| Tumor is a severe disease which affects human health. A prominent component of solid tumors is represented by non-tumoral cells, including stromal cells (fibroblasts and endothelial cells) and leukocytes. Among the latter, macrophages are the major component. Macrophages infiltrated in the tumors, which are also called tumor-associated macrophages (TAMs), have been studied extensively on their relationship with tumor cells and their multi-faceted functions in the tumor microenvironment. TAMs are able to exert pro- and anti-tumor effects in the tumor microenvironment depending on their activation states. TAMs can not only exert immune surveillance and anti-tumor function by phagocytosing, killing tumor cells and presenting tumor antigens, but also promote tumor development and blood vessel angiogenesis by secreting many chemokines and cytokines, and promote tumor invasion and metastasis by degrading matrix.As we know, there are two types of Macrophages: Ml macrophages, also termed as classically activated macrophages, and M2 macrophages, also termed as alternatively activated macrophages. Ml macrophages activated in response to microorganisms or microbial products (such as Lipopolysaccharides (LPS)) or interferon-γ(IFN-γ) are characterized by: high capacity to present antigen; high interleukin-12 (IL-12) and IL-23 production and consequent activation of a polarized type I response; high production of toxic intermediates (nitric oxide (NO), reactive oxygen intermediates (ROI)). Based on these, Ml macrophages are generally considered to be potent effector cells that kill microorganisms and tumor cells and produce copious amounts of pro-inflammatory cytokines. In stark contrast, M2 macrophages induced by IL-4, IL-13, glucocorticoid, transforming growth factor-β(TGF-β), and prostaglandin E2 (PGE2) and so on, are characterized by: poor antigen-presenting capability, producing factors that suppress T-cell proliferation and activity (such as IL-10), and generally be better adapted to scavenging debris, promoting angiogenesis, and repairing and remodeling wounded/damaged tissues.Increasing evidences have shown that TAMs do not exert anti-tumor effect, while are involved in the process of tumorigenesis, tumor development, invasion and metastasis, especially are closely related to tumor angiogenesis and lymphangiogenesis. According to the functions of the TAMs in the tumors and their activation states, we speculated that TAMs exerted immune surveillance and anti-tumor function at the early stage of the tumors and most of them were M1 macrophages, while TAMs could promote tumor development, invasion and metastasis at the later stage of the tumors, and mostly were M2 macrophages; the types of TAMs at the later stage of the tumors were changed comparing with that at the early stage of the tumors, and the type-shift of the macrophages influences the development, invasion and metastasis of the tumors. Do TAMs change their types as we speculated in the development of tumors? How do the different types of macrophages influence the development of the tumors? To confirm it, we observed the types of macrophages at the early stage and the later stage of the tumor in 4T1 -bearing BALB/c mice model. Then we studied whether different types of the macrophages affected the development of the tumor with above experiment data and the mechanisms that different types of macrophages influenced the development of 4T1 tumor. These studies would provide theoretical basis and experimental proof in the treatment of tumors with TAMs as therapeutic targets, and would be meaningful for the study of tumor immunity and tumor therapeutics.1. The classification and identification of the macrophages.We used RAW264.7 cell line, which is a macrophage cell line derived from the tumor induced by Abelson murine leukemia virus in BALB/c mice, as the source of macrophages in vitro. We used LPS to induce M1 macrophages and used IL-4 to induce M2 macrophages in vitro. Using unstimulated RAW264.7 cells as control, macrophages stimulated with LPS or IL-4 were detected for the levels of membrane proteins CD16/32 and CD206 by FACS (fluorescence-activated cell sorting), the chemokine production of CCL3 and CCL22 by RT-PCR, and the gene expression of iNOS (inducible nitric oxide synthase) and Arg-1 by RT-PCR. It was shown that RAW264.7 cells stimulated with LPS expressed higher level of CD16/32 than that of control group, and expressed lower level of CCL22 than that of control group, and nearly did not express Arg-1; while RAW264.7 cells stimulated with IL-4 expressed higher level of CD206 than that of control group, expressed much higher level of Arg-1, and expressed lower levels of CD16/32 and CCL3 than that of control group (P<0.05). These results were in accordance with the features of the M1 and M2 macrophages. The above results indicated that there were two types of Macrophages and we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4.We next investigated the types of the macrophages in different disease models by isolating macrophages from BALB/c mice bearing 4T1 tumor at 4 weeks. To identify the types of infiltrated macrophages from 4 weeks 4T1-bearing mice by density-gradient centrifugation and adhering to disks, we detected the expression of CD16/32 and CD206 by FACS, the expression of CCL3, CCL22, iNOS and Arg-1 by RT-PCR, and the ability of phagocytosis using yeast cells. It was shown that TAMs isolated from 4T1-bearing mice expressed low level of CD16/32, high levels of CD206, CCL22 and Arg-1, and poor capacity to