Hypothesis: are neoplastic macrophages/microglia present in glioblastoma multiforme?

INTRODUCTION

It has recently been suggested that many types of human cancers (lung, breast, colon, kidney, etc.) contain neoplastic cells with mesenchymal/macrophage properties ( Huysentruyt and Seyfried, 2010 ). These neoplastic macrophages are often the most highly invasive and metastatic cells within the tumour. It is not clear if similar kinds of cells are part of the malignant-cell population in human GBM (glioblastoma multiforme). GBM is the most common form of primary brain cancer in adults and represents ∼65% of all newly diagnosed malignant gliomas ( Ohgaki and Kleihues, 2005 ; Stupp et al., 2009 ). GBM portends an extremely poor outcome with only approx. 10% of patients surviving 5 years after diagnosis, and a median survival of ∼12 months ( Krex et al., 2007 ; Preusser et al., 2011 ). The poor prognosis is due largely to the highly invasive nature of this tumour. GBM invades throughout the brain and often produces multi-centric secondary legions at sites distant from the primary tumour ( Scherer, 1940 ; Rubinstein, 1972 ; Laws et al., 1993 ). Complete surgical resection of GBM is extremely rare. Radiation therapy, which enhances the necrotic microenvironment, often results in further tissue damage and more aggressive tumours ( Lakka and Rao, 2008 ; Kargiotis et al., 2010 ; Seyfried et al., 2010 , 2011 ). Thus, effective therapeutic options are desperately needed for GBM patients.

GBM is classified as either primary or secondary. Primary GBM arise de novo without any prior evidence of a low-grade tumour, whereas secondary GBM arises from malignant progression of a lower grade glioma ( Ohgaki and Kleihues, 2009 ). A defining characteristic of GBM is the ‘secondary structures of Scherer’ which include diffuse parenchymal invasion, perivascular growth, subpial surface growth and invasion along white matter tracks ( Scherer, 1940 ; Shelton et al., 2010b ). While systemic metastasis of GBM is uncommon due to early patient death, GBM can be extremely metastatic especially if the neoplastic cells gain access to extraneural sites ( Hoffman and Duffner, 1985 ; Rubinstein, 1972 ; Ng et al., 2005 ; Taha et al., 2005 ; Frank et al., 2009 ; Zhen et al., 2010 ; Gotway et al., 2011 ). These findings indicate that GBM is not only highly invasive within the CNS (central nervous system), but can also invade outside the CNS.

GBM generally contain multiple morphologically diverse cell types that express neural, glial and myeloid markers ( Rubinstein, 1972 ; Wood and Morantz, 1979 ; Seyfried, 2001 ; Yuan et al., 2004 ; Huysentruyt et al., 2008 ). In fact, mesenchymal cells with characteristics of TAM (tumour-associated macrophages) and/or microglia can comprise up to 70% of some GBM ( Morantz et al., 1979 ). It has been difficult to determine with certainty, however, the origin of all the mesenchymal and macrophage-like cells that appear within the GBM ( Tso et al., 2006 ; Ricci-Vitiani et al., 2010 ). Based on numerous similarities between macrophages and invasive neoplastic cells in non-CNS tumours ( Huysentruyt and Seyfried, 2010 ), we propose that some neoplastic cells within the GBM arise from transformed macrophage/microglia. As cells of the myeloid/macrophage lineage naturally embody the capacity to invade ( Mallat et al., 2005 ; Banaei-Bouchareb et al., 2006 ), we suggest that some of the highly invasive mesenchymal-type cells within GBM may arise from resident or infiltrating myeloid cells of the tumour stroma that then become neoplastic during disease progression ( Huysentruyt and Seyfried, 2010 ).

Our hypothesis comes from information on the VM (vasculogenic mimicry) mouse model of GBM which consists of highly invasive tumours ( Shelton et al., 2010b ). The VM tumours arise spontaneously in the brains of inbred VM mice ( Fraser, 1971 , 1986 ; Shelton et al., 2010b ). The neoplastic cells in the invasive VM brain tumours express multiple properties of macrophages and microglial cells and the VM-M3 and VM-M2 tumours have been established as a model system for human GBM ( Huysentruyt et al., 2008 ; Huysentruyt et al., 2010 ; Huysentruyt and Seyfried, 2010 ; Shelton et al., 2010b ). We recently reviewed evidence showing that many invasive and metastatic human cancers also express multiple properties of mesenchymal myeloid cells ( Huysentruyt and Seyfried, 2010 ). If many non-neural metastatic tumours arise from mesenchymal myeloid type cells, what about malignant tumours of the CNS? The goal of this commentary is to highlight similarities between myeloid cells and the invasive-cell populations of GBM, and to discuss possible mechanisms by which neoplastic cells could express these properties.

