Tumor-associated macrophages: Effectors of angiogenesis and tumor progression

Tumor-associated macrophages (TAMs) are a prominent inflammatory cell population in many tumor types residing in both perivascular and avascular, hypoxic regions of these tissues. Analysis of TAMs in human tumor biopsies has shown that they express a variety of tumor-promoting factors and evidence from transgenic murine tumor models has provided unequivocal evidence for the importance of these cells in driving angiogenesis, lymphangiogenesis, immunosuppression, and metastasis. This review will summarize the mechanisms by which monocytes are recruited into tumors, their myriad, tumor-promoting functions within tumors, and the influence of the tumor microenvironment in driving these activities. We also discuss recent attempts to both target/destroy TAMs and exploit them as delivery vehicles for anti-cancer gene therapy.     

Tumor necrosis factor-alpha promotes the accumulation of neutrophils and macrophages in skeletal muscle.

Tumor necrosis factor-alpha (TNF-alpha) has been associated with cachexia and is known to regulate multiple inflammatory cell (neutrophil and macrophage) responses. We tested the hypothesis that neutrophils and macrophages accumulate in the extensor digitorum longus (EDL) and soleus muscles of mice after chronic TNF-alpha administration. Murine recombinant TNF-alpha (approximately 100 microg x kg(-1) x day(-1)) in vehicle solution or vehicle solution alone (sham) was administered to C57BL/6 mice for 7 days via osmotic minipumps. In EDL muscles from TNF-alpha-treated mice, neutrophil and macrophage concentrations were elevated seven- and threefold, respectively, compared with sham mice. Neutrophil and macrophage concentrations were also elevated five- and twofold, respectively, in solei of TNF-alpha- relative to sham-treated mice. Treatment with TNF-alpha elevated ubiquitin content by approximately 25% relative to sham values for both the EDL and soleus muscles; however, these elevations were not statistically significant. No differences were observed between TNF-alpha- and sham-treated mice in body weight, food consumption, muscle mass, myofiber cross-sectional area, carbonyl groups, total protein content, or relative abundance of myosin heavy chain protein. Furthermore, no overt signs of muscle injury or regeneration were observed in muscles from TNF-alpha-treated mice in either the EDL or soleus muscles. These observations suggest that 7 days of TNF-alpha administration promote muscle inflammation as indicated by the accumulation of neutrophils and macrophages without overt signs of atrophy, injury, or regeneration.     

Autophagy regulation in macrophages and neutrophils

Autophagy is a conserved proteolytic mechanism that degrades cytoplasmic material including cell organelles. Accumulating evidence exists that autophagy also plays a major role in immunity and inflammation. Specifically, it appears that autophagy protects against infections and inflammation. Here, we review recent work performed in macrophages and neutrophils, which both represent critical phagocytes in mammalians.     

Tumor-associated neutrophils (TAN) develop pro-tumorigenic properties during tumor progression

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines—Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H₂O₂. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway.     

Tumor cell-mediated neovascularization and lymphangiogenesis contrive tumor progression and cancer metastasis

Robust neovascularization and lymphangiogenesis have been found in a variety of aggressive and metastatic tumors. Endothelial sprouting angiogenesis is generally considered to be the major mechanism by which new vasculature forms in tumors. However, increasing evidence shows that tumor vasculature is not solely composed of endothelial cells (ECs). Some tumor cells acquire processes similar to embryonic vasculogenesis and produce new vasculature through vasculogenic mimicry, trans-differentiation of tumor cells into tumor ECs, and tumor cell–EC vascular co-option. In addition, tumor cells secrete various vasculogenic factors that induce sprouting angiogenesis and lymphangiogenesis. Vasculogenic tumor cells actively participate in the formation of vascular cancer stem cell niche and a premetastatic niche. Therefore, tumor cell-mediated neovascularization and lymphangiogenesis are closely associated with tumor progression, cancer metastasis, and poor prognosis. Vasculogenic tumor cells have emerged as key players in tumor neovascularization and lymphangiogenesis and play pivotal roles in tumor progression and cancer metastasis. However, the mechanisms underlying tumor cell-mediated vascularity as they relate to tumor progression and cancer metastasis remain unclear. Increasing data have shown that various intrinsic and extrinsic factors activate oncogenes and vasculogenic genes, enhance vasculogenic signaling pathways, and trigger tumor neovascularization and lymphangiogenesis. Collectively, tumor cells are the instigators of neovascularization. Therefore, targeting vasculogenic tumor cells, genes, and signaling pathways will open new avenues for anti-tumor vasculogenic and metastatic drug discovery. Dual targeting of endothelial sprouting angiogenesis and tumor cell-mediated neovascularization and lymphangiogenesis may overcome current clinical problems with anti-angiogenic therapy, resulting in significantly improved anti-angiogenesis and anti-cancer therapies.     