lick up yeast cells (P<0.05). These results showed us that tumor mass of the BALB/c mice bearing 4T1 tumor at 4 weeks were infiltrated with abundant M2 macrophages.The above results suggested that we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, and there were a great deal of M2 macrophages in tumor-bearing mice in vivo.2. Type-shift of macrophages in the process of tumor development.To investigate the type-shift of macrophages in the process of tumor development, we used 4T1-bearing BALB/c mice as tumor animal model. We firstly defined the early stage and the later stage of the 4T1 tumor by examining the size and the weight of the tumors, and detecting the metastasis of the tumor to lymph nodes and lung. The size and weight of 4T1 tumors were increased with time. In the 4 weeks 4T1 -bearing mice, we observed one or more tumor metastases in the lungs of 4T1-bearing mice after fixation by Bouin's fixative, and found tumor metastases in the oxter lymph node at the same side of the tumor and the lung in pathologic sections, but we did not find tumor metastases in the oxter lymph node at the same side of the tumor and the lung in the 1 week 4T1-bearing mice. Therefore, we confirmed with the size and weight of the tumor that the early stage of the tumor was within 1 week after innoculating tumor cells, and the later stage of the tumor was 3 to 4 weeks after innoculating tumor cells.We investigated the type-shift of macrophages in the process of tumor development by Immunofluorescence in the pathologic sections of the early or the later stage tumors from the 4T1-bearing BALB/c mice doublestaining with PE-CD16/32 mAb and FITC-F4/80 mAb or with PE-CD206 mAb and FITC-F4/80 mAb. Labeled cells were counted automatically by Image-Pro Plus 6.0 software. It was shown that most of the TAMs were M1 macrophages (F4/80~+CD16/32~+) at the early stage of the tumor, while most of the TAMs were M2 macrophages (F4/80~+CD206~+) at the later stage of the tumor. We also investigated the relationship between the types of the macrophages and the stages of the tumor. It was shown that the types of the macrophages were correlated with the process of tumor development.The above results suggested that the types of the TAMs were changed with the development of the tumors, and most of the TAMs were M1 macrophages at the early stage of the tumors, while most of the TAMs were M2 macrophages at the later stage of the tumors.3. The effects of different types of macrophages in tumor development.To investigate the effects of different types of macrophages in tumor development, we induced M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, using unstimulated RAW264.7 cells as control, mixed them with 4T1 cells at the ratio of 1:2, and subcutaneously injected the mixture at the abdominal skin of the BALB/c mice to get 4T1-bearing mice. After tumor innoculation, we observed daily the tumors in several groups appearing sooner or later and the size of the tumors since the second day, and got splenocytes from a mice of each group to examine the change of CD4~+ and CD8~+ T cells and detect the cytotoxic capacity of the splenocytes against 4T1 cells at day 7 and day 28. It was shown that tumors of the group of the M2 macrophages induced by IL-4 appeared earlier than other groups and were bigger than that of other groups at the early stage, tumors of the group of the M1 macrophages induced by LPS were smaller than that of other groups, and the size of the tumors from the group of the M2 macrophages induced by IL-4 were similar with that of the group of unstimulated macrophages and the group of 4T1 cells; at the early stage of the tumor, the percentage of the CD4~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 37.01%, similar with that of the group of M1 macrophages induced by LPS (34.50%) and the group of unstimulated RAW264.7 cells (36.74%) and the group of tumor-free mice (35.78%), slightly higher than that of the group of 4T1-bearing mice (30.91%), and the percentage of the CD8~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 17.33%, similar with that of the group of M1 macrophages induced by LPS (17.15%) and the group of unstimulated RAW264.7 cells (17.00%) and the group of tumor-free mice (16.84%), slightly higher than that of the group of 4T1 -bearing mice (15.14%); at the later stage of the tumor, the percentage of the CD4~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 8.16%, lower than that of the group of unstimulated RAW264.7 cells (11.26%) and the group of 4T1-bearing mice (18.19%), much lower than that of the group of M1 macrophages induced by LPS (32.41%) and the group of tumor-free mice (36.16%)(P < 0.05), and the percentage of the CD8~+ T cells in the splenocytes in the group of M2 macrophages induced by IL-4 was 5.07%, lower than that of the group of unstimulated RAW264.7 cells (5.86%) and the group of 4T1-bearing mice (7.78%), much lower than that of the group of M1 macrophages induced by LPS (16.19%) and the group of tumor-free mice (21.30%)(P < 0.05); at the early stage of the tumor, the cytotoxic capacity of the splenocytes against 4T1 cells in the group of M2 macrophages induced by IL-4 was 45.39%, slightly lower than that of the group of M1 macrophages induced by LPS (50.59%), much lower than that of the group of unstimulated RAW264.7 cells (75.61%), the group of 4T1-bearing mice (82.54%) and the group of tumor-free mice (64.29%), while at the later stage of the tumor, the cytotoxic function of the splenocytes against 4T1 cells in the group of M2 macrophages induced by