MYELOID CELLS AND INVASIVE CANCER

Myeloid cells have long been considered the origin of human metastatic cancers; however, this hypothesis has received little attention in the cancer field ( Munzarova and Kovarik, 1987 ; Rachkovsky et al., 1998 ; Huysentruyt and Seyfried, 2010 ; Pawelek, 2000 ; Vignery, 2005 ; Pawelek and Chakraborty, 2008 ). Although large numbers of cells with macrophage and myeloid properties are found in most malignant cancers including GBM, these cells are generally considered part of the tumour stroma and not part of the neoplastic population ( Seyfried, 2001 ; Mantovani et al., 2002 ). These cells are often referred to as TAM, and are thought to facilitate tumour development and malignant progression ( Seyfried, 2001 ; Bingle et al., 2002 ; Lewis and Pollard, 2006 ; Talmadge et al., 2007 ; Pollard, 2008 ). The tumour stroma is a complex microenvironment that generally comprises malignant tumour cells and host infiltrating cells that include immune and epithelial cells ( Bissell and Hines, 2011 ). The most commonly accepted view is that tumour-derived chemoattractants signal monocytes to extravasate out of the bloodstream and infiltrate the tumour mass ( Bottazzi et al., 1983 ; Murdoch et al., 2004 ). Once in the tumour microenvironment, enhanced inflammation and angiogenesis establish the pre-metastatic niche, thus contributing to tumour progression ( Seyfried, 2001 ; Bingle et al., 2002 ; Lewis and Pollard, 2006 ; Talmadge et al., 2007 ; Pollard, 2008 ). However, little consideration has been given to the possibility that microglial or TAM subsets might actually be part of the neoplastic cell population within GBM ( Graeber et al., 2002 ; Watters et al., 2005 ). As cells of macrophage/microglial origin can express multiple mesenchymal morphologies depending on the microenvironment, how is it possible to be sure that none of these stromal cells are part of the neoplastic cell population?

Macrophages and microglia are among the most versatile cells of the body with respect to their ability to migrate, to change shape and to secrete growth factors and cytokines ( Gordon, 1999 ; Stossel, 1999 ; Burke and Lewis, 2002 ; Huysentruyt et al., 2008 ). Macrophages fluctuate between two activation states depending on the microenvironment. Macrophages can become classically activated (M1 activation) in response to pathogens and pro-inflammatory molecules, resulting in the generation of NO and ROS (reactive oxygen species) ( Sica et al., 2002 , 2006 ; Biswas et al., 2008 ; Mantovani and Sica, 2010 ; Qian and Pollard, 2010 ). In contrast, macrophages can become alternatively activated (M2 activation) in response to anti-inflammatory molecules such as IL-4 (interleukin-4) and IL-10 and are the major wound-healing cells ( Gordon, 2003 ; Biswas et al., 2008 ). Interestingly, the two macrophage subpopulations play separate roles in tumorigenic processes ( Biswas et al., 2008 ). M1 macrophages, located at sites of chronic inflammation, contribute to neoplastic transformation through the release of cytotoxic and DNA damaging pro-inflammatory molecules ( Biswas et al., 2008 ). Macrophages with an M2 phenotype contribute to angiogenesis and metastasis within the established tumour ( Mantovani et al., 2002 ; Sica et al., 2006 ; Biswas et al., 2008 ; Huysentruyt and Seyfried, 2010 ; Mantovani and Sica, 2010 ). However, recent evidence suggests that TAMs may be a more complex subtype that can share features of both activation states ( Ojalvo et al., 2009 , 2010 ; Qian and Pollard, 2010 ).

Ineffective anti-tumour immunity resulting from reduced T-cell function is a hallmark of many cancers including GBM ( Waziri, 2010 ; Raychaudhuri et al., 2011 ). Recently, it has been suggested that another myeloid cell type, MDSCs (myeloid-derived suppressor cells) are partially responsible for GBM-related immune suppression ( Raychaudhuri et al., 2011 ). MDSCs are a heterogeneous population of immature myeloid cells that accumulate in tumour-bearing hosts where they suppress T-cell function, resulting in ineffective anti-tumour immunity ( Gabrilovich and Nagaraj, 2009 ; Ostrand-Rosenberg, 2010 ; Raychaudhuri et al., 2011 ). This cell type accumulates in the blood, lymph nodes and spleen in patients with solid tumours and has been reported to represent approx. 5% of the total tumour mass in various experimental tumour-model systems ( Yang et al., 2004 ). Raychaudhuri et al. demonstrated that patients with GBM have increased MDSC counts in their blood when compared with healthy controls and these cells likely promote T-cell immune suppression in this patient population ( Raychaudhuri et al., 2011 ). While the phenotype and function of MDSCs are believed to be distinct from those of TAMs, MDSCs can differentiate into TAMs within the tumour microenvironment thus further contributing to tumour progression (reviewed in Gabrilovich and Nagaraj, 2009 ). To our knowledge, it has not been demonstrated if MDSCs contribute to GBM tumour mass.

