Aluminum nanoparticles enhance anticancer immune response induced by tumor cell vaccine
© Springer-Verlag 2010
Received: 28 January 2010
Accepted: 18 February 2010
Published: 7 May 2010
The application of nanomaterial in cancer treatment is promising and intriguing. Anti-tumor immunotherapy has the potential to significantly improve the prognosis of cancer treatment, though the efficacy of immunotherapy generally needs further improvement. One way to improve the efficacy is using immune adjuvants, but the adjuvants for anticancer immunotherapy have to be more potent than for prophylactic vaccines. Here, we report that compared to conventional alum adjuvant, aluminum oxide nanoparticles (nano-alum) may further enhance the anticancer effects of an immunotherapy that employs tumor cell vaccine (TCV). The average tumor size tends to be lower in animals that receive the combinational treatment of nano-alum and TCV. The anticancer cytotoxicity by the lymphocytes was also significantly higher in the treatment group that received both TCV and nano-alum. These results suggest that nano-alum may potentially serve as a potent immune adjuvant and have prospective applications in anticancer immunotherapy.
Nanotechnology may have important applications in multiple biomedical fields (Williams et al. 2002; Heller et al. 2006; Elder et al. 2008; Jain 2008; Cai et al. 2005; Fenske and Cullis 2008; Bianco et al. 2005a; Martin and Kohli 2003; Liu et al. 2005; Ni et al. 2005; Sun et al. 2002; Zanello et al. 2006; Bianco et al. 2005b; Hartman et al. 2008), and the use of nanomaterial in cancer treatment is particularly intriguing (Fifis et al. 2004; Farokhzad et al. 2006). One important issue in cancer immunology is the search of novel immune adjuvants that can enhance the efficacy of anti-tumor immunotherapy (Mesa and Fernandez 2004). Immunotherapy is an important form of adjunctive cancer treatment that may significantly improve the prognosis (Vermorken et al. 1999; Schirrmacher 2005; Takayama et al. 2000). Prior clinical trials of immunotherapy have achieved promising results in treating malignancies such as melanoma, malignant glioma, or renal cell carcinoma (Berd et al. 2004; Yu et al. 2004; Rosenberg et al. 1994), which tend to respond poorly to chemotherapies. Nevertheless, the efficacy of current anticancer immunotherapy generally needs further improvement (Emens 2006). Because tumor antigens are usually self-derived and are, therefore, poorly immunogenic, the adjuvants for anticancer immunotherapy have to be more potent than for prophylactic vaccines (Mesa and Fernandez 2004). One way to increase the efficacy is using immune adjuvants, which are defined as products that increase the immune response toward antigens. The identification of novel and more efficacious adjuvants is thus of practical importance for anticancer immunotherapy (Mesa and Fernandez 2004; Emens 2006).
Aluminum nanoparticle (nano-alum) may serve as a candidate of prospective adjuvant for anticancer immunotherapy, which needs to be both safe and efficacious. Alum (aluminum hydroxide, phosphate, or hydroxyphosphate) has an excellent safety record for systemic vaccination. It has been used as adjuvant for more than 60 years and is at present the most widely used adjuvant in both veterinary and human vaccines (Mesa and Fernandez 2004; Lindblad 2004). However, alum is mostly a Th2 stimulator of the immune system and primarily enhances the antibody-mediated reaction (Lindblad 2004). It has limited influence on the cytotoxic T cell-mediated response, which is considered the main mechanism of anticancer immune effects (Mesa and Fernandez 2004; Berd et al. 2004; Lindblad 2004). Since nanosized materials often exhibit unique properties that may offer significant advantage for biomedical applications (Jain 2008; Martin and Kohli 2003), it is sensible to investigate if nano-alum will be more efficacious in enhancing anticancer immune response than conventional alum. Here, we studied whether nano-alum would influence the efficacy of an immunotherapy that employs tumor cell vaccine (TCV), to evaluate nano-alum’s applicability as an adjuvant in a commonly used anticancer immune regimen.
2 Materials and methods
Nano-alum (Al2O3, MW 101.96) was purchased from Degussa Nanotechnology of Evonik Industries. Scanning electron microscopy (SEM) was performed to evaluate the size of the nanoparticles. Conventional alum in the form of Al(OH)3 (analytical grade, MW 78.0) was purchased from Beijing Chemical Reagents Company.
BALB/c mice were purchased from the Experimental Animal Institute of the Chinese Academy of Medical Sciences (Beijing, China), bred and maintained under defined flora conditions in individually ventilated (high-efficiency particle-arresting filtered air) sterile microisolator cages (Techniplast, Milan, Italy). All animal handling and experimental procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences.
2.3 Preparations of alum or nano-alum adjuvants
Conventional alum in the form of Al(OH)3 was mixed with D-Hanks solution at a concentration of 500 µg per ml (6.4 mM/l) and fully sonicated, yielding a suspension ready for further use. Nano-alum was mixed with D-Hanks solution at the concentration of 326.9 µg per ml, to ensure that the final molar concentration of nano-alum (6.4 mM/l) is the same as that of the conventional alum adjuvant. The nano-alum mixture was then sonicated immediately prior to application, yielding a semi-clear solution ready for immune studies.
