DC-SIGN (CD209)-mediated interactions between bacteria, lung cancer tissues, and macrophages promote cancer metastasis

Abstract

One of the hallmarks of lung cancers is the earlier metastasis resulting from the dissemination of cancer cells. Although accumulating evidence suggests that bacterial infection may be involved in the development of the metastasis of lung cancer, few studies have explored the molecular mechanisms of bacterial infection in the dissemination of lung cancer cells. A series of studies have indicated that certain Gram-negative bacteria are able to hijack antigen-presenting cells (APCs) via interaction with DC-SIGN (CD209) receptors to facilitate the dissemination of pathogens, including viruses, bacteria, fungi, and parasites. Therefore, in the present work, it was hypothesized that bacterial infection may promote the dissemination of cancer cells via the utilization of a similar mechanism. It was first discovered that human lung cancer tissues contain a very high diversity of bacterial DNAs, indicating the co-existence of lung cancer tissues and microbial organisms. It was then found that lung cancer tissues express DC-SIGN, leading to binding with a Gram-negative bacterium, Shigella sonnei. Further, this bacterium was found to be able not only to induce the expression of DC-SIGN on macrophages but also to enhance the migration ability of lung cancer cells in vitro. The in vivo experiments supported these observations, showing that in wild-type (WT) mice, Shigella sonnei infection significantly increased tumor size, weight, and metastatic nodules compared to SIGNR1 knockout (KO) mice. These observations were associated with increasing DC-SIGN expression in WT mice. Finally, these results suggest that bacterial infections could play a significant role in promoting lung cancer progression and metastasis via DC-SIGN-mediated mechanisms.

Keywords: DC-SIGN; Gram-negative bacteria; Lung cancer; Metastasis.

Fig. 1

Microbiota determination in lung tissues. This figure displays differences in the abundance of microbiome members in various lung cancer tissue and adjacent normal tissue samples. The 7000 selected sequences of lung cancer tissue and adjacent normal tissue specimens were sequenced, and the bacterial OTUs of the lung cancer tissue and adjacent normal tissue specimens were ranked by abundance. The proportion of each out was determined by the column length

Fig. 2

DC-SIGN expression in normal lung tissue, lung cancer adjacent tissue, and lung cancer tissue. DC-SIGN expression was examined in normal lung tissue, lung cancer tissue, and adjacent normal tissue. Immunostaining for DC-SIGN via the brown immunoperoxidase 3,3'-diaminobenzidine (DAB) technique was described in the materials and methods section. Left, DC-SIGN was detected in normal lung tissue. Middle, DC-SIGN was detected in lung cancer adjacent tissue. Right, DC-SIGN was detected in lung cancer tissue. Magnification, 200 ×

Fig. 3

Gram-negative bacteria interact with human lung cancer tissues via DC-SIGN. The sets of bacteria, E. coli K-12 CS180, and CS1861 strains. A Shigella and Shigella-pAY100.1, B P. mirabilis and P. mirabilis-pAY100.1, and C Yersinia pseudotuberculosis cultured at 37 °C and 26 °C were respectively used in an adhesion assay to determine the rate of adherence to the lung tissue. The data presented were pooled from three independent experiments. *P < 0.05, **P < 0.005, ***P < 0.0001. Shigella O + : Shigella-pAY100.1; Pmi: P. mirabilis; Pmi O + : P. mirabilis-pAY100.1; Y1 37: Yersinia pseudotuberculosis cultured at 37 °C; Y1 26: Yersinia pseudotuberculosis cultured at 26 °C

Fig. 4

Inhibition of Shigella sonnei adherence to lung cancer Tissues by anti-hDC-SIGN Antibody and mannan. Shigella sonnei were incubated with lung cancer for 2 h, either with or without anti-hDC-SIGN and mannan. E. coli K12 CS180 served as the control strain. These results are based on data pooled from three separate experiments. ***P < 0.001, ****P < 0.0001

Fig. 5

SIGNR1 is upregulated by Gram-negative bacterial infection. A RAW264.7 cells were incubated in the presence and absence of lung cancer cell supernatant and were infected with live or heat-killed Shigella or treated with IL-4. The level of SIGNR1 expression was then determined using flow cytometry analysis. B The quantification of the positive expression of SIGNR1 on RAW264.7 cells (n = 5). The experiment was performed in triplicate. *P < 0.05, **P < 0.005, compared with non-treated RAW 264.7 cells

Fig. 6

S. sonnei bacteria-infected SIGNR1 + macrophage enhances the migration and invasion of lung cancer cells in vitro. A Transwell assay. PC-9 and LLC were cultured with LLCS-CM and Shigella-CM, respectively. Scale bar, 200 µm. *P < 0.05, **P < 0.01. (B) and C Wound-healing assay. PC-9(B) and LLC(C) cells were treated with Shigella-CM, and the cell monolayers were scratched with 200 μL yellow pipette tips. Images were recorded at 0 and 24 h after the wound scratch (left, representative pictures; right, quantitative migrating distance). The yellow lines mark the original wound; the cells moved closer to the wound. The y-axis of the graph is the distance the cells moved. **P < 0.005. LLCS-CM: the conditioned medium of LLC supernatant-treated RAW264.7 cells; Shigella-CM: the conditioned medium of Shigella-infected RAW264.7 cells

Fig. 7

S. sonnei bacteria can promote the invasion and metastasis of lung cancer cells in vivo. A All the mice were sacrificed, and the lungs were collected. Top, wild-type mice. Bottom, Shigella-infected mice, showing visible lung metastases. The same scale (mm³) was applied to both groups to eliminate size discrepancies. B Quantitative summary of the lung weights (n = 10 mice per group). ****P < 0.0001, *** P < 0.005, ** P < 0.01. C Quantitative summary of the lung cancer sizes (n = 10 mice per group). ****P < 0.0001, *** P < 0.005 D Hematoxylin and eosin (H&E) staining in the lung metastatic site of the LLC cells. Top, 40 × magnification. Bottom, 100 × magnification. E Immunohistochemical analysis of 20 WT murine lung cancers stained with anti-SIGNR1 antibodies. Representative images of cancer tissues show the expression level of SIGNR1 in the two groups (left, n = 10; right, n = 10)