CpG uptake into endosomal compartments may enhance its stimulatory functions. Accordingly, nanoparticles have been employed to augment CpG uptake and function (32
). Lipid nanoparticles, for example, can not only improve CpG uptake and immunopotency (32
), but as demonstrated recently, are critical in sequestering TLR9 migration from endoplasmic reticulum to the late endosomal compartment where it can interact with CpG (35
). Although we did not correlate subcellular colocalization of CNT-CpG with TLR9 in this study, rapid CNT-CpG sequestration into cytoplasmic compartments seen here suggests that CNTs were responsible for augmenting CpG prostimulatory function by facilitating its uptake.
CNTs can enter cells through several mechanisms including phagocytosis, diffusion, or receptor-mediated endocytosis (43
). This internalization process not only depends on the target cell type, but also on a variety of CNT characteristics like size, clustering, and surface charge. Here, we noted CNTs to enhance CpG uptake by both monocytes and gliomas in vitro
. CNT uptake, however, was more efficient in phagocytic BMM and resulted in upregulation of pro-inflammatory cytokines/chemokines. Similarly, in i.c. tumors, CNT-mediated CpG uptake was more efficient in TAMs suggesting active phagocytosis/endocytosis of CNT-sCpG complexes by these antigen-presenting cells. In fact, MG and MP collectively accounted for the highest number of CNT-sCpG positive cells in tumors. Early rise in tumor MG/MP, followed by sustained elevation of circulating NK cells indicated that stimulation of TAM’s into M1 phenotype by CNT-sCpG may have triggered induction of anti-glioma NK and CD8 cells. However, since NK cells also internalized CNTs very efficiently, it’s not clear if their stimulation was entirely dependent on TAM activation or due to CNT-sCpG uptake. Future studies will evaluate the differential contribution of MG/MP and NK cells to CNT-sCpG immune responses.
Another potential mechanism that may have accounted for enhanced CpG immunopotency is the unique CNT-sCpG biodistribution within the CNS. Because of the blood-brain barrier and first-pass clearance by liver and lungs, CNTs typically do not penetrate normal brain tissue after i.v. injections (13
). Biodistribution of CNTs following i.c. injections has not been studied before. But recently, we demonstrated that fCNTs are efficiently phagocytosed by TAMs in i.c. gliomas where they can remain for nearly one week after direct i.t. injections (12
). Similarly, CNT-sCpG complexes studied here remained within i.c. tumors longer than free CpG. Slow clearance of CNT-sCpG complexes from the CNS may have provided a “depot” effect, allowing enough time for migration of inflammatory cells into the tumor environment and inducing a stronger anti-glioma effect.
Toxicity has been raised as a potential limitation of CNT biomedical application. While CNTs can be biodegraded through enzymatic catalysis (44
), pristine (non-modified/non-functionalized) CNTs have been shown to persist for several months raising concerns of potential negative health effects (45
). CNT toxicity, however, is strongly related to physicochemical properties (such as structure, length, surface area, degree of metallic contamination, and surface coating) and can be minimized with functionalization techniques (11
). PEG-functionalized CNTs, for example, have been shown to be nontoxic, and cleared from biliary and renal pathways within 2 months of i.v. injection in mice (47
). Consistent with our previous report, fCNTs studied here were non toxic after i.c. injections in these short-term experiments, thereby supporting their utilization in cancer immunotherapy.
CNTs may have various potential advantages over other nanomaterials in biomedical applications. fCNTs can be used to deliver siRNA into ‘hard-to-transfect’ cells such as MPs and T cells that are usually inert to conventional liposomal transfection agents (48
). They can also be used as contrast agents in multimodality optical imaging, and since they do not contain heavy metals, have a safer chemical composition as compared to gold/silver-containing quantum dots (49
). Finally, the high optical absorbance of CNTs can be used in photothermal therapy, which may potentially be combined with CNT-based chemotherapy and gene therapy of cancer (24
In summary, we demonstrated CNT delivery system to significantly enhance CpG immunotherapy, eradicate i.c. gliomas at low doses, and induce immunity against tumor rechallenge without inducing toxicity. Our findings have direct implications to the development of CNT-based treatments for malignant brain tumors. Considering that fCNTs can be used to carry other oligonucleotiedes such as siRNA (51
), this strategy can be employed to overcome local immunosuppressive microenvironment through modulations of tumor inflammatory responses.
Statement of Translational Relevance
The prognosis of patients with malignant gliomas, the most common type of primary brain neoplasm, remains dismal even after aggressive multimodality treatment. Although immunotherapy is being investigated as an adjunct treatment, the ability of gliomas to escape immune response will continue to be a significant obstacle to this strategy. One approach to overcome the local immunosuppressive tumor microenvironment is the activation of the innate immune system by toll-like receptor (TLR) agonists such as CpG oligonucleotides (CpG). Because TLR9, CpG receptor, is located intracellularly, we hypothesized that methods that enhance CpG internalization may also potentiate its immunostimulatory response. Here, we demonstrate that Carbon nanotubes (CNTs) enhanced CpG uptake by tumor-associated phagocytic cells and resulted in their activation both in vitro and in vivo. Furthermore, a single injection of low-dose CNT-CpG complexes eradicated intracranial gliomas through activation of NK and CD8 cells. These findings demonstrate that CNTs are nontoxic vehicles that can improve CpG uptake into tumor-associated inflammatory cells, leading to a more robust anti-tumor response. These findings have direct application to the design of future CpG immunotherapy trials.