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ARTICLE IN PRESS Immunology Letters xxx (2014) xxx–xxx
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Macrophages in multiple myeloma Domenico Ribatti a,b,∗ , Michele Moschetta c , Angelo Vacca c,∗∗ a
Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy National Cancer Institute “Giovanni Paolo II”, Bari, Italy c Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy b
a r t i c l e
i n f o
Article history: Received 11 November 2013 Accepted 13 December 2013 Available online xxx Keywords: Angiogenesis Anti-angiogenesis Inflammation Macrophages Multiple myeloma
a b s t r a c t Tumor associated macrophages (TAMs) are a rich source of pro-angiogenic cytokines and growth factors, and a relationship between the TAMs content, the rate of tumor growth and the extent of vascularization has been shown in several tumors. In this article, we have summarized the literature and our data concerning the involvement of TAMs in angiogenesis occurring in multiple myeloma. Finally, therapeutic aspects concerning the potential role of molecules which inhibit macrophage recruitment in the tumor side are also discussed. © 2013 Elsevier B.V. All rights reserved.
1. Tumor-associated macrophages Firstly in 1863, Rudolf Virchow described the infiltration of leukocytes into malignant tissues and suggested a link between cancer and chronic inflammation [1]. Macrophages were initially described by Elie Metchnikoff who won the Nobel Prize in 1905 for their identification and the phagocytosis theory. Tumor associated macrophages (TAMs) derive from circulating monocytes and are recruited at the tumor site by several chemo attractants, including colony stimulating factor-1 (CSF-1), the CC chemokines (CCL-2, CCL-3, CCL-4, CCL-5 and CCL-8), and vascular endothelial cell growth factor (VEGF) secreted by both tumor and stromal elements [2]. Overexpression of CSF-1 enhances the TAMs recruitment and accelerates tumor progression in the mammary epithelium of MMTV-PyMT mice [3]. After inhibition of the expression of CSF-1 or its receptor with short interfering RNA (siRNA) in a mice model, macrophages infiltration and vascularity are decreased compared to controls [4]. Macrophages co-cultured with tumor cells secrete several factors which facilitate tumor cell proliferation [5] and extensive TAMs infiltration correlates with poor prognosis in breast, prostate, cervix, and bladder cancer [6].
∗ Corresponding author at: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Policlinico, Piazza G. Cesare, 11, 70124 Bari, Italy. Tel.: +39 080 5478326; fax: +39 080 5478310. ∗∗ Corresponding author at: Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, Policlinico, Piazza Giulio Cesare, 11, I-70124 Bari, Italy. Tel.: +39 080 5478895; fax: +39 080 559 21 89. E-mail addresses: [email protected] (D. Ribatti), [email protected] (A. Vacca).
The term “macrophage activation” is used to describe the process of recruitment of these cells, which exhibit many differences from resident tissue macrophages. Activated macrophages are categorized into two types: M1 (classically activated) and M2 (alternatively activated) [2]. M1 macrophages are induced by interferon gamma (IFN-␥), microbial stimuli, cytokines [tumor necrosis factor alpha (TNF-␣) and interleukin-12 (IL-12)], reactive nitrogen and oxygen intermediates (RNI, ROI) [7]. M1 secrete high levels of pro-inflammatory cytokines, including TNF-␣, IL-1, IL-6, IL-12 and IL-23, and exhibit high levels of superoxide anions, oxygen and nitrogen radicals [7]. The M2 form of macrophage activation is induced by IL-4 and IL-13 [8]. The molecular mechanisms that promote M1 or M2 subsets within the tumor microenvironment are not completely understood. TAMs are mainly constituted by the M2 elements, which promote angiogenesis, repairing and remodeling and suppressing adaptive immunity [9]. In regressing tumors, TAMs resemble the M1 type, which exhibits anti-tumor activity, while in advanced tumors, TAMs are oriented toward the M2 type that favors tumor growth and progression [10]. In the tumor mass, TAMs can express pro-tumoral or anti-tumoral activities in function of their activation state. 2. Macrophages and tumor angiogenesis TAMs are a rich source of pro-angiogenic cytokines and growth factors, such as VEGF, TNF-␣, IL-8 and fibroblast growth factor-2 (FGF-2). In addition, TAMs express a broad array of angiogenesismodulating enzymes, including matrix metalloproteinases (MMP)2, -7, -9, -12, and cycloxygenase-2 (COX-2) [11].
