Hedgehog Signaling Regulates The Migration Of Breast Cancer Cells By Altering Macrophage Polarization

Breast cancer (breast cancer BRC) is a malignant tumor in breast epithelial tissue. The incidence rates of malignant tumors among women.Macrophage is one of the important immune cells, the immune surveillance and immune clearance, clearance of pathogens, maintenance of internal environment homeostasis. In this process, macrophages can exhibit different phenotypes to regulate inflammation in physiological and pathological condition of different. Which is defined as different polarization states. Years of research found that there is a close relationship between the occurrence and development of diseases related to inflammation and macrophage polarization. Further research also shows that various signaling molecules regulate macrophage polarization process.Hedgehog (Hh) signaling pathway is an important signaling pathway in vivo and in breast cancer plays an important role in the development of. Cyclopamine is a small molecule, and Hedgehog signaling pathway specific antagonist, on the pathway of Smo receptor. This study aimed to investigate the effect of Hedgehog pathway on macrophage polarization in breast cancer and explore its role in the development of the mechanism, to provide experimental basis for the research on molecular mechanism of immune therapy. The specific contents are as follows:1. The Changed of M2-type macrophages in patients with breast cancer after chemotherapyTo investigate the role of macrophages in tumor development, one pair of cancer tissue and para-cancer tissue samples were examined. Expression of Ml-type (iNOS) and M2-type (Arg-1) macrophage-associated cytokines was used were used to determine macrophage distribution. Results of IHC analysis showed that the number of macrophages, irrespective of their subtype, was increased in breast cancer tissues compared with that in para-cancer tissues, suggesting that a number of macrophages reached cancer tissues during tumor growth, which led to incomplete tumor clearance. To investigate the distribution of tumor-associated macrophages during breast cancer treatment, tissues from 9 patients with breast cancer were collected before and after chemotherapy and were compared. Results of qPCR showed that expression of M2-type macrophage-associated cytokines increased significantly after chemotherapy, suggesting that patients achieved effective tumor growth inhibition after chemotherapy. This may be because tumor-associated macrophages (M2-type) promoted tumor regrowth and did not kill tumor cells.2. Cyclopamine reduce the secretion of iNOS in M1 macrophageUsing LPS (100ng/ml) and IFN-y (20ng/ml) stimulated RAW264.7 into the Ml-type for 24h, treated with IL-4 (20ng/ml) into the M1-type for 24h, QPCR analysis of the relative expression levels of iNOS, CD86, ARG-1, CD206, GLI1, Ptchl in various types; the mRNA level of iNOS were determined by QPCR after joining Cyclopamine in the Ml-type, the protein expression of iNOS was assessed by western blot and immunofluorescence after joining Cyclopamine in the M1-type; The relative expression of iNOS, CD86 was significantly higher in Ml-type than that in M2-type at mRNA level (P<0.01), the relative expression of ARG-1,CD206 was significantly higher in M2-type than that in M1-type at mRNA level (P<0.01). After 40 nmol/L Cyclopamine stimulation, the mRNA level of Ptchl was significantly enhanced in M1 macrophages and reached the maximum at 100 nmol/L.Treatmented of Ml-type with Cyclopamine at 1000 nmol/L, which effectively reduced the expression of iNOS at mRNA and protein levels, maybe can promote the macrophages polarization to M2-type.3. Cyclopamine inhibited M1-type macrophage polarizationWe selected Cyclopamine, a specific inhibitor of the Hedgehog signaling pathway. Cells in the experimental group were treated with 1 μmol Cyclopamine and those in the control group were treated with an equal volume of anhydrous ethanol before the induction of macrophage polarization. Results of qPCR showed that expression of M1-type macrophage-associated cytokines (CD86, IL-1, iNOS, IL-6, and IL-12b) was lower in cells in the experimental group than in those in the control group. In contrast, expression of M2-type macrophage-associated cytokines (Fizz, YM-1, IL-10, Arg-1, and CD206) was unchanged in cells in the experimental group compared with that in cells in the control group. Results of western blottingand NO quantification showed that iNOS expression decreased in cells in the experimental group. These results indicated that inhibition of the Hedgehog signaling pathway inhibited M1-type macrophage polarization.4. Effect of Cyclopamine