| Section1The role of hypoxia in the selective promotion of M2macrophage polarization and its underlying mechanismsObjective:Macrophages display phenotypic and functional plasticity, as they can shift between different modes of activation and perform divergent functions according to the microenvironmental cues they receive. The macrophages have two well-established polarized phenotypes, the classically activated macrophages (M1) and alternatively activated macrophages (M2). The macrophages within the tumor are often called tumor-associated macrophages (TAMs). The activation and function of TAM is related to the microenvironment of tumor. As the hallmark feature of malignant tumors, hypoxia has been proved to play an important role in TAM infiltration into tumor tissue. However, the impact of hypoxia on the phenotype shift of TAM is still largely unknown. Thus, this study attempts to characterize the effect of hypoxia on macrophage phenotype shift and functional responses, as well as the underlying mechanisms involved.Methods:(1) Flow cytometric analysis was performed to analyze the expression of cell surface markers.(2) Immunostaining analysis was used to detect the expression of related proteins in tumor tissue.(3) The expression of M2phenotype genes was measured by RT-PCR.(4) A gene microarray approach was used to compare the expression of differential genes.(5) Western blot assay was employed to detect protein expression.Results:The previous study in LLC model has showed that nearly half of the macrophages (F4/80+) exhibited a M2phenotype (CD209+) and preferentially situated at hypoxic regions (PIMO+), indicating that tumor hypoxia is associated with M2macrophage polarization. To further clarify this issue, C57BL/6mice bearing LLC tumor were exposed to normoxia or normobaric hypoxia for4h every day. As expected, intermittent hypoxia exposure significantly increased the infiltration of macrophages (F4/80+) as well as the ratio of CD209+macrophages in primary tumor tissue. By using the in vitro co-culture model of hypoxia, we observed that hypoxia selectively promoted the expression of CD209and CD206(markers for M2macrophage) but not CD86(a marker for M1macrophage) in macrophages induced by stimulation (LLC-CM, IL6, IL4or IL13). Moreover, the mRNA expression of ARG1and YM1was significantly elevated in HC+IL6macrophages compared with that of NC+IL6macrophages. The microarray results showed that HC+IL6macrophages expressed low levels of M1phenotype related genes and high levels of M2phenotype related genes. We subsequently extended our study using a primary macrophages model based on the generation of BMDMs. Once again, IL6-treated BMDMs exposed to hypoxia expressed higher levels of CD209compared with the ones exposed to normoxia. The data above clearly demonstrate that hypoxia selectively promotes the M2polarization of macrophages triggered by IL6. Of note, HIF activity and STAT3signaling pathway did not participate in the M2macrophage polarization in response to IL6plus hypoxia. Furthermore, data from microarray assay and western blot analysis suggest that MAPK cascade is operative in our experimental model. While all three MAPKs, ERK, JNK and p38, are activated under hypoxia conditions, only the inhibition of ERK activation can block hypoxia-promoted M2macrophage polarization.Conclusion:Tumor hypoxia selectively promotes M2macrophage polarization through the activation of ERK. These observations highlight a novel concept of tumor hypoxia involved phenotype shift of macrophages.Section2Effects of hypoxia-promoted M2macrophages on tumor metastasisObjective:Hypoxia is a common phenomenon occurring in the majority of human tumors including NSCLC, and has been proved to play an important role in tumor progression. Most studies of tumor hypoxia focus on the effect of hypoxia on the inherent adhesive and invasive ability of tumor cells, but the possibility that hypoxia may aggravate tumor behaviors via effecting non-cancer cells is largely unknown. As the most prominent component of non-cancer cells, TAMs are associated with poor prognosis. Together with the fact that hypoxia selectively promotes the M2polarization of macrophages. In this section, we attempt to characterize the effect of hypoxia-promoted M2macrophages on tumor metastasis and angiogenesis, in the hope to determine whether the action of hypoxia on TAM is involved in hypoxia-driven tumor behaviors.Methods:(1) Immunostaining analysis was used to detect the expression of related proteins in tissue biopsies.(2) Wound healing assay and transwell assay were used to evaluate the effect of polarized macrophages on LLC cell migration.(3) The tube formation assay was employed to investigate the effect of polarized macrophages on tumor angiogenesis.(4) LLC spontaneous metastasis model was introduced to investigate the rate of metastasis.