Macrophages in the cancer microenvironment

The role of tumor-infiltrating macrophages (TIMs, TAM- tumor associated macrophages, TEM- tumor educated macrophages) in cancer development seems to be a dual one. On the one hand, these cells can stimulate tumor growth; on the other hand, they can control tumor rejection (23). A positive correlation has been found between the number of tumor-infiltrating macrophages and a poor prognosis for cases of various types of malignant tumors. Moreover, macrophages are present within the tumor stroma from the earliest stages of the tumor’s development, alongside hyperplasia and atypical cells appearance (24, 25); this confirms their role in the initiation of tumor development. The tumor and its stroma express chemo-attractive factors for macrophages while macrophages secrete growth and the pro-angiogenic factors (26) mentioned above, thus influencing the cancer microenvironment and modifying tumor growth (27). The suppression of macrophage infiltration in the tumor correlates with the inhibition of tumor growth in vivo (28) while the induction of cytokines activating macrophages promotes macrophage infiltration of the tumor and the promotion of tumor growth (29,30). Macrophages/monocytes that infiltrate the tumor and its stroma are stimulated by the stroma and acquire one of two phenotypes, M1 or M2 (21). The M1 phenotype is related to antigen-presenting cell activity in inflammation and the activity against infection, while phenotype M2 is associated with tissue remodeling and the pro-angiogenic activity of these cells. The M2 phenotype seems to be crucial for tumor development and is related to IL-12lowIL-10high and the production of TGF-β.

As a result of the interaction between the tumor and macrophages, macrophages release cytokines, chemokines, growth factors, and activity factors (such as GM-CSF, IL-8, and EGF), which in turn increase immune system cell infiltration to the area of the tumor stroma, aggravating the inflammatory response in the cancer microenvironment (1). Chemokines play an important role in coordinating the stromal response to tumor growth. They polarize the response of the immune system cells to the tumor, regulate the type of cellular infiltration, and initiate angiogenesis. The receptors for chemokines were identified on cancer cells and their ligands were demonstrated in primary malignant tumors and metastatic cells which together suggest the important role of chemokines in tumor growth and the development of metastases (1).

The participation of macrophages in the process of angiogenesis is also dual. On the one hand, macrophages produce pro-angiogenic factors; on the other hand, they secrete anti-angiogenic factors which inhibit angiogenesis and destroy the integrity of the blood vessels. The interaction between the tumor and macrophages induces the pro-angiogenic function in macrophages, and the accumulation of macrophages is associated with the secretion of VEGF and PDGF (1). The hypoxemic regions of the tumor induce the migration of macrophages and evoke the pro-angiogenic program in these cells. Among the factors undergoing expression in macrophages under the hypoxemia, the following were identified: VEGF, TNF-a, bFGF, CXCL8 (IL-8), and glycolytic enzymes, the transcription of which is controlled by HIF-1 and HIF-2 transcription factors (31-33). Macrophages infiltrating the cancer stroma along with directly acting factors secreted by tumor cells (e.g., CXCL12 acts as a chemokine for various endothelial cells typified by CXCR4, IL-8-CXCL8, VEGF, and bFGF) regulate the process of angiogenesis in another mode as well. Macrophages also control the lymphangiogenesis process in the stroma and lymphangiogenesis is regulated by VEGF-C and VEGF-D acting through the receptor VEGFR3. Recently, it has been shown that VEGF-A chemo-attracts monocytes and increases lymphangiogenesis through the induction of monocyte infiltration (31-35). In cervical cancer, the production of VEGF-C by macrophages plays an important role in lymphangiogenesis and cancer dissemination through the lymphatic route. Additionally, macrophages induce angiogenesis in the cancer stroma through the production of thymidine phosphorylase (TP), the pro-angiogenic factor stimulating the migration of endothelial cells in vitro, and its high expression has been correlated with tumor neovascularization (34,35).

Local tumor development depends on the controlled degradation of the extracellular matrix. It has been shown that macrophages participate in initiating the creation of metastases and an increased number typically indicates a poor prognosis. Furthermore, genetic studies on mice have demonstrated that a decreased number of macrophages correlates with a lower incidence of metastases (36). Macrophages are characterized by proteolytic activity that results in the degradation of the basal membrane in pre-invasive cancer (in situ) and enables the spread of local cancer cells to the microenvironment (37).