Integrin signalling in neutrophils and macrophages.

Integrins have been characterized extensively as adhesion receptors capable of transducing signals inside the cell. In myelomonocytic cells, integrin-mediated adhesive interactions regulate different selective cell responses, such as transmigration into the inflammatory site, cytokine secretion, production or reactive oxygen intermediates, degranulation and phagocytosis. In the last few years, great progress has been made in elucidating mechanisms of signal transduction by integrins in neutrophils and macrophages. This review summarises the current information on the role of integrins in regulating myelomonocytic cell functions and highlights the signalling pathways activated by integrin engagement in these cells. Also, exploiting the current knowledge of mechanisms of integrin signal transduction in other cell types, we propose a model to explain how integrins transduce signals inside neutrophils and macrophages, and how signaling pathways leading to regulation of selective cell functions may be coordinated.     

Tumor progression: defining the soil round the tumor seed

The tumor microenvironment, or stroma, is known to contribute to tumor progression. Two recent studies have shown that the stromal protein matrix metalloproteinase MMP-9 has a role in the early stages of tumor growth and angiogenesis.     

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.     

Tumor progression despite efficient tumor antigen cross-presentation and effective "arming" of tumor antigen-specific CTL

To determine whether APC function or "arming" of CTL for lytic function are the points at which Ags from a nonimmunogenic tumor fail to induce an effective immune response, we established a murine tumor model that expressed intracellular OVA and selected a clone (cOVA-9) that remained susceptible to lysis by specific CD8(+) T cells throughout tumor growth. Viable cOVA-9 tumor cells grew in normal mice at a rate similar to the parental tumor, and vaccination with irradiated cOVA-9 cells did not induce protection against itself or the parental line, confirming its nonimmunogenic status. In vivo evaluation during tumor growth demonstrated persisting tumor Ag cross-presentation accompanied by the generation of potent, specific CTL which were detectable when tumors were barely palpable. Despite the presence of highly active CTL in the tumor-draining lymph nodes, there was no apparent lysis of tumor-associated APC. These data show that tumor-draining APC are not dysfunctional with regard to two crucial processes, in vivo tumor Ag cross-presentation and specific CTL arming, and that failure to prevent tumor growth is not in the induction phase, but in the effector phase and occurs within the tumor itself before the tumor matrix is established.     

Effect of mesenchymal stem cells-derived exosomes on tumor microenvironment: Tumor progression versus tumor suppression

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into different cell types. Owing to their immunosuppressive and anti-inflammatory properties, they are widely used in regenerative medicine, but they have a dual effect on cancer progression and exert both growth-stimulatory or -inhibitory effects on different cancer types. It has been proposed that these controversial effects of MSC in tumor microenvironment (TME) are mediated by their polarization to proinflammatory or anti-inflammatory phenotype. In addition, they can polarize the immune system cells that in turn influence tumor progression. One of the mechanisms involved in the TME communications is extracellular vesicles (EVs). MSCs, as one of cell populations in TME, produce a large amount of EVs that can influence tumor development. Similar to MSC, MSC-EVs can exert both anti- or protumorigenic effects. In the current study, we will investigate the current knowledge related to MSC role in cancer progression with a focus on the MSC-EV content in limiting tumor growth, angiogenesis, and metastasis. We suppose MSC-EVs can be used as safe vehicles for delivering antitumor agents to TME.     