(5) SRB assay was used to examine the cell proliferation.Results:Through the analysis of microarray data (GSE1987) from36samples obtained from human lung tissue and the infiltration of M2macrophages in primary tumor tissues isolated from55NSCLC patients including20patients with metastasis, we observed that high density of M2TAM is associated with metastasis in NSCLC patients. Based on this finding, we prompted to evaluate the effect of hypoxia-promoted M2macrophages on the metastasis of LLC tumor both in vitro and in vivo. LLC cells or HUVEC cells were co-incubated with indicated macrophage-conditioned mediums, by employing cell proliferation assay, wound-healing assay, transwell assay and tube formation assay, the results suggested that the supernatant of hypoxia-promoted M2macrophages enhanced the formation of capillary-like structures in HUVEC cells and the migration of LLC cells, whereas showed no effect on LLC cell proliferation. Additionally, co-inoculation of hypoxia-promoted M2macrophages with LLC cells increased the metastasis of LLC transplanted tumors (from28.6%to100%) and the proportion of CD31-positive cells in tumor tissue. These results demonstrate that hypoxia-promoted M2macrophages could enhance the tumor metastasis and angiogenesis. Given that hypoxia-promoted M2macrophages exhibited similar function in cancer promotion as hypoxia, we thus hypothesized that hypoxia-promoted M2macrophage polarization might be involved in tumor hypoxia-driven tumorigenesis. By applying the in vivo hypoxia model, the results suggested that intermittent hypoxia significantly promoted the metastasis of LLC (from20%to60%), accompanied with increased CD209+macrophages infiltration in primary tumor tissue. Interestingly, by targeting to macrophages rather than tumor cells, the well-known ERK inhibitor PD98059could suppress the migration of LLC cells.Conclusion:Hypoxia-promoted M2macrophages enhance LLC metastasis and angiogenesis both in vitro and in vivo. By targeting macrophage polarization, the blockage of ERK could serve as a promising lung cancer therapeutic strategy. This study unveils a novel concept of tumor hypoxia and provides evidence for lung cancer intervention through modulating the phenotype of macrophages.Section3Compound M selectively inhibits TAM M2polarization and its effects on tumor metastasisObjective:Since TAM depolarization from M2phenotype is considered to be a promising anti-metastasis strategy, the development of specific small-molecule inhibitors for TAM M2polarization have been taken into concern. We have previously screened series of compounds and found that compound M can selectively reduce M2macrophage polarization. Based on this finding, we will further investigate the role of compound M in anti-metastasis and evaluate its underlying mechanisms.Methods:(1) SRB assay was used to examine the cell proliferation.(2) Flow cytometric analysis was performed to analyze the expression of cell surface markers.(3) Immunostaining analysis was used to detect the expression of related proteins in tumor tissues.(4) The expression of M1/M2phenotype genes was measured by RT-PCR.(5) Transwell assay was used to evaluate the migration of LLC cell.(6) LLC spontaneous metastasis model was introduced to investigate the tumor metastasis.(7) HE staining was used to evaluate lung metastasis.(8) Western blot assay was employed to detect protein expression.Results:By using IL13induced-M2macrophages, we successfully found compound M from series of small-molecule compounds, which could significantly suppress the M2polarization of macrophages. In addition, compound M decreased the M2polarization of macrophages induced by IL4and IL6. Subsequently, we extended our study into IFN-y and LPS-mediated Ml macrophage polarization model. Intriguingly, compound M has no effect on IFN-γ and LPS-triggered M1polarization. The data above clearly demonstrate that compound M selectively inhibits the M2polarization of macrophage. Immunostaining derived from LLC tumor tissues further validate that compound M can also inhibit M2macrophage polarization in vivo. Next, in order to clarify the role of compound M in inhibition of metastasis, transwell assay and LLC spontaneous metastasis assay were performed. As expected, compound M blocked the migration-promotion effect of M2macrophages on LLC cells and effectively prevented the metastasis of LLC in vivo. Furthermore, C57BL/6mice were depleted of macrophages by clodronate liposomes2days before inoculation with LLC cells, and then treated with or without compound M. The results from lung metastasis suggested that the anti-metastasis effect of compound M is through the inhibition of M2polarization. In addition, compound M could reduce the formation of new blood vessels in LLC-transplantated tumor tissue. We further demonstrated that reeducation of M2macrophage polarization by compound M relies substantially on activation of AMPK signaling.Conclusion:Compound M selectively suppresses the M2polarization of TAM through the activation of AMPK to prevent tumor metastasis. The study confirms the feasibility of small molecule compounds to inhibit tumor metastasis by targeting macrophage ploarization and provides potential therapeutic targets for anti-metastasis therapy. |
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