Correlation between PD-1/PD-L1 expression and polarization in tumor-associated macrophages: A key player in tumor immunotherapy

Tumor immunotherapy, such as PD-1/PD-L1 blockade, has shown promising clinical efficacy in patients with various types of tumors. However, the response to PD-1/PD-L1 blockade in a majority of malignancies is limited, indicating an urgent need for a deeper understanding of the mechanisms of PD-1/PD-L1 axis-mediated tumor tolerance. As the most abundant immune cells in the tumor stroma, macrophages display multiple phenotypes and functions in response to the stimuli of the tumor microenvironment. PD-1/PD-L1 has been demonstrated to be highly expressed in tumor-associated macrophages (TAMs), and TAM polarization has been shown to be important during tumor progression. In this review, we outline the relationship between TAM PD-1/PD-L1 expression and polarizations, summarize the involvement of M2 TAMs in PD-1/PD-L1-mediated T-cell exhaustion, and discuss improved approaches for overcoming PD-1/PD-L1 blockade resistance by inducing M2/M1 switching of TAMs.     

Tumor cell apoptosis polarizes macrophages role of sphingosine-1-phosphate

Macrophage polarization contributes to a number of human pathologies. This is exemplified for tumor-associated macrophages (TAMs), which display a polarized M2 phenotype, closely associated with promotion of angiogenesis and suppression of innate immune responses. We present evidence that induction of apoptosis in tumor cells and subsequent recognition of apoptotic debris by macrophages participates in the macrophage phenotype shift. During coculture of human primary macrophages with human breast cancer carcinoma cells (MCF-7) the latter ones were killed, while macrophages acquired an alternatively activated phenotype. This was characterized by decreased tumor necrosis factor (TNF)-alpha and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2-overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-kappaB signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization.     

Sulforaphene targets NLRP3 inflammasome to suppress M1 polarization of macrophages and inflammatory response in rheumatoid arthritis

This work aimed to explore the therapeutic effect and target of sulforaphene (LF) in mice with rheumatoid arthritis (RA). Lipopolysaccharide (LPS) and IFN-γ were added to induce the M1 polarization of SMG cells, and later cells were pretreated with 5 μM and 15 μM LF. M1 cell proportion was detected by flow cytometry (FCM), inflammatory factors were measured by enzyme-linked immunosorbent assay, and protein levels were analyzed by western blotting (WB) assay. Besides, small molecule-protein docking and pull-down assays were carried out to detect the binding of LF to NLRP3. After the knockdown of NLRP3 in SMG cells, the effect of LF was further detected. The RA mouse model was induced with collagen antibody and LPS, after LF intervention, H&E staining was performed to detect the pathological changes in mouse synovial membrane, whereas safranin O-fast green staining was performed to detect cartilage injury, NLRP3 inflammasome and inflammatory factor levels in tissues. LF suppressed M1 polarization of macrophages, reduced M1 cell proportion and inflammatory factor levels, and suppressed the activation of NLRP3 inflammasome. After NLRP3 knockdown, LF did not further suppress the M1 polarization of macrophages. Pull-down assay suggested that LF bound to NLRP3. As revealed by mouse experimental results, LF inhibited bone injury in mice, decreased M1 cell infiltration and inflammatory response in tissues, and inhibited NLRP3 inflammasome expression in tissues. LF targets NLRP3 to suppress the M1 polarization of macrophages and decrease tissue inflammation in RA.     

Tumor associated macrophages and neutrophils in tumor progression

Tumor‐associated macrophages (TAMs) are a key component of the tumor microenvironment and orchestrate various aspects of cancer. Diversity and plasticity are hallmarks of cells of the monocyte–macrophage lineage. In response to distinct signals macrophages undergo M1 (classical) or M2 (alternative) activation, which represent extremes of a continuum in a spectrum of activation states. Metabolic adaptation is a key component of macrophage plasticity and polarization, instrumental to their function in homeostasis, immunity and inflammation. Generally, TAMs acquire an M2‐like phenotype that plays important roles in many aspects of tumor growth and progression. There is now evidence that also neutrophils can be driven towards distinct phenotypes in response to microenvironmental signals. The identification of mechanisms and molecules associated with macrophage and neutrophil plasticity and polarized activation provides a basis for new diagnostic and therapeutic strategies. J. Cell. Physiol. 228: 1404–1412, 2013. © 2012 Wiley Periodicals, Inc.     

Emerging role of tumor-associated macrophages as therapeutic targets in patients with metastatic renal cell carcinoma

Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial–mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.     