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TAMs accumulate in hypoxia regions of tumors and their adaption to this condition is achieved by the increased expression of hypoxia-inducible and pro-angiogenic genes, including VEGF, FGF2 and CXCL-8, as well as glycolytic enzymes [12]. A relationship between the TAMs counts, the rate of tumor growth and the density of vascularization has been demonstrated in several tumors, including breast carcinoma malignant uveal melanoma, glioma, squamous-cell carcinoma of the esophagus, bladder carcinoma and prostate carcinoma [13]. 3. Angiogenesis in multiple myeloma Angiogenesis is a constant hallmark of multiple myeloma (MM) progression and has prognostic potential [14]. It is induced by plasma cells via angiogenic factors with the transition from monoclonal gammopathies of undetermined significance (MGUS) to MM, and probably with loss of angiostatic activity on the part of MGUS. The pathophysiology of MM-associated angiogenesis is complex and involves both direct production of angiogenic cytokines by plasma cells and their induction within the microenvironment cells. Bone marrow stroma cells (BMSCs) increase the concentration of angiogenic factors and matrix degrading enzymes in the bone marrow microenvironment by direct secretion or following stimulation by myeloma cells or by endothelial cells through paracrine interactions [15,16]. BMSCs, osteoclasts, osteoblasts and endothelial cells secrete several factors, including VEGF, FGF-2, TNF-␣, IL-6, B-cell activating factor, stromal cell-derived factor 1␣ (SDF1-␣, also known as CXCL-12), and various Notch family members, which are further up-regulated by tumor cell adhesion to extracellular matrix proteins and/or BMSCs. Finally, circulating endothelial cells and endothelial precursor cells (EPCs) contribute to the neovascularization, and the presence of EPCs suggests that vasculogenesis may also contribute to the full MM vascular tree [14]. 4. Multiple myeloma-associated macrophages Multiple myeloma is characterized by the accumulation of monoclonal plasma cells in the bone marrow, suggesting the importance of the bone marrow microenvironment in supporting the MM cell growth and survival. The MM microenvironment is formed by clonal plasma cells, extracellular matrix proteins, and BMSCs, and reciprocal positive and negative interactions between plasma cells and BMSCs are mediated by any array of cytokines, receptors and adhesion molecules. Interactions between these cell components
determine the proliferation, migration and survival of plasma cells as well as their acquisition of drug resistance and the development of a progressive behavior [15]. In MM, macrophages are an important component of the stromal cells, and in active MM (at diagnosis, at relapse, on refractory phase), plasma cells secrete VEGF and FGF-2 that induce inflammatory cells to secrete their own VEGF, FGF-2, and hepatocyte growth factor (HGF), able to recruit and activate the MM-associated macrophages; these pathways are irrelevant in nonactive MM (at complete/partial remission) [17]. Moreover, bone marrow macrophages protect MM cells from spontaneous and melphalaninduced apoptosis [18]. Chen et al. [19] demonstrated that monocytes induce vascular endothelial cell gene expression and develop tube-like structures when they are cultured with the bone marrow from patients with MM that express an angiogenic factor, namely pleiotrophin; the tubulogenesis was specifically blocked by anti-pleiotrophin antibodies. Moreover, when co-injected with human MM cells into SCID mice, green fluorescent protein-marked human monocytes were found to be incorporated into tumor blood vessels and expressed human vascular endothelial cell protein markers and genes that were blocked by anti-pleiotrophin antibodies. In patients with active MM, fluorescence activated cell sorting (FACS) analysis on isolated bone marrow mononuclear cells revealed higher percentages of CD68+ macrophages than in patients with nonactive disease or those with MGUS [20]. Bone marrow macrophages in patients with active MM were functionally, phenotypically, and morphologically different from those of patients with nonactive disease and MGUS. Indeed, macrophages of these patients were similar to paired endothelial cells and contributed to angiogenesis through a vasculogenic mimicry, in parallel with progression of plasma cell tumors [20]. When macrophages from active MM patients were exposed in vitro to VEGF and FGF-2, they transformed into cells functionally and phenotypically similar to MM endothelial cells because they were able to generate capillary-like networks [20]. Moreover, bone marrow biopsies of active MM patients examined on confocal laser microscopy displayed macrophages with both endothelial cell-like (i.e., CD68/FVIII-RA double positive) and apparently typical (i.e., CD68 positive/FVIII-RA negative) features located in the microvessel wall. Figures of this type were rare in nonactive MM patients and absent in MGUS (Fig. 1) [20]. The MM macrophages give increased levels of VEGF-A and VEGF-C mRNAs [21]. It has been assumed that mesenchymal stromal cells (MSCs) are the major source of the VEGF isoforms [22],