PHAGOCYTOSIS: A DEFINING BEHAVIOUR OF MACROPHAGES AND INVASIVE CELLS

Phagocytosis, involving engulfment and ingestion of extracellular material, is a defining property of macrophages/microglia and of other professional phagocytes ( Burke and Lewis, 2002 ). In order to maintain tissue homoeostasis, macrophages phagocytose cellular debris, apoptotic cells and pathogens ( Burke and Lewis, 2002 ). Phagocytosis is also expressed in numerous invasive and metastatic cancers including breast, lung, liver, pancreatic, skin and brain (reviewed in Huysentruyt and Seyfried, 2010 ). It has been suggested that invasive tumour cells use phagocytosis to facilitate migration through the extracellular matrix and into surrounding tissues ( Bjerknes et al., 1987 ; Coopman et al., 1998 ).

The phagocytic/cannibalistic behaviour of tumour cells was first described over a century ago when foreign cell bodies were identified within human cancer cells (reviewed in Steinhaus, 1981 ). These cells were commonly described as ‘signet-ring’ and ‘birds-eye’ cells due to the peripheral displacement of their nuclei from engulfed materials ( Fais, 2007 ). Although numerous micro-organisms use cannibalism as a feeding mechanism, cellular cannibalism is an exclusive property of malignant tumour cells ( Fais, 2007 ). Both human and murine cancers have been shown to phagocytose tumour cells, erythrocytes, leucocytes, platelets, apoptotic cells and extracellular particles (reviewed in Huysentruyt and Seyfried, 2010 ). Interestingly, phagocytic tumour cells are observed in malignant gliomas, especially in GBM ( Figure 1 ) ( Youness et al., 1980 ; Bjerknes et al., 1987 ; Nitta et al., 1992 ; Zimmer et al., 1995 ; Chang et al., 2000 ; Persson and Englund, 2009 ; van Landeghem et al., 2009 ).

We previously identified two spontaneous brain tumours (VM-M2 and VM-M3) in the inbred VM mouse strain that are highly invasive in brain ( Huysentruyt et al., 2008 ; Shelton et al., 2010b ). These VM brain tumours express multiple properties of macrophages including phagocytic activity and are a model for human GBM ( Huysentruyt et al., 2008 ; Shelton et al., 2010b ). The phagocytic phenotype in these cells is remarkably similar to that of observed in the RAW 264.7 macrophage cell line ( Figure 1A ) ( Huysentruyt et al., 2008 ). In a similar study to ours that examined the phagocytic activity of four rat glioma cell lines with varying degrees of in vivo invasiveness, Bjerknes et al. demonstrated that the invasive glioma cells phagocytosed bacteria, red blood cells, zymosan particles and glia cell fragments ( Bjerknes et al., 1987 ). Furthermore, the two most invasive rat glioma cell lines displayed the highest level of phagocytosis. The authors noted that the phagocytic behaviour correlated with the amount of rat brain destruction during tumour cell invasion, suggesting that phagocytic activity may be connected to the excretion of lysosomal enzymes ( Bjerknes et al., 1987 ). Lysosomal enzyme secretion and phagocytosis are macrophage-specific behaviours. Invasive glioma cell lines that display macrophage/microglia-like behaviours suggest a myeloid origin. Chang et al. (2000) also demonstrated that the glioma cell lines U87, U251 and SF268 exhibit phagocytic behaviour against apoptotic glioma cells. These findings further demonstrate that invasive glioma cells can exhibit macrophage-like phagocytic behaviour.

Although astrocytes have been described as semi-professional phagocytes, their phagocytic ability is limited. It could be suggested that the phagocytic behaviour observed in the GBM might arise from transformed astrocytes. However, the recent findings of Persson and Englund (2009) suggest that the phagocytic activity observed in human GBM are properties of malignant macrophages/microglia, rather than properties of malignant astrocytes. Utilizing paraffin-embedded GBM material, they identified numerous CD68-positive phagocytic macrophage/microglia throughout brain tumour specimens. Double-immunostaining with CD68 and the tumour cell marker hTERT (human telomerase reverse transcriptase) was used to verify that the CD68-positive cells were in fact part of the malignant GBM cell population ( Persson and Englund, 2009 ). These findings support the hypothesis that neoplastic macrophages/microglia can exist within GBM.