Metformin's antitumour and anti‐angiogenic activities are mediated by skewing macrophage polarization

Beneficial effects of metformin on cancer risk and mortality have been proved by epidemiological and clinical studies, thus attracting research interest in elucidating the underlying mechanisms. Recently, tumour‐associated macrophages (TAMs) appeared to be implicated in metformin‐induced antitumour activities. However, how metformin inhibits TAMs‐induced tumour progression remains ill‐defined. Here, we report that metformin‐induced antitumour and anti‐angiogenic activities were not or only partially contributed by its direct inhibition of functions of tumour and endothelial cells. By skewing TAM polarization from M2‐ to M1‐like phenotype, metformin inhibited both tumour growth and angiogenesis. Depletion of TAMs by clodronate liposomes eliminated M2‐TAMs‐induced angiogenic promotion, while also abrogating M1‐TAMs‐mediated anti‐angiogenesis, thus promoting angiogenesis in tumours from metformin treatment mice. Further in vitro experiments using TAMs‐conditioned medium and a coculture system were performed, which demonstrated an inhibitory effect of metformin on endothelial sprouting and tumour cell proliferation promoted by M2‐polarized RAW264.7 macrophages. Based on these results, metformin‐induced inhibition of tumour growth and angiogenesis is greatly contributed by skewing of TAMs polarization in microenvironment, thus offering therapeutic opportunities for metformin in cancer treatment.     

Decoy receptor 3 enhances tumor progression via induction of tumor-associated macrophages

Tumor-associated macrophages (TAMs) are the major component of tumor-infiltrating leukocytes. TAMs are heterogeneous, with distinct phenotypes influenced by the microenvironment surrounding tumor tissues. Decoy receptor 3 (DcR3), a member of the TNFR superfamily, is overexpressed in tumor cells and is capable of modulating host immunity as either a neutralizing decoy receptor or an effector molecule. Upregulation of DcR3 has been observed to correlate with a poor prognosis in various cancers. However, the mechanisms underlying the DcR3-mediated tumor-promoting effect remain unclear. We previously demonstrated that DcR3 modulates macrophage activation toward an M2-like phenotype in vitro and that DcR3 downregulates MHC class II expression in TAMs via epigenetic control. To investigate whether DcR3 promotes tumor growth, CT26-DcR3 stable transfectants were established. Compared with the vector control clone, DcR3-transfectants grew faster and resulted in TAM infiltration. We further generated CD68 promoter-driven DcR3 transgenic (Tg) mice to investigate tumor growth in vivo. Compared with wild-type mice, macrophages isolated from DcR3-Tg mice displayed higher levels of IL-10, IL-1ra, Ym1, and arginase activity, whereas the expression of IL-12, TNF-α, IL-6, NO, and MHC class II was downregulated. Significantly enhanced tumor growth and spreading were observed in DcR3-Tg mice, and the enhanced tumor growth was abolished by arginase inhibitor N-ω-hydroxy-l-norarginine and histone deacetylase inhibitor sodium valproate. These results indicated that induction of TAMs is an important mechanism for DcR3-mediated tumor progression. Our findings also suggest that targeting DcR3 might help in the development of novel treatment strategies for tumors with high DcR3 expression.     

AQP9 transports lactate in tumor-associated macrophages to stimulate an M2-like polarization that promotes colon cancer progression

Macrophages play a major role in the immune defense against pathogenic factors; however, they can lead to tumor exacerbation and metastasis, as the tumor microenvironment (TME) polarizes tumor-associated macrophages (TAMs) into the M2 subtype. Lactate, a metabolite produced by carcinoma cells at high concentrations in the TME, induces an M2-polarization in macrophages, which ultimately leads to the secretion of factors, such as vascular endothelial growth factor (VEGF), and promotes tumor progression. However, the effect of TAM lactate import on tumor progression has not been fully elucidated. Aquaporin 9 (AQP9) is a transporter of water and glycerol expressed in macrophages. Here, we used a tumor allograft mouse model to show that AQP9 knockout (AQP9^?/?) mice were more resistant against tumor cell growth and exhibited a suppressive M2-like polarization in tumor tissue than wild-type mice. Moreover, we discovered that the primary bone marrow-derived macrophages from AQP9^?/? mice were less sensitive to lactate stimulation and exhibited reduced M2-like polarization as well as decreased VEGF production. To further investigate the role of AQP9 in macrophage polarization, we overexpressed AQP9 in Chinese hamster ovary cells and found that AQP9 functioned in lactate import. In contrast, primary AQP9^?/? macrophages and AQP9 knockdown RAW264.7 cells exhibited a reduced lactate transport rate, suggesting the involvement of AQP9 in lactate transport in macrophages. Together, our results reveal the mechanism by which the TME modifies the polarization and function of tumor-infiltrating macrophages via AQP9 transport function.     