Fig. 1. On the left, microvessel lined by flattened FVIII-RA positive multiple myeloma endothelial cells (arrow), an FVIII-RA positive macrophage (arrowhead) showing protrusions connected to multiple myeloma endothelial cells, and another macrophage containing double-labeled CD68 (red arrowhead) and FVIII-RA (green arrowhead) granules in the cytoplasm and connected to multiple myeloma endothelial cells by an FVIII-RA positive cytoplasmic protrusion (double arrow). Erythrocytes (orange) are well recognizable inside the lumen. Another microvessel formed by FVIII-RA positive (green) multiple myeloma endothelial cells and CD68 positive (red, arrowheads) tracts that belong to the cytoplasmic protrusions (double arrow) of macrophages, some of which are arrowed (middle panel). On the right, the MGUS microvessels are formed by only FVIII-RA positive endothelial cells: macrophages (arrows) are randomly scattered in the tissue and independent of them. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Source: Reproduced from Ref. [20].
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but the above results also suggest that macrophages might be another major contributors of VEGFs. VEGF receptor-1 (VEGFR-1) mediates macrophage chemotaxis [23], and exposure of active MM macrophages to VEGF plus FGF-2 leads to enhanced expression of Tie2/Tek and VEGFR-2, and a slight decrease in FGF receptor-2 (FGFR-2). The intense expression of VEGFR-2 and Tie2/Tek, together with the decreased expression of VE-cadherin, is indicative of ongoing neovascularization [20]. Moreover, MM macrophages synthesize and release inducible nitric oxide synthase, which increases blood flow and promotes angiogenesis [24]. The MM macrophages are characterized by high expression of factors that stimulate plasma cell proliferation and survival, including IL-6 and IL-10, and lower expression of IL-12 and TNF-␣. IL-6 stimulates the proliferation of MM cells isolated from patients in IL-6 deprived cultures [22]. Additionally, both IL-12 and TNF-␣ are considered to retain anti-tumor effects [23]; hence, a lower expression of these cytokines by macrophages could favor the growth of malignant plasma cells. Human bone marrow-derived MSCs change the secretory profile of macrophages to a phenotype characterized by higher expression of IL-6 and IL-10, and lower expression of IL-12 and TNF-␣ [24]. Kim et al. [21] have investigated the effect of interactions between MSCs and macrophages on the growth of MM tumor cell lines in the presence of either MSCs or macrophages compared to cell line alone, and demonstrated that co-culture of MSCs and macrophages induced the greatest degree of proliferation and the lowest rate of apoptosis of MM cells. IL-10 stimulates the proliferation of MM cells [22] and both IL-12 and TNF-␣ retain anti-tumor effects [23,25]. Airoldi et al. [26] demonstrated both in vitro and in vivo the ability of IL-12 to modulate the expression of chemokines/cytokines involved in MM angiogenesis. Supernatants from primary MM cells exposed to IL-12 showed a significant reduction of their angiogenic potential in comparison with untreated cells in the chorioallantoic membrane (CAM) assay [26]. In addition, mice injected with MM cell lines and treated with IL-12 developed tumors significantly smaller than those treated with PBS [26]. Molecular analysis demonstrated that angiogenic activators including Ang-2, FGF-2, VEGF, IL-1 and IL-6 was down-regulated by IL-12; on the contrary, inhibitors of angiogenesis, including CXCL-4, IFN-␣, IFN-␥, and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were up-regulated by IL-12 [26]. TAMs promote tumor growth not only by supporting angiogenesis but also by inducing immunosuppression [27]. In MM, myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells that lack cell surface markers of monocytes, macrophages, or dendritic cells and potently suppress T cells, are involved in immunosuppression associated to MM [28]. Indeed, MDSCs are significantly increased in patients with MM compared to patients with MGUS and healthy controls [28].