In addition to GBM, other invasive CNS tumours contain neoplastic phagocytic cells. For example, phagocytic cells were observed in the bone marrow of a patient with a highly invasive and metastatic medulloblastoma, which involved invasion and metastasis to brainstem, bone marrow, lymph nodes and spinal cord ( Youness et al., 1980 ). After extensive characterization of the phagocytic cells via histopatholgy, electron microscopy and cytogenetics, the authors stated that the phagocytic cells were unquestionably metastatic medulloblastoma cells. Interestingly, this tumour metastasized outside the CNS after excision of the primary tumour from the right cerebellar region ( Youness et al., 1980 ). While extracranial metastasis of CNS tumours is uncommon, invasive CNS tumours can metastasize throughout the body if they gain access to extraneural sites ( Rubinstein, 1972 ; Hoffman and Duffner, 1985 ; Vural et al., 1996 ; Taha et al., 2005 ). In fact, several reports show that GBM can be highly metastatic ( Hoffman and Duffner, 1985 ; Ng et al., 2005 ; Taha et al., 2005 ; Frank et al., 2009 ; Zhen et al., 2010 ; Gotway et al., 2011 ). It therefore appears that this case of metastatic medulloblastoma exhibited the macrophage/microglia characteristics seen in some GBM.

While it might be difficult to demonstrate a myeloid origin of invasive GBM, the evidence reviewed here suggests that subpopulations of neoplastic GBM cells display the phagocytic behaviour of macrophages/microglia. As microglia are the resident macrophages of the brain, we propose that subpopulations of the malignant GBM cells could arise from microglia/macrophages ( Morantz et al., 1979 ; Seyfried, 2001 ). Human GBM contain mixtures of numerous neoplastic cell types, many of which have mesenchymal properties, do not express GFAP (glial fibrillary acidic protein) and are of unknown origin ( Duffy, 1983 ; Ohgaki and Kleihues, 2009 ; Han et al., 2010 ). Indeed, the original 19th century observations of Virchow (1863/1865) described glioblastomas as gliosarcomas of mesenchymal origin ( Scherer, 1940 ; Zagzag et al., 2008 ). While numerous mesenchymal cells are frequently seen in GBM, the specific classification of all tumour cell types within human GBM remains ambiguous at best ( Yates, 1992 ; Tso et al., 2006 ; Fan et al., 2007 ). According to our hypothesis, some of these neoplastic mesenchymal cells could arise from transformed macrophages/microglia.

Novel therapeutic approaches to GBM management become possible if the phagocytic behaviour can be targeted. Gollapudi and co-workers demonstrated that tumour cells undergo apoptosis after ‘feeding’ tumour cells with anti-tumour agents ( Ghoneum and Gollapudi, 2004 ; Ghoneum et al., 2005 , 2007 , 2008 ). They showed that various tumour cells, grown either in vitro or in vivo, underwent a yeast-induced apoptosis after engulfing Saccharomyces cerevisiae . Recent studies indicate that similar approaches could have therapeutic efficacy in GBM. Glioblastoma patients underwent treatment with a magnetic nanoparticle-based immunotherapy where nanoparticles were engulfed by cells within the GBM ( van Landeghem et al., 2009 ). To identify the phagocytic cells within the tumour, these investigators stained post-mortem fixed GBM sections with either the macrophage marker, CD68, or with the astrocyte/CNS stem cell marker, GFAP ( van Landeghem et al., 2009 ). The authors reported that most of the cells engulfing the magnetic nanoparticles were CD68-positive. It was not possible from their study, however, to determine if the CD68-positive cells were also neoplastic since CD68 would stain both transformed macrophages/microglia as well as non-neoplastic TAM.

FUSOGENICITY

Fusogenicity involves the merging of two distinct plasma membranes and is a defining characteristic of macrophages and microglia ( Vignery, 2000 ; Duelli and Lazebnik, 2003 ; Huysentruyt and Seyfried, 2010 ). Cell fusion is a highly regulated process that is essential for fertilization, skeletal muscle and placenta formation ( Duelli and Lazebnik, 2003 ). Outside of normal developmental processes, fusion events are normally restricted to cells of myeloid origin (reviewed in Duelli and Lazebnik, 2003 ). Macrophages often fuse during osteoclast formation and wound healing in order increase cell volume to facilitate engulfment of large extracellular materials ( Vignery, 2000 , 2005 ). Macrophages also undergo heterotypic fusions with numerous cell types. It is currently believed that macrophage heterotypic fusion is a mechanism by which macrophages can ‘heal’ a defective cell or tissue ( Vignery, 2000 ; Camargo et al., 2004a , 2004b ; Vignery, 2005 ; Powell et al., 2011 ). There is also considerable evidence demonstrating that macrophages fuse with tumour cells (reviewed in Huysentruyt and Seyfried, 2010 ). These properties would also be expected in microglia since microglia are the resident macrophages of the CNS.