In sum, macrophages express factors that modulate cell proliferation, angiogenesis, and the degradation of connective tissue. These cells are also able to stimulate the creation of the new cancer microenvironment by secreting PDGF, acting together with TGF-β secreted by the tumor cells (37). Macrophages release the matrix metaloproteinases, MMP-2 and MMP-9 that degrade the extra-cellular matrix, as well as MMP activators, such as chemokines, and other factors that facilitate matrix degradation and cancer cell invasion and migration (TGF-β, PDGF, IL-6, and tissue type plasminogen activators u-PA and t-PA) (38-41). Macrophages also secrete factors that encourage cancer cells to home the tissue (e.g., EGF), while cancer cells release factors that chemoattract  macrophages (e.g., M-CSF or macrophage colony stimulating factor) (24-43).

  

B7-H4 positive macrophage

Antigen-presenting cells are important for initiating and maintaining the tumor-associated antigen-specific T-cell immunity. Tumor-infiltrating macrophages significantly counter the number of other antigen-presenting cells within the cancer microenvironment (25,37,44-46). In mice it was found that macrophages associated with the tumor were involved in promoting tumor growth and had direct metastatic action on cancer cells (25, 37, 44-46). B7-H4 (B7x and B7S1) has recently been described as a member of B7 molecules co-stimulating T lymphocytes, a negative regulator of T-cell response. This molecule inhibits T-cell proliferation, cell cycle progression, and the production of cytokines in vitro. Antigenic specific T-cell responses in mice are disturbed by B7-H4 protein; in humans, however, the expression, regulation, and function of B7-H4 protein remain unknown. Kryczek et al. have demonstrated the presence of B7-H4-positive macrophages in the cancer microenvironment in patients with ovarian cancer and have confirmed the suppressive activity of these cells, which is comparable to that of Treg cells. Moreover, B7-H4 macrophages interact with CD4+Treg cells, and CD4+Treg cells stimulated B7-H4 expression in macrophages, enabling the suppressive function of these cells. Through Il-6 and IL-10 cytokine secretion, the cancer microenvironment stimulated B7-H4 expression by macrophages. Additionally, Treg cells induce Il-10 production by APC which stimulates the suppressive activity of macrophages through B7-H4 (44, 47-48). In our study, B7-H4-positive macrophages were identified in almost all the patients with uterine cervical carcinoma. A significantly higher number of B7-H4-positive cancer cells were identified in the tumor front of those patients in whom lymph node metastases were present than those patients without such metastases. A significant increase in B7-H4-positive macrophage infiltration within the tumor microenvironment was observed in those patients who did have lymph node metastases (49).

RCAS1-positive macrophages

RCAS1 (receptor-binding cancer antigen expressed on SiSo cells) is a II type trans-membrane protein with a gene EBAG9 or estrogen receptor-binding fragment-associated antigen 9, located at 8q23gen (51). This protein was first described by Sonoda et al. in 1996 on cervical cancer cells. It has been demonstrated that RCAS1 is a ligand for the putative receptor expressed by lymphocytes T, B, and NK cell. It has been shown both in vitro and in vivo that the interaction of RCAS1 with the receptor inhibits the growth of receptor-expressing cells and induces their apoptosis through the activation of FADD and the caspase pathway (51-52). It was noted that RCAS1 is secreted to the supernatant in the cancer cell line as a soluble form of RCAS1 (sRCAS1) during ectodomain shedding (53-55). It was determined that sRCAS1 was able to inhibit the growth of receptor expressing cells and induce their apoptosis; it thus possessed the same biological function as a membrane form (53-55). The level of sRCAS1 increased in the sera of patients with cervical and head and neck cancers as the tumor progressed (53-56); in patients with head and neck cancer, it was observed that the sRCAS1 level decreased in blood sera following surgical treatment and increased again in those patients with a recurrence of the disease (56). RCAS1 protein is therefore responsible for tumor escape from host immunological surveillance and participates in the creation of immune tolerance for the tumor cells and maternal immune tolerance for fetal antigens during pregnancy (51-64). RCAS1 expression has been found in various malignant neoplasms, and it is significantly higher in patients with advanced tumors, high tumor grade, and the presence of lymph node metastases, and in cases with a poor prognosis (51-64). It has furthermore been suggested that in patients with cervical cancer RCAS1 participates in cancer microenvironment remodeling (53-55).