KDM6B regulates M2 polarization of macrophages by modulating the stability of nuclear β-catenin

Accumulating evidences suggest that the epigenetic regulation plays a pivotal role in establishing phenotype and function of tumor associated macrophages (TAMs). KDM6B is an epigenetic enzyme responsible for the H3K27me3 and reported to influence macrophage polarization. However, the underlying mechanism remains to be determined. Here, we demonstrated that inhibition of KDM6B in TAMs increased M2 polarization induced by coculture of breast cancer cells. Furthermore, we identified that KDM6B downregulation activated β-catenin/c-Myc signaling, and thus promoted the M2-like phenotype. KDM6B accelerated the intranuclear ubiquitination degradation of β-catenin, which depended on its demethylase activity. Therapeutically, our data showed that activated vitamin D analog paricalcitol upregulated the expression of KDM6B and decreased the M2 polarization, consequently protected against tumor progress in the xenograft mouse model of breast cancer. Taken together, our data reveal that epigenetic regulator KDM6B prevents M2 polarization via promoting the intranuclear degradation of β-catenin. Active vitamin D analog induces KDM6B and suppresses tumor progress, suggesting a novel therapeutic potential of epigenetic modulation for the tumor treatment.     

Anti-tumor effect of aquaporin 3 monoclonal antibody on syngeneic mouse tumor model

Aquaporin-3 (AQP3), a water channel protein, has been found to be involved in cancer progression via water and small molecule transport function. However, drug development targeting AQP3 has not yet begun.Here, we showed that a recently established anti-AQP3 monoclonal antibody (mAb) suppresses tumor growth in allograft mouse colorectal tumor models produced using CT26 or MC38 cancer cells. Administration of the anti-AQP3 mAb to BALB/c mice with transplanted CT26 cells increased the M1/M2 ratio of tumor-associated macrophages (TAM) and improved the mitochondrial function of T cells in the tumor microenvironment (TME). Administration of anti-AQP3 mAb also restored the TAM-induced decrease in T cell proliferation. Macrophage depletion in wild-type mice counteracted the antitumor effect of anti-AQP3 mAb in the mouse tumor model, suggesting that one of the primary targets of anti-AQP3 mAb is macrophages. In in vitro studies using mice bone marrow monocytes and human monocyte THP-1 cells, anti-AQP3 mAb attenuated carcinoma cell-mediated polarization of monocytes into M2-like TAMs.These data suggest that anti-AQP3 mAb suppresses tumor growth by attenuating immunosuppressive M2-like TAMs, which in turn maintains the antitumor function of T cells in the TME. Thus, the anti-AQP3 mAb is a potential cancer therapy that functions by targeting TAMs.     

Prostate cancer cells modulate the differentiation of THP‐1 cells in response to etoposide and TLR agonists treatments

The aim of this study was to determine the polarization of macrophages in the tumor microenvironment, as well as the effect of soluble factors secreted from these polarized macrophages on etoposide‐induced cancer cell apoptosis. We investigated the effect of soluble factors secreted from the supernatant of PC3 cells treated with TLR4 and TLR8 agonists, and etoposide on macrophage polarization at the protein level through flow cytometry and enzyme‐linked immunosorbent assay. We further explored the cell cycle distribution and phagocytic activity of THP‐1 cells by flow cytometry. To imitate the relationship between cancer cells and tumor‐associated macrophages (TAMs), we cocultured macrophages with etoposide‐treated PC3 cells. After the incubation, the apoptosis in cancer cells was assessed through FACS analysis and by annexin V and PI staining. Our results demonstrate that protein expression of M1 and M2 markers confirmed the upregulation of M1 markers upon etoposide treatment, and mixed M1/M2 phenotype upon treatment with TLR agonists‐treated PC3 supernatant. In coculture methods, our results demonstrate that the apoptosis of etoposide‐treated cancer cells increases in the presence of M0 macrophages and THP‐1 cells incubated with the supernatant of TLR4 agonists‐treated PC3 cells. These results indicate clear protective effects of M0 macrophages and THP‐1 cells incubated with the supernatant of PC3 cells treated with TLR4 agonists (THP‐1 + SUP + TLR4a) on etoposide‐induced cancer cell apoptosis.     