2.4 Preparations of tumor cell vaccine
TCV was prepared by incubating H22 liver cancer cells in D-Hanks solutioin containing Mitomycin C (80 mg/l) for 60 min, followed by thorough wash with D-Hanks for five times.
2.5 Experiments of immunotherapy
Immunotherapy experiments were conducted using female adult BALB/c mice, 8 weeks of age, weighing 18 to 22 g. Twenty-eight mice were randomly divided into four groups: (1) the control group, (2) the TCV treatment group, (3) the TCV plus conventional alum (TCV + alum) treatment group, and (4) the TCV plus nano-alum (TCV + nano-alum) treatment group. On day zero, every mouse was inoculated with 2 × 106 live H22 cells subcutaneously in the right hind leg. On day 7 and 14, the treatment groups also received two subcutaneous doses of designed immune treatment agents in the left hind leg for triggering anti-tumor reactions. Specifically, the TCV group received 2 × 106 TCV cells in 200 µl D-Hanks; the TCV + alum group received 2 × 106 TCV cells in 100 µl D-Hanks mixed with 100 µl of alum adjuvant; and the TCV + nano-alum group received 2 × 106 TCV cells in 100 µl D-Hanks mixed with 100 µl of nano-alum adjuvant. On day 7 and day 14, the control group also received injections of 200 µl D-Hanks that did not contain TCV or adjuvant. Tumor dimensions were monitored using calipers at right angles every 3 to 4 days. The tumor size was measured using the product of the two longest dimensions perpendicular to each other.
2.6 Immune cytotoxicity studies
2.7 Histological studies of the tumor tissue
Tumor tissues were fixed and imbedded in paraffin. Tumor sections of 5 μm were cut from the embedded tissue and stained by standard hematoxylin and eosin procedure (HE stain), for evaluation of tumor necrosis and lymphocyte infiltration.
Statistical analysis was performed with the statistical SPSS 13.0 software. The nonparametric test was used to calculate the probability of significant differences among the groups. Statistical significance was defined as p < 0.05.
3.1 Characterization of the nano-alum
3.2 Tumor size study
3.3 Immune cytotoxicity studies
3.4 Histological studies of the tumor tissue
The aim of this study was to investigate if nano-alum would be a more potent adjuvant than conventional alum in an anti-tumor immunotherapy employing TCV. The result showed that nano-alum enhanced lymphocytes’ cytotoxicity against the H22 liver cancer cells, whereas conventional alum failed to generate a positive immune stimulating effect (Fig. 3). The average tumor size in the TCV + nano-alum group also tended to be lower (Fig. 2), presumably because of the enhanced anti-tumor immune cytotoxicity in this group. In addition, histological study also revealed increased lymphocyte infiltration and tumor necrosis in the TCV + nano-alum group (Fig. 4), again suggesting that nano-alum boosted the anti-tumor response.
Frey et al. reported that aluminum nanoparticles chemically conjugated to the C4 domain of the gp120 HIV protein can generate relatively high antibody response toward gp120 (Frey et al. 1999). Here, we showed that nano-alum alone, without conjugation to antigen, could enhance the TCV-induced anticancer immune response, and that conventional alum failed to generate a similar effect (Fig. 3). Both studies suggest that nano-alum is a more potent immune adjuvant than conventional alum. This presumably could be attributed to the unique physical and chemical properties of nano-alum, which might be more favorable for immune stimulation. It should be noted, however, that the detailed mechanisms of both conventional alum and nano-alum adjuvants remain to be unveiled to date (Lindblad 2004), and much further research is still warranted.
TCV was used in this study for triggering immune response. TCV carries a mixture of tumor proteins and is multivalent in terms of tumor antigens (Copier and Dalgleish 2006). The major advantage of TCV is that it may be applied in treatment of multiple malignancies, irrespective of the genetic makeup of the cancer. However, the lack of clearly defined tumor antigens in TCV therapy renders the antibody responses difficult to assess. Because the nature of the immunogens was not known, the efficacy of TCV therapy was generally measured by tumor growth retardation or prolonged survival, and not by antibody production (Copier and Dalgleish 2006; Levy and Colombetti 2006). Nevertheless, some clinical studies based on such vaccines have generated promising results (Vermorken et al. 1999; Schirrmacher 2005). In this study, nano-alum was found to enhance the anti-tumor immunity induced by TCV. Since TCV can often be made from surgically resected tumors, nano-alum thus may have potential application in immune treatment of multiple types of tumors that are surgically accessible.
In summary, this study showed that nano-alum enhanced the anticancer immune response induced by TCV. The results suggest that nano-alum may potentially serve as an effective adjuvant in anticancer immunotherapies.
The authors thank Professors Hui Li and Yan Shen of the Chinese Academy of Medical Sciences for insightful suggestions. This work was made possible by the funding support from the China Medical Board, the Ministry of Science and Technology (2006CB933204), and Natural Science Foundation of Beijing (Z0005190043511).
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