Anti‐tumor activity of CpG‐ODN aerosol in mouse lung metastases

Abstract

Studies in preclinical models have demonstrated the superior anti-tumor effect of CpG oligodeoxynucleotides (CpG-ODN) when administered at the tumor site rather than systemically. We evaluated the effect of aerosolized CpG-ODN on lung metastases in mice injected with immunogenic N202.1A mammary carcinoma cells or weakly immunogenic B16 melanoma cells. Upon reaching the bronchoalveolar space, aerosolized CpG-ODN activated a local immune response, as indicated by production of IL-12p40, IFN-c and IL-1b and by recruitment and maturation of DC cells in bronchoalveolar lavage fluid of mice. Treatment with aerosolized CpG-ODN induced an expansion of CD41 cells in lung and was more efficacious than systemic i.p. administration against experimental lung metastases of immunogenic N202.1A mammary carcinoma cells, whereas only i.p. delivery of CpG-ODN provided anti-tumor activity, which correlated with NK cell expansion in the lung, against lung metastases of the poorly immunogenic B16 melanoma. The inefficacy of aerosol therapy to induce NK expansion was related to the presence of immunosuppressive macrophages in B16 tumor-bearing lungs, as mice depleted of these cells by clodronate treatment responded to aerosol CpG-ODN through expansion of the NK cell population and significantly reduced numbers of lung metastases. Our results indicate that tumor immunogenicity and the tumor-induced immunosuppressive environment are critical factors to the success of CpG therapy in the lung, and point to the value of routine sampling of the lung immune environment in defining an optimal immunotherapeutic strategy.

Poly(I:C) and CpG-ODN combined aerosolization to treat lung metastases and counter the immunosuppressive microenvironment

“…2B). Consistent with our previous results, 7 CpG-ODN aerosol treatment alone failed to modify the number of NK cells in lungs bearing the B16 tumor, while the reduction of M2 macrophages induced by the CpG-ODN/Poly(I:C) combination allowed locally delivered CpG-ODN to expand NK cell numbers, as revealed by flow cytometric analysis of CD45C tumor-infiltrating cells in lungs of mice injected i.v. with B16 cells and treated with aerosolized CpG-ODN alone or combined with Poly(I:C) (mean % DX5C/ CD3-/CD45C § SD: 22.8 § 1.5% in untreated; 23.9 § 2.5% in CpG-ODN-treated; 25.5 § 1.6 in Poly(I:C)-treated; 32.7 § 2.1% in CpG-ODN/Poly(I:C)-treated; 4 mice/group; pD0.036 CpG-ODN/Poly(I:C) vs. CpG-ODN; p D 0.0094) (Fig.…”

“…The enhanced antitumor effect of the CpG-ODN/Poly(I:C) aerosol combination correlated with a significant reduction in the number of macrophages producing arginase and/or IL-10 and/or CD163 expressing macrophages, three different markers of the M2 phenotype, which are selectively recruited by B16 melanoma cells in the lung and responsible for the low efficacy of locally delivered CpG-ODN alone. 7 Besides blocking conversion of TAM to the M2 phenotype, Poly(I:C) treatment was recently shown to promote the maturation of myeloid-derived suppressive cells (MDSC), immature cells often elevated in tumor-bearing hosts, rendering them competent for NK cell activation. Indeed, incubation of Poly(I:C)-treated CD11bCGr1C MDSC cells with NK cells induced the upregulation of CD69 expression on the NK cell surface and increased their interferon-g production.…”

“…1,2,5,6 For lung tumors, repeated inhalation of TLR agonists represents a convenient and practical approach to induce frequent replenishment of innate immune effectors at the tumor site, avoiding toxic effects of systemic treatment. Our studies of aerosol delivery of CpG-ODN as a strategy for local administration of immunostimulators 7 showed that aerosolized CpG-ODN reached the bronchoalveolar space, locally activated an immune response without signs of toxicity, and was more efficacious than systemic administration against lung metastases of N202.1A mammary carcinoma cells. In contrast, aerosol delivery of CpG-ODN was minimally effective against metastases of B16 melanoma cells, which selectively recruit CD68 C macrophages with an M2 phenotype and induce an immunosuppressive environment in the lung.…”

“…Aerosol administration was performed using a mouse whole-body exposure system (EMMS) as described. 7 Poly(I:C) (15 mg) or CpG-ODN (1.5 mg) were dissolved in 5 mL saline and placed in the nebulizer unit to treat up to 10 mice placed in the aerosol box; mice were exposed to aerosol for 15 min, with the 5 mL volume of liquid in the nebulizer nearly consumed in 10 min. At the end of the experiments, all mice were euthanized and macroscopic lung metastases were counted.…”

“…In contrast, aerosol delivery of CpG-ODN was minimally effective against metastases of B16 melanoma cells, which selectively recruit CD68 C macrophages with an M2 phenotype and induce an immunosuppressive environment in the lung. 7,8 Thus, the tumor microenvironment is a critical factor for successful use of these immunotherapeutics, and strategies to shift a tumor-supporting milieu to a host-friendly one might lead to improved antitumor activity of CpG-ODN.…”