Abstract:

Objective To investigate the key mechanism of transforming growth factor-β (TGF-β) inducing the expression of interleukin-17D (IL-17D) in lung cancer-associated fibroblast (CAF) and promoting the recruitment of myeloid-derived suppressor cells (MDSCs). Methods C57BL/6 mice were established for B16 lung melanoma metastasis model (tumor model group), and control group was set up, 6 mice in each group. Flow cytometry (FACS) was used to detect the lung CAF and the changes of its ability to secrete IL-17D and the proportion of MDSCs in tumor mice. The changes of TGF-β level in lung tumor were examined by ELISA and quantitative real-time PCR (RT-qPCR). Lung fibroblasts were screened by FACS, and the effects of TGF-β on the secretion of IL-17D, C-C motif chemokine ligand (CCL)2 and CCL7 in fibroblasts were detected by RT-PCR. The migration of MDSCs under the condition of TGF-β stimulating fibroblasts was detected by Transwell. Results The proportion of CAF (CD45^-CD326^-CD31^-) in the tumor model group was higher than that in the control group [(28.02±2.23)% vs. (7.35±2.14)%, t=9.956, P<0.001]. The ability of CAF to secrete IL-17D in the tumor model group was significantly higher than that in the control group [(38.27±2.93)% vs. (19.04±3.16)%, t=5.995, P=0.001]. The proportion of MDSCs in the tumor model group was significantly higher than that in the control group [(12.93±1.27)% vs. (8.21±1.40)%, t=4.804, P=0.009]. Compared with the control group, the protein and transcription levels of TGF-β in lung of the tumor model group were significantly increased [(1 685.07±135.61) ng/L vs. (1 047.98±68.50) ng/L, t=5.051, P=0.002; 2.17±0.03 vs. 1.00±0.05, t=51.237, P<0.001]. In vitro, lung fibroblasts were stimulated with different concentrations of TGF-β (0, 5 and 10 μg/L) for 24 hours, the relative expressions of IL-17D mRNA secreted by stimulated fibroblasts were 0.42±0.01, 0.67±0.01 and 0.84±0.04 respectively, the relative expressions of CCL2 mRNA in each group were 0.89±0.08, 1.08±0.04, 1.19±0.01 and CCL7 were 0.53±0.05, 0.65±0.04, 0.74±0.03 respectively. With the increase of TGF-β concentration, the expression levels of IL-17D, CCL2 and CCL7 in fibroblasts were significantly increased (F=57.384, P<0.001; F=15.802, P=0.004; F=14.544, P=0.005). In addition, compared with the control group (0 μg/L TGF-β), fibroblasts treated with 10 μg/L TGF-β for 24 hours could promote the migration of MDSCs in spleen of tumor mice [(9.59±0.21)% vs. (2.14±0.24)%, t=6.585, P<0.001]. Conclusion TGF-β can induce high expression of IL-17D in lung CAF, which is an important factor in promoting the expressions of CCL2 and CCL7 and the migration of MDSCs in tumor microenvironment.

Key words: Tumor microenvironment, Transforming growth factor beta, Cancer-associated fibroblasts, Interleukin-17D, Myeloid-derived suppressor cells