IL-4 was 49.18%, much lower than that of the group of M1 macrophages induced by LPS (78.53%) and the group of tumor-free mice (73.50%)( P < 0.05).The above results suggested that different types of macrophages in the course of tumor progression could affect the development of the tumor, the subpopulations and the functions of the T cells in the tumor-bearing mice. M2 macrophages could promote the development of the tumors. At the early stage of the tumor, the different types of macrophages had little effect on the subpopulations and the functions of the T cells in the tumor-bearing mice, while at the later stage of the tumor, M1 macrophages could resume the subpopulations and the functions of the T cells in the tumor-bearing mice to normal levels of the tumor-free mice, which could favor anti-tumor immune response.4. The investigation of the mechanisms affecting tumor immunity by different types of macrophages—M2 macrophages induced the regulatory T cellsIt has been shown that regulatory T cells (Tregs) played an important role in the tumor escape and the tumor immunity. To investigate whether Treg cells changed in the early and the later stage of the tumors, we isolated tumor infiltrating lymphocytes (TILs) from 4T1-bearing mice, and detected the change of the percentage and the number of the CD4~+CD25~+ Treg cells in the TILs by FACS. We found that the percentage of the CD4~+CD25~+ Treg cells in the tumors in the later stage of the tumor was obviously increased than that in the early stage of the tumor (7.36% vs 4.47%, P<0.05), and the number of the CD4~+CD25~+ Treg cells in the TILs in the later stage of the tumor was obviously increased than that in the early stage of the tumor (2×10~4 vs 1×10~3,P<0.05).When we did the experiment in part 3, we also detected the change of Treg cells in the early and the later stage of the tumor at the same time. It was shown that at the early stage of the tumor the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ splenocytes in the group of M2 macrophages induced by IL-4 was 9.37%, similar with that of the group of M1 macrophages induced by LPS (11.04%), the group of unstimulated RAW264.7 cells (10.46%), the group of 4T1-bearing mice (10.24%) and the group of tumor-free mice (10.87%), while at the later stage of the tumor, the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ splenocytes in the group of M2 macrophages induced by IL-4 was 22.43%, much higher than that of the group of M1 macrophages induced by LPS (14.31%) and the group of tumor-free mice (13.90%), slightly higher than that of the group of unstimulated RAW264.7 cells (20.42%) and the group of 4T1-bearing mice (18.57%)(P < 0.05). These results suggested that different types of macrophages affected the course of tumor progression by inducing the Treg cells in the tumor. At the early stage of the tumor, the different types of macrophages had little effect on the level of the Treg cells in the tumor-bearing mice, while at the later stage of the tumor, M2 macrophages could increase the level of the Treg cells in the tumor-bearing mice which were harmful to anti-tumor immune response.To observe the influence of different types of macrophages on the Treg cells, we induced M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, using unstimulated RAW264.7 cells as control, co-cultured them with splenocytes or lymph node cells isolated from 4T1-bearing mice for 48 hours, then detected the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ cells by FACS. It was shown that the percentage of the CD4~+Foxp3~+ Treg cells in the CD4~+ cells in the group of M2 macrophages induced by IL-4 was obviously higher than that of the group of M1 macrophages induced by LPS and the group of unstimulated splenocytes or lymph node cells(P < 0.05). At the same time, we detected the expression of TGF-βat gene level in the macrophages of different types, which could induce Treg cells. Results showed that the gene level of TGF-βin the group of M2 macrophages induced by IL-4 was higher than that of the group of M1 macrophages induced by LPS and the group of unstimulated RAW264.7 cells (P < 0.05). These results indicated that different types of macrophages could influence Treg cells, subsequently affected the anti-tumor immunity.In summary, we succeeded in inducing M1 macrophages with LPS and M2 macrophages with IL-4 in vitro, and based on these results we studied the relationship between macrophages and tumors. It was shown that the types of TAMs were changed with the development of the tumors by examining the pathologic sections, most of the TAMs were M1 macrophages in the early stage of the tumor and most of the TAMs were M2 macrophages in the later stage of the tumor, and the types of the macrophages were correlated with the process of tumor development. We further studied the effects of different types of macrophages on tumor development and their mechanisms, and found that M2 macrophages could promote the development of the tumors and expand Treg cells, while M1 macrophages could make the subpopulations and the functions of the T cells in the tumor-bearing mice resuming to normal levels of the normal mice, which could favor anti-tumor immune response. Therefore, changing the types of the macrophages could affect host tumor immunity. These studies would provide the theoretical basis and experimental proof in the treatment of tumors with TAMs as therapeutic targets, and would be meaningful for the study of tumor immunity and tumor therapeutics.
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