RCAS1-positive macrophages were first described in cervical cancer tissue  (51); later, these cells were also detected in the bone marrow where they play an important regulative role in erythropoiesis through the expression of RCAS1 (65-67). It has been shown that RCAS1 is expressed by activated monocytes or stimulated by lipopolysaccharide (65). RCAS1-positive macrophages have been observed in various types of hepatitis, including acute viral hepatitis, chronic viral hepatitis, primary cirrhosis, and immune-mediated hepatitis. RCAS1-positive macrophages have been identified in patients with very high ALT (alanine aminotransferase) and surrounding massive hepatocellular necrosis. The observed RCAS1-positive macrophages indicated that these cells might represent the M2 phenotype of macrophages and might play a regulatory role in chronic inflammation. Chronic inflammation may induce the expression of RCAS1 on macrophages so that the M2 phenotype of a suppressive activity is induced in turn (65-67). The interaction of RCAS1 protein with the tumor and its microenvironment is presented in Figure 1. In our previous

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studies, RCAS1-expression and RCAS1-positive macrophages have been observed in various types of malignant neoplasms and their microenvironments as well as in conditions of chronic inflammation, such as chronic tonsillitis and nasal polyps (68-75)). The expression of RCAS1 was identified in both squamous cell carcinoma and adenocarcinoma cells from the tissues of patients with head and neck, cervical, and ovarian cancer; it was also found in hydatidiform mole tissue and in the cancer microenvironments of these patients. The tissue remodeling of the cancer microenvironment was marked by the vimentin and MT expression, and the suppressive cancer microenvironment was confirmed by a decreased number of TIL with lower immunoreactivity of such antigens as CD56 and CD57; moreover, this cancer microenvironment was infiltrated by RCAS1-expressing macrophages (68-73). In our studies on head and neck cancer in both histological types of tumors (squamous carcinoma and adenocarcinoma), the number of RCAS1-positive macrophages infiltrating the cancer microenvironment was significantly higher in patients with the presence of lymph node metastases than in patients without such metastases (68-73). Moreover, we detected tumor-infiltrating macrophages and RCAS1-positive macrophages infiltrating the tumor and stroma of malignant B-cell lymphomas originating from palatine tonsils (Figure 2, Figure 3). In patients with hydatiform mole, RCAS1 immunoreactivity was identified in both trophoblast and decidual cells as well as in the stroma. Significantly lower RCAS1 levels were found in those patients who were treated by surgery alone than in the patients who also required chemotherapy. Since strongly RCAS1-positive macrophages were found dispersed in the stroma, we concluded that RCAS1 staining might provide information about the intensity of the immune suppressive microenvironment in the molar lesion and endometrium (72) and could serve as a marker of the need for more aggressive treatment. In ovarian cancer, the cancer microenvironment was characterized by the presence of the excessive infiltration of RCAS1-positive macrophages. A statistically significantly higher number of RCAS1-positive macrophages were identified in patients with the presence of lymph node metastases than in patients without such metastases. Moreover, the cytoplasmic RCAS1 expression and the number of RCAS1-positive macrophages was higher in the border part of the tumor (which was defined as a younger part of the tumor with signs of dynamic growth) derived from the patients with the lymph node metastases in comparison to those patients without such metastases (71). Furthermore, RCAS1-positive macrophages were identified in the cancer microenvironment of all the patients in the study with uterine cervical carcinoma. No correlation, however, was seen between the presence of these cells and the particular stage of the disease. A correlation might be observed if patients with operable cervical cancer and with less advanced stages of the disease (I and II) were to be included in the study (49-50).

In patients who had had their palatine tonsils removed due to chronic tonsillitis, RCAS1 immunoreactivity was detected in the crypts epithelium, a very specialized tissue responsible for immune interactions with foreign antigens (Figure 4). Single epithelial exfoliated cells and macrophages positive for RCAS1 were also observed in the crypts lumen. Nasal polyps are a symptom of chronic rhinitis and sinusitis, and the presence of RCAS1-positive macrophages has been noted within the stroma of such polyps (69-70, 74-75). These observations would seem to confirm a very important immunoregulatory role for RCAS1-positive macrophages in various types of clinical situations. It seems that RCAS1-positive macrophages belong to the main regulatory mechanisms of the immune system. 

We would therefore propose that RCAS1-positive macrophages represent the M2 macrophage phenotype, participate in microenvironment remodeling, enable the local and distant spread of the tumor, and negatively regulate the anti-tumor immune response, and so are responsible for the creation of the suppressive cancer microenvironment in these tumors.

In sum, tumor-associated macrophages, expressing RCAS1, B7-H4 molecules, and inhibiting the anti-tumor immune response, are all responsible for the development of the suppressive phenotype of the cancer microenvironment and are also related to poor prognosis.