Aerosol 1,25-dihydroxyvitamin D3 supplementation: A strategy to boost anti-tumor innate immune activity

Abstract

Background: 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] plays a role in calcium homeostasis but can also exert immunomodulatory effects. In lungs, characterized by a particular immunosuppressive environment primarily due to the presence of alveolar macrophages (AM), 1,25(OH)2D3 has been shown to favor the immune response against pathogens. Here, we explored the ability of aerosolized 1,25(OH)2D3 to locally promote an anti-tumor phenotype in alveolar macrophages (AM) in the treatment of lung metastases.

Methods: Cytotoxicity assay has been used to assess the capability of AM, in vitro treated of not with 1,25(OH)2D3, to stimulate NK cells. Sulforhodamine B (SRB) assay has been used to assess the effect of 1,25(OH)2D3 on MC-38 and B16 tumor cells in vitro growth. 1,25(OH)2D3 was aerosolized in immunocompetent mouse models to evaluate the effect of local administration of 1,25(OH)2D3 on in vivo growth of MC-38 and B16 tumor cells within lungs and on infiltrating immune cells.

Results: In vitro incubation of naïve AM with 1,25(OH)2D3 improved their ability to stimulate NK cell cytotoxicity. In vivo aerosolized 1,25(OH)2D3 significantly reduced the metastatic growth of MC-38 colon carcinoma, a tumor histotype that frequently metastasizes to lung in human. Immune infiltrate obtained from digested lungs of 1,25(OH)2D3-treated mice bearing MC-38 metastases revealed an increased expression of MHCII and CD80 on AM and an up-modulation of CD69 expression on effector cells that paralleled a strong increased ability of these cells to kill MC-38 tumor in vitro.

Conclusions: Together, these data show that aerosol delivery can represent a feasible and novel approach to supplement 1,25(OH)2D3 directly to the lungs promoting the activation of local immunity against cancer.

Fig 1. 1,25(OH)2D3 increased the ability of murine alveolar macrophages to stimulate in vitro NK cell cytotoxicity.

NK cells, from spleen of C57BL/6 healthy mice, co-cultured with 1,25(OH)2D3-pretreated lung macrophages significantly increased the percentage of lysis of YAC-1 cells, as compared to NK cells co-cultured with untreated lung macrophages. Unpaired t test; *p<0.05.

Fig 2. Aerosolized supplementation of 1,25(OH)2D3 reduced metastatic tumor growth in the lung.

MC-38 colon cancer cells were injected in C57BL/6 mice and mice were treated with aerosolized saline or 1,25(OH)2D3 as described in Materials and Methods. At the end of the experiment was evaluated (A) in saline-treated group (n = 5) and in 1,25(OH)2D3-treated group (n = 6) mean number of lung metastases and (B) in saline-treated group (n = 6) and in 1,25(OH)2D3-treated group (n = 6) metastatic lung weight. Unpaired t test; *p<0.05.

Fig 3. Anti-proliferative in vitro activity of 1,25(OH)2D3 against cancer cells.

MC-38 colon cancer cell (A) and B16 melanoma cells (B) were exposed to different concentration of 1,25(OH)2D3 for 72 h. Cells proliferation was evaluated by SRB assay at the end of the experiment as the optical density (OD) of 1,25(OH)2D3-treated cells/ the optical density (OD) of 1,25(OH)2D3-untreated cells *100; bars represent the mean of cells proliferation ± SD; Unpaired t test ** p<0.005; ***p<0.0001.

Fig 4. 1,25(OH)2D3 aerosolization induces activation of innate immune effectors in the lung.

Lungs were recovered from tumor-bearing mice treated with 1,25(OH)2D3 or saline. Flow cytometry analysis of the expression of MHCII and CD80 maturation markers on AM of lungs digestion (A). Real time PCR analysis of IL-12 mRNA expression in adherent fraction from lung suspensions (B); count of cells in the non-adherent fraction containing effector cells from lung suspensions (C); flow cytometry analysis of CD69 activation marker on the non-adherent fraction (D); percentage of MC-38 lysated cells evaluated by in vitro cytotoxic assay performed with non-adherent fractions from lung suspensions (E); Unpaired t test; ***p<0.0001.