5. The role of erythropoietin in the angiogenic activity of macrophages in multiple myeloma
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NFkB pathways, important in activation of cytokine secretion and phagocytosis. More recently, we have demonstrated that erythropoietin receptor (EpoR) is overexpressed in bone marrow macrophages of MM patients with active disease as compared to MGUS patients [33]. The treatment of bone marrow macrophages with human Epo (huEpo) significantly increased the expression and secretion of angiogenic factors including VEGF, HGF, MCP-1/CCL-2, through activation of JAK2/STAT5 and PI3K/Akt pathways. Moreover, conditioned media from huEpo-treated macrophages enhanced bone marrow-derived endothelial cell migration and capillary morphogenesis in vitro, and induced angiogenesis in the in vivo CAM assay. Finally, we found an increase in the circulating levels of several proangiogenic cytokines in serum of MM patients with anemia under treatment with huEpo [33]. 6. Therapeutic aspects Macrophages has become an appealing target for antiangiogenic therapies through two approaches: compounds that suppress secretion of angiogenic molecules by macrophages or compounds that inhibit macrophage infiltration into the tumor mass. Although chemotherapy is now the most effective treatment for MM, plasma cells often fail to respond to the drugs. Coculture of MM plasma cells with macrophages protected these cells from melphalan-induced apoptosis by inhibiting the activation and cleavage of caspase-3 and poly(ADP-ribose) polymerization (PARP) and maintaining the levels of Bcl-XL. These results suggest that macrophages protect MM cells from apoptosis via inhibiting BclXL-dependent caspase activation [18]. Bortezomib and zoledronic acid synergistically impact MM macrophage proliferation, adhesion, and migration, as well as VEGF, FGF-2, HGF, and platelet derived growth factor (PDGF) secretion [34]. These drugs synergistically inhibit macrophage vasculogenesis on the matrigel surface and the expression of FVIII-RA, Tie2/Tek, and VEGFR-2/VE-cadherin, indicative of cell transdifferentiation into endothelial-like cells. Both drugs reduce phospho-activation of VEGFR-2 and ERK1/2 and NFKB activity. These data provide evidence that the exposure of bone marrow macrophages during the treatment with bortezomib and zoledronic acid impacts their angiogenic and vasculogenic properties, suggesting that these cells may be considered as a target of both drugs in MM patients. Kim et al. [21] demonstrated that when macrophages pre-treated with bortezomib were co-cultured with MM cell lines, the proliferative index of these latter decreased significantly. Addition of defibrotide, an agent that could modulate macrophage-endothelial cell interactions to other chemotherapeutics resulted in encouraging results both in pre-clinical models [35] and in a multicentre phase I/II clinical trial [36]. Acknowledgments
Transgenic TG6 mice over-expressing human erythropoietin (Epo) have increased populations of liver and duodenal mucosa macrophages [29,30]; expression of Epo receptor (EpoR) was shown in wound healing macrophages and Epo enhanced the activity of these cells [31]. Lifshitz et al. [32] demonstrated that macrophages isolated form bone marrow expressed in vitro EpoR transcripts. In vitro treatment of these cells enhanced their phagocytic activity as well as IL-12 and nitric oxide secretion, while decreased IL-10 secretion in response to lipopolysaccharide. Following Epo treatment the bone marrow macrophages also exhibited activation of PI3k, MAPK, and
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The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/20072013) under grant agreement no. 278570 to DR and no. 278706 to AV. References [1] Virchow R. Die Krankenaften Geschwulste. Berlin: August Hirschwald; 1863. p. 1–255. [2] Mantovani A, Sozzani S, Locati M, Allavena P, Sica A. Macrophage polarization: tumor associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 2002;23:549–55.