Sunitinib suppresses M2 polarization of macrophages in tumor microenvironment to regulate hepatocellular carcinoma progression

This work aimed to investigate the role and mechanism of Sunitinib (Sun) in suppressing M2 polarization of macrophages in tumor microenvironment (TME). IL-4 was applied to induce the M2 polarization of RAW264.7 cells, followed by treatment with Sun at 50 and 100 nM. Flow cytometry (FCM) was conducted to detect the proportion of F4/80 + CD206 + cells. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of IL-10, Arg-1 and VEGF. Immunofluorescence (IF) staining was carried out to detect the expression of CD206 and Arg-1. Besides, western-Blot (WB) assay was performed to measure the levels of p-JAK1 and p-STAT6 proteins. After polarization, the macrophage culture medium was employed to culture hepatocellular carcinoma (HCC) Hca-F cells. Thereafter, Transwell assays were conducted to examine cell invasion and migration, whereas plate clone formation assay was carried out to detect the clone forming capacity. In further experiments, cells were treated with the STAT6 inhibitor, or STAT6 inhibitor + Sun. Then, the polarization levels of RAW264.7 cells were detected. Moreover, this study established the xenograft tumor mouse model. Later, CD206 and Ki67 expression, IL-10, Arg-1 and VEGF expression levels in tissues, and p-JAK1 and p-STAT6 protein levels were detected by histochemical staining. Sun suppressed the M2 polarization of RAW264.7 cells. Compared with IL-4 treatment, the proportion of F4/80 + CD206 + cells decreased. Meanwhile, the levels of IL-10, Arg-1 and VEGF were downregulated, and the phosphorylation level of JAK1-STAT6 signaling was suppressed. After being cocultured with Hca-F, the malignant behaviors of HCC cells were suppressed after Sun treatment. Similarly, STAT6 inhibitor treatment suppressed the M2 polarization, while the combined application of Sun did not further restrain the polarization level. In the mouse model, Sun suppressed the expression of CD206 and Ki67, simultaneously inhibiting the polarization of JAK1-STAT6 signaling. Sunitinib can suppress the M2 polarization of macrophages to exert the anti-HCC effect, which is its another anticancer mechanism     

Serum CXCL5 level is associated with tumor progression in penile cancer

Chemokine (C-X-C motif) ligand 5 is an important regulator of tumor progression in many cancers, and could serve as potential serum cancer biomarker. Our initial analysis identified CXCL5 as a cancer-related gene highly expressed in PC. Patients with PC exhibited markedly higher preoperative serum CXCL5 levels compared with that in healthy individuals (P<0.001). The area under the curve (AUC) was 0.880 with the sensitivity of 84.0%, and specificity of 80.4% to distinguish PC. Serum CXCL5 levels were also significantly decreased following tumor resection in patients with PC (P=0.001). Preoperative serum CXCL5 level was significantly associated with clinicopathological characteristics including T stage (P=0.001), nodal status (P<0.001), and pelvic lymph node metastasis (P=0.018). Cox regression analysis showed that serum CXCL5 level could serve as an independent prognostic factor for disease-free survival with a HR of 6.363 (95% CI: 2.185–18.531, P=0.001). CXCL5 and its receptor CXCR2 exhibited correlated expression pattern in PC tissues. Differential CXCL5 expression was observed in normal penile tissues, PC cell lines, and their culture supernatants. Furthermore, knockdown of CXCL5 or CXCR2 expression markedly suppressed malignant phenotypes (cell proliferation, clonogenesis, apoptosis escape, migration, and invasion), attenuated STAT3 and AKT signaling, and reduced MMP2/9 secretion in PC cell lines. In conclusion, our findings revealed that serum CXCL5 level might serve as a potential diagnostic and prognostic cancer biomarker for penile cancer. Autocrine CXCL5/CXCR2 signaling might activate multiple downstream oncogenic signaling pathways (STAT3, AKT, MMP2/9) to promote malignant progression of PC, which may warrant further investigation in the future.     

