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

Abstract

The immunostimulatory ability of synthetic oligonucleotides containing CpG motifs (CpG-ODN), agonists of Toll-like receptor 9 (TLR9), can be harnessed to promote antitumor immunity by their application at the tumor site to stimulate local activation of innate immunity; however, particularly in the lung, tumor-associated immunosuppression can subvert such antitumor innate immune responses. To locally maintain continuous activation of innate subpopulations while inhibiting immunosuppressive cells, we evaluated aerosol delivery CpG-ODN combined with Poly(I:C), a TLR3 agonist able to convert tumor-supporting macrophages to tumoricidal effectors, in the treatment of B16 melanoma lung metastases in C57BL/6 mice. Aerosolization of CpG-ODN with Poly(I:C) into the bronchoalveolar space reduced the presence of M2-associated arginase- and IL-10-secreting macrophages in tumor-bearing lungs and increased the antitumor activity of aerosolized CpG-ODN alone against B16 lung metastases without apparent signs of toxicity or injury of the bronchial-bronchiolar structures and alveolar walls. Moreover, CpG-ODN/Poly(I:C) aerosol combined with dacarbazine, a therapeutic agent used in patients with inoperable metastatic melanoma able to exert immunostimulatory effects, led to a significant increase in antitumor activity as compared to treatments with aerosolized CpG-ODN/Poly(I:C) or dacarbazine alone. This effect was related to an enhanced recruitment and cytotoxic activity of tumor-infiltrating NK cells in the lung. Our results point to aerosol delivery as a convenient approach for repeated applications of immunostimulants in patients with lung metastases to maintain a continuous local activation of innate immune cells while suppressing polarization of tumor-infiltrating macrophages to an M2 phenotype.

Keywords: TLR agonists; aerosol delivery; dacarbazin; lung metastases; mice.

Figure 1.

Recruitment of innate immune cells in lungs of mice treated with aerosolized TLR3 agonist Poly(I:C). Percentage and representative dot plots of macrophages, identified as CD11c-F4/80+ cells among CD45+ cells (A), and dendritic cells, identified as CD11c+F4/80- cells gated on CD11b+ cells (B), obtained by enzymatic digestion of lungs from mice (four mice/group) treated three times at 24-h intervals with aerosolized TLR3 agonist poly(I:C) (15 mg) or saline. *p < 0.05.

Figure 2.

Arginase- and IL-10-secreting macrophages in lungs of B16 tumor-bearing mice treated with CpG-ODN or CpG-ODN/Poly(I:C) aerosol. Immunohistochemical staining for arginase I-expressing and CD163-expressing (A) macrophages in formalin-fixed, paraffin-embedded lung tissue collected after i.v. injection of 5 × 10⁵ B16 melanoma cells from mice treated with aerosolized CpG-ODN alone or combined with Poly(I:C) or left untreated (3–4 mice/group). CpG-ODN/Poly(I:C) was more effective than CpG-ODN alone in reducing the number of arginase I-positive and CD163-positive M2 macrophages populating the interstitium as compared to untreated controls. Original magnification x400; Inset in upper right panel is a higher-magnification (x630) showing the cell morphology of macrophages infiltrating the lungs of untreated mice. Histograms in the bottom show the mean number of arginase I-expressing or CD163-expressing macrophages in lung tissue evaluated on 10 fields/group. *p < 0.05; ***p < 0.001.Representative immunofluorescence images of lung samples showing M2-polarized macrophages as CD68 (red)-positive cells also expressing IL-10 (green) (B). Original magnification x400.

Figure 3.

Effects of aerosolized CpG-ODN/Poly(I:C) on the growth of B16 lung metastases and on lung parenchyma of tumor-free mice. (A) Number of macroscopic lung metastases after i.v. injection of B16 melanoma cells in mice untreated (7 mice) or treated with CpG-ODN aerosol (12 mice), Poly(I:C) aerosol (12 mice) orCpG-ODN/Poly(I:C) aerosol (14 mice)*p <0.05; ***p <0.001. (B) Histopathological evaluation of hematoxylin and eosin-stained lung tissue sections from aerosolized CpG-ODN,Poly(I:C),or CpG-ODN/Poly(I:C) mice and untreated mice. Note focal areas of mononuclear and granulocytic infiltrate (magnification x400).

Figure 4.

Up-modulation of NKG2D ligand expression on dacarbazine-treated B16 melanoma cells. (A) MULT1 and RAE1 expression on the cell surface of B16 melanoma cells analyzed by flow cytometry after 24-h culture in complete medium alone (PBS) or supplemented with dacarbazine (DTIC) at different concentrations. The mean fluorescence intensity (MFI) was normalized to the isotype control. One representative experiment of 2 conducted is shown. (B) Increase in Mult1 and Rae1 mRNA expression in lungs of mice injected i.v. with B16 melanoma cells and untreated or treated for 3 weeks with DTIC (80 mg/kg administered i.p. 5 days/week) (3 mice/group). Data represent mean relative expression (normalized to GAPDH) ± SD from three independent real-time PCR analyses. ***p <0.001.

Figure 5.

Antitumor activity of aerosol CpG-ODN/Poly(I:C) combined with dacarbazine (DTIC) on B16 experimental lung metastases. Number of macroscopic B16 melanoma lung metastases in mice untreated (7 mice) or treated with aerosol CpG-ODN/Poly(I:C) (8 mice), DTIC (8 mice), or aerosol CpG-ODN/Poly(I:C) plus DTIC (8 mice) starting 4 d after tumor injection (A), and in mice untreated (7 mice) or treated with aerosol CpG-ODN/Poly(I:C) (8 mice), DTIC (7 mice), or aerosol CpG-ODN/Poly(I:C) plus DTIC (16 mice) starting 1 week after tumor injection (B). *p<0.05; ** p < 0.01 ***p < 0.001.

Figure 6.

Recruitment and activation of NK cells in B16 metastases-bearing lungs of mice treated with CpG-ODN/Poly(I:C) aerosol combined with dacarbazine. Percentage and representative dot plots of NK cells, evaluated as DX5⁺ cells gated on FSClowSSClowCD45+CD3- cells (A), and of CD69+ (B) and NKG2D+ (C) NK cells, gated on DX5+ cells, obtained by enzymatic digestion of B16 metastases-bearing lungs of mice (4 mice/group) treated with aerosol CpG-ODN/Poly(I:C), DTIC, aerosol CpG-ODN/Poly(I:C) plus DTIC or left untreated. Percentage and representative dot plots of degranulating NK cells, evaluated as CD107a+ cells gated on CD45+CD3-DX5+ cells, after co-culture with B16 melanoma cells (D), and of CFSE-labeled B16 dead cells, evaluated as 7AAD+ cells gated on CFSE+ cells, after co-culture with cells obtained from lung enzymatic digestion (E). *p < 0.05, ***p < 0.001.