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Macrophages
in
multiple
myeloma.
Immunol
Lett
(2014),
ARTICLE IN PRESS Immunology Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Macrophages in multiple myeloma Domenico Ribatti a,b,∗ , Michele Moschetta c , Angelo Vacca c,∗∗ a
Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Bari, Italy National Cancer Institute “Giovanni Paolo II”, Bari, Italy c Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, University of Bari Medical School, Bari, Italy b
a r t i c l e
i n f o
Article history: Received 11 November 2013 Accepted 13 December 2013 Available online xxx Keywords: Angiogenesis Anti-angiogenesis Inflammation Macrophages Multiple myeloma
a b s t r a c t Tumor associated macrophages (TAMs) are a rich source of pro-angiogenic cytokines and growth factors, and a relationship between the TAMs content, the rate of tumor growth and the extent of vascularization has been shown in several tumors. In this article, we have summarized the literature and our data concerning the involvement of TAMs in angiogenesis occurring in multiple myeloma. Finally, therapeutic aspects concerning the potential role of molecules which inhibit macrophage recruitment in the tumor side are also discussed. © 2013 Elsevier B.V. All rights reserved.
1. Tumor-associated macrophages Firstly in 1863, Rudolf Virchow described the infiltration of leukocytes into malignant tissues and suggested a link between cancer and chronic inflammation [1]. Macrophages were initially described by Elie Metchnikoff who won the Nobel Prize in 1905 for their identification and the phagocytosis theory. Tumor associated macrophages (TAMs) derive from circulating monocytes and are recruited at the tumor site by several chemo attractants, including colony stimulating factor-1 (CSF-1), the CC chemokines (CCL-2, CCL-3, CCL-4, CCL-5 and CCL-8), and vascular endothelial cell growth factor (VEGF) secreted by both tumor and stromal elements [2]. Overexpression of CSF-1 enhances the TAMs recruitment and accelerates tumor progression in the mammary epithelium of MMTV-PyMT mice [3]. After inhibition of the expression of CSF-1 or its receptor with short interfering RNA (siRNA) in a mice model, macrophages infiltration and vascularity are decreased compared to controls [4]. Macrophages co-cultured with tumor cells secrete several factors which facilitate tumor cell proliferation [5] and extensive TAMs infiltration correlates with poor prognosis in breast, prostate, cervix, and bladder cancer [6].
∗ Corresponding author at: Department of Basic Medical Sciences, Neurosciences and Sensory Organs, University of Bari Medical School, Policlinico, Piazza G. Cesare, 11, 70124 Bari, Italy. Tel.: +39 080 5478326; fax: +39 080 5478310. ∗∗ Corresponding author at: Department of Biomedical Sciences and Human Oncology, Section of Internal Medicine and Clinical Oncology, Policlinico, Piazza Giulio Cesare, 11, I-70124 Bari, Italy. Tel.: +39 080 5478895; fax: +39 080 559 21 89. E-mail addresses: [email protected] (D. Ribatti), [email protected] (A. Vacca).
The term “macrophage activation” is used to describe the process of recruitment of these cells, which exhibit many differences from resident tissue macrophages. Activated macrophages are categorized into two types: M1 (classically activated) and M2 (alternatively activated) [2]. M1 macrophages are induced by interferon gamma (IFN-␥), microbial stimuli, cytokines [tumor necrosis factor alpha (TNF-␣) and interleukin-12 (IL-12)], reactive nitrogen and oxygen intermediates (RNI, ROI) [7]. M1 secrete high levels of pro-inflammatory cytokines, including TNF-␣, IL-1, IL-6, IL-12 and IL-23, and exhibit high levels of superoxide anions, oxygen and nitrogen radicals [7]. The M2 form of macrophage activation is induced by IL-4 and IL-13 [8]. The molecular mechanisms that promote M1 or M2 subsets within the tumor microenvironment are not completely understood. TAMs are mainly constituted by the M2 elements, which promote angiogenesis, repairing and remodeling and suppressing adaptive immunity [9]. In regressing tumors, TAMs resemble the M1 type, which exhibits anti-tumor activity, while in advanced tumors, TAMs are oriented toward the M2 type that favors tumor growth and progression [10]. In the tumor mass, TAMs can express pro-tumoral or anti-tumoral activities in function of their activation state. 2. Macrophages and tumor angiogenesis TAMs are a rich source of pro-angiogenic cytokines and growth factors, such as VEGF, TNF-␣, IL-8 and fibroblast growth factor-2 (FGF-2). In addition, TAMs express a broad array of angiogenesismodulating enzymes, including matrix metalloproteinases (MMP)2, -7, -9, -12, and cycloxygenase-2 (COX-2) [11].