Spontaneous rejection of intradermally transplanted non-engineered tumor cells by neutrophils and macrophages from syngeneic strains of mice

It is not surprising that tumors arising spontaneously are rarely rejected by T cells, because in general they lack molecules to elicit a primary T-cell response. In fact, cytokine-engineered tumors can induce granulocyte infiltration leading to tumor rejection. In the present study, we i.d. injected seven kinds of non-engineered tumor cells into syngeneic strains of mice. Three of them (i.e. B16, KLN205, and 3LL cells) continued to grow, whereas four of them (i.e. Meth A, I-10, CL-S1, and FM3A cells) were spontaneously rejected after transient growth or without growth. In contrast to the i.d. injection of B16 cells into C57BL/6 mice, which induces infiltration of TAMs into the tumors, the i.d. injection of Meth A cells into BALB/c mice induced the invasion of cytotoxic inflammatory cells, but not of TAMs, into or around the tumors leading to an IFN-γ-dependent rejection. On day 5, the cytotoxic activity against the tumor cells reached a peak; and the effector cells were found to be neutrophils and macrophages. The i.d. Meth A or I-10 cell-immunized, but not non-immunized, mice rejected i.p.- or i.m.-transplanted Meth A or I-10 cells without growth, respectively. The main effector cells were CTLs; and there was no cross-sensitization between these two kinds of tumor cells, suggesting specific rejection of tumor cells by CTLs from i.d. immunized mice. These results indicate that infiltration of cytotoxic myeloid cells (i.e. neutrophils and macrophages, but not TAMs) into or around tumors is essential for their IFN-γ-dependent spontaneous rejection.     

Expression of cyclo-oxygenase-2 in macrophages associated with cutaneous melanoma at different stages of progression

The biological significance of the almost constant presence of macrophages in the tumoral microenvironment is an issue debated by several authors. The major difficulty in understanding the role played by tumor-associated macrophages (TAMs) in tumor progression is due to the contrasting effects of TAMs found in different studies. In addition, there is a limited information on which of the many biological activities expressed by TAMs are critical in inducing stimulatory or inhibitory effect on tumor growth. The aim of our study was: (a) to explore to what extent cyclo-oxygenase-2 (COX-2) in TAMs associated with human melanoma is expressed at different stages of tumor progression; and (b) to explore whether COX-2 expression in TAMs is stimulated by melanoma cells. In order to answer this question, we determined COX-2 positive TAMs associated with cutaneous melanocytic nevi, in situ, invasive and metastatic melanoma. In addition, we investigated whether COX-2 is expressed in peritoneal thioglycollate-elicited macrophages after co-cultivation with murine B16 melanoma cells. We found that COX-2-positive TAMs, as revealed by immunohistochemical analysis, were rare in common nevi and "dysplastic nevi", but present in a high percentage in in situ and thin melanoma. COX-2-positive TAMs were also found in more advanced tumors and metastatic melanoma, although at a significantly lower percentage in these latter. The in vitro protocol revealed that COX-2 was expressed in peritoneal macrophages upon contact with B16 murine melanoma cells, but not with normal murine fibroblasts. On the whole, the results of in vivo and in vitro studies suggest that COX-2 expressed in TAMs appears to act as an effective biomarker of melanoma progression, and melanoma cells themselves might stimulate COX-2 in macrophages.     

The peripheral Ca pump activity of macrophages and neutrophils

Exposure of either alveolar macrophages or blood neutrophils to 0.2 – 1 μM ionophore A23187 in the presence of 0.1 – 1 mM CaCl₂ causes a rapid extracellular release of Ca²⁺, which can be measured by a Ca²⁺-selective electrode. The initial rate at which the cation is extruded from the cells is about 0.1 – 0.2 μg-ions/min/ml of cell water. ATP depletion, but not replacement of extracellular Na⁺ with choline, produces a marked inhibition of Ca²⁺ release from macrophages. When the movements of Ca²⁺ between neutrophils and the incubation medium are followed by an isotopic technique, a transient increase in cell-associated ⁴⁵Ca²⁺ is detected a few seconds after the addition of the ionophore. We suggest that the ionophore A23187 mobilises Ca²⁺ from intracellular stores, with a subsequent cell extrusion of the bivalent cation catalysed by a pump localised at the cell surface. These and other data are consistent with the conclusion that the peripheral Ca²⁺ pump system of macrophages and neutrophils is very similar to the well know Ca²⁺ pump of the red cells with regard to mechanism and capacity.     