AIM2 promotes non-small-cell lung cancer cell growth through inflammasome-dependent pathway

The human absent in melanoma 2 (AIM2) is considered as a DNA recognizer. AIM2 has been described as a tumor suppressor gene in the early years. But recent studies suggested that it functions as an oncogene in several cancers. However, its roles in non-small-cell lung cancer (NSCLC) remain unclear. Here we reported that AIM2 highly expressed in NSCLC cells and exhibited a tumor-promoting property both in vitro and in vivo. Besides, AIM2 short hairpin RNA (shRNA)-mediated suppression of cell proliferation was triggered by the accumulation of cells at the G2/M phase. Knockdown of AIM2 reduced the inflammasome formation, while overexpression of AIM2 or stimulation by poly(dA:dT) induced the inflammasome formation. Interestingly, blockade of the inflammasome by caspase-1 inhibitor VX-765 or ASC small interfering RNA (siRNA) abolished the effects brought by AIM2 shRNA and AIM2 plasmid. In summary, our results revealed that AIM2 functioned as an oncogene in NSCLC in an inflammasome-dependent way.     

CDDO-Me Redirects Activation of Breast Tumor Associated Macrophages

Tumor-associated macrophages can account for up to 50% of the tumor mass in breast cancer patients and high TAM density is associated with poor clinical prognosis. Because TAMs enhance tumor growth, development, and metastatic potential, redirection of TAM activation may have significant therapeutic benefit. Our studies in primary human macrophages and murine breast TAMs suggest that the synthetic oleanane triterpenoid CDDO-methyl ester (CDDO-Me) reprograms the activation profile of TAMs from tumor-promoting to tumor-inhibiting. We show that CDDO-Me treatment inhibits expression of IL-10 and VEGF in stimulated human M2 macrophages and TAMs but increases expression of TNF-α and IL-6. Surface expression of CD206 and CD163, which are characteristic of M2 activation, is significantly attenuated by CDDO-Me. In contrast, CDDO-Me up-regulates surface expression of HLA-DR and CD80, which are markers of M1 activation, and importantly potentiates macrophage activation of autologous T cells but inhibits endothelial cell vascularization. These results show for the first time that CDDO-Me redirects activation of M2 macrophages and TAMs from immune-suppressive to immune-stimulatory, and implicate a role for CDDO-Me as an immunotherapeutic in the treatment of breast and potentially other types of cancer.     

Tumor‐associated macrophages (TAMs) depend on ZEB1 for their cancer‐promoting roles

Accumulation of tumor‐associated macrophages (TAMs) associates with malignant progression in cancer. However, the mechanisms that drive the pro‐tumor functions of TAMs are not fully understood. ZEB1 is best known for driving an epithelial‐to‐mesenchymal transition (EMT) in cancer cells to promote tumor progression. However, a role for ZEB1 in macrophages and TAMs has not been studied. Here we describe that TAMs require ZEB1 for their tumor‐promoting and chemotherapy resistance functions in a mouse model of ovarian cancer. Only TAMs that expressed full levels of Zeb1 accelerated tumor growth. Mechanistically, ZEB1 expression in TAMs induced their polarization toward an F4/80^low pro‐tumor phenotype, including direct activation of Ccr2. In turn, expression of ZEB1 by TAMs induced Ccl2, Cd74, and a mesenchymal/stem‐like phenotype in cancer cells. In human ovarian carcinomas, TAM infiltration and CCR2 expression correlated with ZEB1 in tumor cells, where along with CCL2 and CD74 determined poorer prognosis. Importantly, ZEB1 in TAMs was a factor of poorer survival in human ovarian carcinomas. These data establish ZEB1 as a key factor in the tumor microenvironment and for maintaining TAMs’ tumor‐promoting functions.     

Tumor-derived lactate induces M2 macrophage polarization via the activation of the ERK/STAT3 signaling pathway in breast cancer

Tumor-associated macrophages (TAM) are prominent components of tumor microenvironment (TME) and capable of promoting cancer progression. However, the mechanisms for the formation of M2-like TAMs remain enigmatic. Here, we show that lactate is a pivotal oncometabolite in the TME that drives macrophage M2-polarization to promote breast cancer proliferation, migration, and angiogenesis. In addition, we identified that the activation of ERK/STAT3, major signaling molecules in the lactate signaling pathway, deepens our molecular understanding of how lactate educates TAMs. Moreover, suppression of ERK/STAT3 signaling diminished tumor growth and angiogenesis by abolishing lactate-induced M2 macrophage polarization. Finally, research data of the natural compound withanolide D provide evidence for ERK/STAT3 signaling as a potential therapeutic strategy for the prevention and treatment of breast cancer. These findings suggest that the lactate-ERK/STAT3 signaling pathway is a driver of breast cancer progression by stimulating macrophage M2-like polarization and reveal potential new therapeutic targets for breast cancer treatment.