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TAMs accumulate in hypoxia regions of tumors and their adaption to this condition is achieved by the increased expression of hypoxia-inducible and pro-angiogenic genes, including VEGF, FGF2 and CXCL-8, as well as glycolytic enzymes [12]. A relationship between the TAMs counts, the rate of tumor growth and the density of vascularization has been demonstrated in several tumors, including breast carcinoma malignant uveal melanoma, glioma, squamous-cell carcinoma of the esophagus, bladder carcinoma and prostate carcinoma [13]. 3. Angiogenesis in multiple myeloma Angiogenesis is a constant hallmark of multiple myeloma (MM) progression and has prognostic potential [14]. It is induced by plasma cells via angiogenic factors with the transition from monoclonal gammopathies of undetermined significance (MGUS) to MM, and probably with loss of angiostatic activity on the part of MGUS. The pathophysiology of MM-associated angiogenesis is complex and involves both direct production of angiogenic cytokines by plasma cells and their induction within the microenvironment cells. Bone marrow stroma cells (BMSCs) increase the concentration of angiogenic factors and matrix degrading enzymes in the bone marrow microenvironment by direct secretion or following stimulation by myeloma cells or by endothelial cells through paracrine interactions [15,16]. BMSCs, osteoclasts, osteoblasts and endothelial cells secrete several factors, including VEGF, FGF-2, TNF-␣, IL-6, B-cell activating factor, stromal cell-derived factor 1␣ (SDF1-␣, also known as CXCL-12), and various Notch family members, which are further up-regulated by tumor cell adhesion to extracellular matrix proteins and/or BMSCs. Finally, circulating endothelial cells and endothelial precursor cells (EPCs) contribute to the neovascularization, and the presence of EPCs suggests that vasculogenesis may also contribute to the full MM vascular tree [14]. 4. Multiple myeloma-associated macrophages Multiple myeloma is characterized by the accumulation of monoclonal plasma cells in the bone marrow, suggesting the importance of the bone marrow microenvironment in supporting the MM cell growth and survival. The MM microenvironment is formed by clonal plasma cells, extracellular matrix proteins, and BMSCs, and reciprocal positive and negative interactions between plasma cells and BMSCs are mediated by any array of cytokines, receptors and adhesion molecules. Interactions between these cell components
determine the proliferation, migration and survival of plasma cells as well as their acquisition of drug resistance and the development of a progressive behavior [15]. In MM, macrophages are an important component of the stromal cells, and in active MM (at diagnosis, at relapse, on refractory phase), plasma cells secrete VEGF and FGF-2 that induce inflammatory cells to secrete their own VEGF, FGF-2, and hepatocyte growth factor (HGF), able to recruit and activate the MM-associated macrophages; these pathways are irrelevant in nonactive MM (at complete/partial remission) [17]. Moreover, bone marrow macrophages protect MM cells from spontaneous and melphalaninduced apoptosis [18]. Chen et al. [19] demonstrated that monocytes induce vascular endothelial cell gene expression and develop tube-like structures when they are cultured with the bone marrow from patients with MM that express an angiogenic factor, namely pleiotrophin; the tubulogenesis was specifically blocked by anti-pleiotrophin antibodies. Moreover, when co-injected with human MM cells into SCID mice, green fluorescent protein-marked human monocytes were found to be incorporated into tumor blood vessels and expressed human vascular endothelial cell protein markers and genes that were blocked by anti-pleiotrophin antibodies. In patients with active MM, fluorescence activated cell sorting (FACS) analysis on isolated bone marrow mononuclear cells revealed higher percentages of CD68+ macrophages than in patients with nonactive disease or those with MGUS [20]. Bone marrow macrophages in patients with active MM were functionally, phenotypically, and morphologically different from those of patients with nonactive disease and MGUS. Indeed, macrophages of these patients were similar to paired endothelial cells and contributed to angiogenesis through a vasculogenic mimicry, in parallel with progression of plasma cell tumors [20]. When macrophages from active MM patients were exposed in vitro to VEGF and FGF-2, they transformed into cells functionally and phenotypically similar to MM endothelial cells because they were able to generate capillary-like networks [20]. Moreover, bone marrow biopsies of active MM patients examined on confocal laser microscopy displayed macrophages with both endothelial cell-like (i.e., CD68/FVIII-RA double positive) and apparently typical (i.e., CD68 positive/FVIII-RA negative) features located in the microvessel wall. Figures of this type were rare in nonactive MM patients and absent in MGUS (Fig. 1) [20]. The MM macrophages give increased levels of VEGF-A and VEGF-C mRNAs [21]. It has been assumed that mesenchymal stromal cells (MSCs) are the major source of the VEGF isoforms [22],