on the invasion of MDA-MB-231 cells was reversed by M2-type macrophagesWe supernatant from the culture of macrophages were incubated with breast cancer cells at a 1:1 ratio of supernatant to culture medium. Vimentin was used as a marker of invasion of MDA-MB-231 cells. Changes in vimentin level were detected in MDA-MB-231 cells. We observed that response of THP-1-derived M2-type macrophages in the experimental group was completely different from that of, macrophages in the control group. Vimentin levels were significantly changed in cells in the experimental group compared with those in cells in the control group. Moreover, treatment with Cyclopamine and supernatant from the culture of THP-1-derived macrophages downregulated vimentin expression in MDA-MB-231 cells. However, a combination of the 2 treatments did not exert a synergistic effect and increased vimentin expression. Based on the indirect coculture, we found that the combined effect of Cyclopamine and M2-type macrophages had no synergistic effect on vimentin expression. Results of qPCR showed that vimentin expression (associated with M2-type macrophages) was significantly increased in the coculture of THP-1/U937 and MDA-MB-231 cells compared with that in cells in the control group. To determine how Cyclopamine treatment reversed the effect of M2-type macrophages on the invasion of MDA-MB-231 cells, we examined the effects of different macrophage subtypes on the proliferation of breast cancer cells. We cocultured DsRed-tagged MDA-MB-231 and THP-1 cells and determined the cell number after 72 h by performing flow cytometry. The total number of MO-and Ml-type macrophages derived from DsRed-tagged MDA-MB-231 cells was significantly lower whereas that of M2-type macrophages derived from DsRed-tagged MDA-MB-231 cells was significantly higher in the coculture than in the control group. This suggested that M2-type macrophages treatment did not inhibit the invasion of breast cancer cells synergistically with Cyclopamine because of the increased proportion of tumor cells in the tumor microenvironment. We performed cell invasion assay to determine whether M2-type macrophages affected the invasion of breast cancer cells. Results of this assay confirmed that that cells in the experimental group showed significantly lower invasion capacity than those in the control group.5. Cyclopamine increased IL-6 expression in monocyte-derived macrophagesWe explored whether the Hedgehog signaling pathway affected the expression of cytokines involved in the invasion of breast cancer cells. Results of qPCR indicated that Cyclopamine treatment upregulated the expression of most M1-and M2-type macrophage-associated cytokines such as IL-6, CD86, and CD206 but downregulated the expression of IL-1 in THP-1-derived macrophages. In addition, we observed that Cyclopamine treatment induced the expression of most M1-and M2-type macrophage-associated cytokines such as IL-6 and IL-1 but downregulated the expression of CD206 in U937-derived macrophages. Comparison of the 2 types of monocyte-derived macrophages indicated that Cyclopamine treatment increased IL-6 expression in M1-type macrophages.6. Macrophage cooperated with the Hedgehog signaling pathway promoted the invasion of breast cancer cells by high expression of IL-6.We explored the possible mechanisms underlying the increase in IL-6 expression in THP-1-and U937-derived macrophages after Cyclopamine treatment. We speculated that THP-1-and U937-derived macrophages acted synergistically to upregulate IL-6 expression in breast cancer cells. To confirm this, we cocultured MDA-MB-231 cells and THP-1-derived macrophages and harvested these cells after 72 h. Results of qPCR showed that IL-6 expression in M2-type macrophages was significantly increased in the experimental group compared with that in the control group. Results of western blotting also showed that IL-6 and vimentin levels were increased in cells in the experimental group compared with those in cells in the control group. To further validate the role of the Hedgehog signaling pathway in macrophage polarization, we constructed a SMO-knockout stable cell line, stimulated it to obtain M2-type macrophages by using methods described above, and cocultured it with MDA-MB-231 cells. Western blotting was performed to detect the expression of IL-6 and vimentin. Results of the cell invasion assay demonstrated that the cells in the experimental group (SMO-shl) showed significantly enhanced invasion capacity compared with those in the control group (NC). These results indicated that macrophage cooperated with the Hedgehog signaling pathway promoted the invasion of breast cancer cells by high expression of IL-6.