Chemokines in tumor development and progression

Chemokines were originally identified as mediators of the inflammatory process and regulators of leukocyte trafficking. Subsequent studies revealed their essential roles in leukocyte physiology and pathology. Moreover, chemokines have profound effects on other types of cells associated with the inflammatory response, such as endothelial cells and fibroblasts. Thus, chemokines are crucial for cancer-related inflammation, which can promote tumor development and progression. Increasing evidence points to the vital effects of several chemokines on the proliferative and invasive properties of tumor cells. The wide range of activities of chemokines in tumorigenesis highlights their roles in tumor development and progression.     

Delineating the roles of neutrophils and macrophages in zebrafish regeneration models

The outcome following injury can be healing, scarring or regeneration, all of which initiate within a resolving inflammatory response. Regeneration, comprising the complete anatomical and functional restoration of lost tissue with minimal residual consequence of injury, is the outcome that most holistically restores prior function. Leukocytes are recognized as playing an important role in determining the balance between fully regenerative or only partially reparative outcomes. Although macrophages have attracted considerable attention for their capacity to direct pro-regenerative outcomes, neutrophils are also key players in initiating inflammation and in influencing its ensuing outcome. In the context of prior studies investigating the role of neutrophils and macrophages in wound healing and in tissue/organ regeneration (mostly wound repair/healing models in mice), we comprehensively review the experimental possibilities that zebrafish models offer for delineating the individual and interactive contributions of neutrophils and macrophages to the regenerative process in embryos and adults. Zebrafish are a highly regenerative vertebrate and have a myeloid system very analogous to that of less-regenerative mammalian models. There are well-characterized reporter lines for imaging and distinguishing neutrophil and macrophage behaviors in vivo, and tools enabling selective, independent manipulation of these two leukocyte lineages for functional studies. Zebrafish are an attractive model for delineating neutrophil and macrophage contributions not only to regeneration, but also to many other pathological processes.This article is part of a directed issue entitled: Regenerative Medicine: the challenge of translation.     

Tumor models for prostate cancer exemplified by fibroblast growth factor 8-induced tumorigenesis and tumor progression

Prostate cancer is a very common malignancy among Western males. Although most tumors are indolent and grow slowly, some grow and metastasize aggressively. Because prostate cancer growth is usually androgen-dependent, androgen ablation offers a therapeutic option to treat post-resection tumor recurrence or primarily metastasized prostate cancer. However, patients often relapse after the primary response to androgen ablation therapy, and there is no effective cure for cases of castration-resistant prostate cancer (CRPC). The mechanisms of tumor growth in CRPC are poorly understood. Although the androgen receptors (ARs) remain functional in CRPC, other mechanisms are clearly activated (e.g., disturbed growth factor signaling). Results from our laboratory and others have shown that dysregulation of fibroblast growth factor (FGF) signaling, including FGF receptor 1 (FGFR1) activation and FGF8b overexpression, has an important role in prostate cancer growth and progression. Several experimental models have been developed for prostate tumorigenesis and various stages of tumor progression. These models include genetically engineered mice and rats, as well as induced tumors and xenografts in immunodeficient mice. The latter was created using parental and genetically modified cell lines. All of these models greatly helped to elucidate the roles of different genes in prostate carcinogenesis and tumor progression. Recently, patient-derived xenografts have been studied for possible use in testing individual, specific responses of tumor tissue to different treatment options. Feasible and functional CRPC models for drug responsiveness analysis and the development of effective therapies targeting the FGF signaling pathway and other pathways in prostate cancer are being actively investigated.