Fig. 1. On the left, microvessel lined by flattened FVIII-RA positive multiple myeloma endothelial cells (arrow), an FVIII-RA positive macrophage (arrowhead) showing protrusions connected to multiple myeloma endothelial cells, and another macrophage containing double-labeled CD68 (red arrowhead) and FVIII-RA (green arrowhead) granules in the cytoplasm and connected to multiple myeloma endothelial cells by an FVIII-RA positive cytoplasmic protrusion (double arrow). Erythrocytes (orange) are well recognizable inside the lumen. Another microvessel formed by FVIII-RA positive (green) multiple myeloma endothelial cells and CD68 positive (red, arrowheads) tracts that belong to the cytoplasmic protrusions (double arrow) of macrophages, some of which are arrowed (middle panel). On the right, the MGUS microvessels are formed by only FVIII-RA positive endothelial cells: macrophages (arrows) are randomly scattered in the tissue and independent of them. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Source: Reproduced from Ref. [20].
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but the above results also suggest that macrophages might be another major contributors of VEGFs. VEGF receptor-1 (VEGFR-1) mediates macrophage chemotaxis [23], and exposure of active MM macrophages to VEGF plus FGF-2 leads to enhanced expression of Tie2/Tek and VEGFR-2, and a slight decrease in FGF receptor-2 (FGFR-2). The intense expression of VEGFR-2 and Tie2/Tek, together with the decreased expression of VE-cadherin, is indicative of ongoing neovascularization [20]. Moreover, MM macrophages synthesize and release inducible nitric oxide synthase, which increases blood flow and promotes angiogenesis [24]. The MM macrophages are characterized by high expression of factors that stimulate plasma cell proliferation and survival, including IL-6 and IL-10, and lower expression of IL-12 and TNF-␣. IL-6 stimulates the proliferation of MM cells isolated from patients in IL-6 deprived cultures [22]. Additionally, both IL-12 and TNF-␣ are considered to retain anti-tumor effects [23]; hence, a lower expression of these cytokines by macrophages could favor the growth of malignant plasma cells. Human bone marrow-derived MSCs change the secretory profile of macrophages to a phenotype characterized by higher expression of IL-6 and IL-10, and lower expression of IL-12 and TNF-␣ [24]. Kim et al. [21] have investigated the effect of interactions between MSCs and macrophages on the growth of MM tumor cell lines in the presence of either MSCs or macrophages compared to cell line alone, and demonstrated that co-culture of MSCs and macrophages induced the greatest degree of proliferation and the lowest rate of apoptosis of MM cells. IL-10 stimulates the proliferation of MM cells [22] and both IL-12 and TNF-␣ retain anti-tumor effects [23,25]. Airoldi et al. [26] demonstrated both in vitro and in vivo the ability of IL-12 to modulate the expression of chemokines/cytokines involved in MM angiogenesis. Supernatants from primary MM cells exposed to IL-12 showed a significant reduction of their angiogenic potential in comparison with untreated cells in the chorioallantoic membrane (CAM) assay [26]. In addition, mice injected with MM cell lines and treated with IL-12 developed tumors significantly smaller than those treated with PBS [26]. Molecular analysis demonstrated that angiogenic activators including Ang-2, FGF-2, VEGF, IL-1 and IL-6 was down-regulated by IL-12; on the contrary, inhibitors of angiogenesis, including CXCL-4, IFN-␣, IFN-␥, and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) were up-regulated by IL-12 [26]. TAMs promote tumor growth not only by supporting angiogenesis but also by inducing immunosuppression [27]. In MM, myeloid-derived suppressor cells (MDSCs), a heterogeneous population of immature myeloid cells that lack cell surface markers of monocytes, macrophages, or dendritic cells and potently suppress T cells, are involved in immunosuppression associated to MM [28]. Indeed, MDSCs are significantly increased in patients with MM compared to patients with MGUS and healthy controls [28].
5. The role of erythropoietin in the angiogenic activity of macrophages in multiple myeloma
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NFkB pathways, important in activation of cytokine secretion and phagocytosis. More recently, we have demonstrated that erythropoietin receptor (EpoR) is overexpressed in bone marrow macrophages of MM patients with active disease as compared to MGUS patients [33]. The treatment of bone marrow macrophages with human Epo (huEpo) significantly increased the expression and secretion of angiogenic factors including VEGF, HGF, MCP-1/CCL-2, through activation of JAK2/STAT5 and PI3K/Akt pathways. Moreover, conditioned media from huEpo-treated macrophages enhanced bone marrow-derived endothelial cell migration and capillary morphogenesis in vitro, and induced angiogenesis in the in vivo CAM assay. Finally, we found an increase in the circulating levels of several proangiogenic cytokines in serum of MM patients with anemia under treatment with huEpo [33]. 6. Therapeutic aspects Macrophages has become an appealing target for antiangiogenic therapies through two approaches: compounds that suppress secretion of angiogenic molecules by macrophages or compounds that inhibit macrophage infiltration into the tumor mass. Although chemotherapy is now the most effective treatment for MM, plasma cells often fail to respond to the drugs. Coculture of MM plasma cells with macrophages protected these cells from melphalan-induced apoptosis by inhibiting the activation and cleavage of caspase-3 and poly(ADP-ribose) polymerization (PARP) and maintaining the levels of Bcl-XL. These results suggest that macrophages protect MM cells from apoptosis via inhibiting BclXL-dependent caspase activation [18]. Bortezomib and zoledronic acid synergistically impact MM macrophage proliferation, adhesion, and migration, as well as VEGF, FGF-2, HGF, and platelet derived growth factor (PDGF) secretion [34]. These drugs synergistically inhibit macrophage vasculogenesis on the matrigel surface and the expression of FVIII-RA, Tie2/Tek, and VEGFR-2/VE-cadherin, indicative of cell transdifferentiation into endothelial-like cells. Both drugs reduce phospho-activation of VEGFR-2 and ERK1/2 and NFKB activity. These data provide evidence that the exposure of bone marrow macrophages during the treatment with bortezomib and zoledronic acid impacts their angiogenic and vasculogenic properties, suggesting that these cells may be considered as a target of both drugs in MM patients. Kim et al. [21] demonstrated that when macrophages pre-treated with bortezomib were co-cultured with MM cell lines, the proliferative index of these latter decreased significantly. Addition of defibrotide, an agent that could modulate macrophage-endothelial cell interactions to other chemotherapeutics resulted in encouraging results both in pre-clinical models [35] and in a multicentre phase I/II clinical trial [36]. Acknowledgments
Transgenic TG6 mice over-expressing human erythropoietin (Epo) have increased populations of liver and duodenal mucosa macrophages [29,30]; expression of Epo receptor (EpoR) was shown in wound healing macrophages and Epo enhanced the activity of these cells [31]. Lifshitz et al. [32] demonstrated that macrophages isolated form bone marrow expressed in vitro EpoR transcripts. In vitro treatment of these cells enhanced their phagocytic activity as well as IL-12 and nitric oxide secretion, while decreased IL-10 secretion in response to lipopolysaccharide. Following Epo treatment the bone marrow macrophages also exhibited activation of PI3k, MAPK, and
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