Nearly a century ago, Aichel first suggested that fusions between somatic cells and leucocytes could result in aneuploidy and malignant hybrids (reviewed in Rachkovsky et al., 1998 ). Sixty years later, Mekler and Warner proposed that fusions between myeloid cells and tumour cells would produce daughter cells endowed with the invasive properties of the myeloid cell as well as the unlimited proliferative potential of the tumour cell (reviewed in Rachkovsky et al., 1998 ). The fusion hybrid hypothesis has emerged as a credible alternative to the epithelial mesenchymal transition for the origin of invasive and metastatic cancers ( Huysentruyt and Seyfried, 2010 ; Pawelek and Chakraborty, 2008 ). Indeed, Pawelek and co-workers showed that fusions between non-metastatic cells and macrophages result in cells with the ability to invade and metastasize ( Rachkovsky et al., 1998 ; Rachkovsky and Pawelek, 1999 ; Chakraborty et al., 2000 , 2001 , 2004 ; Pawelek, 2000 , 2005 ; Handerson et al., 2005 ; Yilmaz et al., 2005 ; Pawelek and Chakraborty, 2008 ). Additionally, they provided evidence demonstrating that these fusion events occur spontaneously both in vitro and in vivo in a variety of animal models and in human cancers ( Rachkovsky et al., 1998 ; Chakraborty et al., 2000 , 2004 ; Yilmaz et al., 2005 ). Fusions between tumour cells and macrophage/microglia, with subsequent nuclear fusion, could produce novel phenotypes in the absence of new mutations since the tumour-macrophage hybrids express genetic and functional traits of both parental cells ( Figure 2A ) ( Rachkovsky et al., 1998 ; Powell et al., 2011 ). Tumour–macrophage hybrids could also account in part for immune surveillance evasion seen in GBM and other metastatic cancers ( Huysentruyt and Seyfried, 2010 ).

GBM is composed of neoplastic cells with features that vary from small round cells to MNGCs (multinucleated giant cells) ( Homma et al., 2006 ). MNGCs arise from the fusion of two or more macrophages ( Vignery, 2000 , 2005 ). Recent reports suggest that MNGCs in GBM could arise through fusions of peripheral macrophages or microglia with glioma cells ( Alvarez-Dolado et al., 2003 ). A recent study demonstrated that CD98, a protein that regulates monocyte fusion events, was expressed on the surface of MNGCs in 15/16 GBMs ( Figure 2B ) ( Takeuchi et al., 2008 ). These findings suggest that MNGC formation in GBM is similar to what occurs during normal monocyte/macrophage fusion events ( Takeuchi et al., 2008 ). Microglia and bone-marrow derived cells can fuse with CNS cells resulting in MNGCs ( Alvarez-Dolado et al., 2003 ; Ackman et al., 2006 ). The MNGCs in GBM often express GFAP, a protein expressed on CNS stem cells and astrocytes, suggesting that this cell population contains macrophage/microglia and CNS stem cell/astrocyte hybrids ( Takeuchi et al., 2008 ). Patient prognosis is generally worse when GBMs contain large numbers of MNGCs and other macrophage-like cells when compared with GBMs containing fewer of these cells ( Deininger et al., 2003 ). This situation is similar to that seen in non-CNS tumours in that prognosis is generally worse for malignant tumours with higher rather than lower numbers of macrophages ( Leek et al., 1996 ; Homma et al., 2006 ; Shabo et al., 2009 ; Huysentruyt and Seyfried, 2010 ).

The large size and unique phenotype of MNGCs make them easily detectable in brain tumour biopsy specimens ( Figure 1B ) ( Rubinstein, 1972 ). However, it can be difficult to detect fusion events among tumour cells and myeloid cells with subsequent nuclear fusions. Utilizing a double immunostaining technique, Deininger et al. (2000) identified a subset of glioma cells that were positive for both GFAP and AIF1 (allograft inflammatory factor 1), a microglial marker also referred to as Iba1. GFAP+/AIF1+ cells were localized near areas of tumour growth and were indicative of spontaneous in vivo fusion events ( Deininger et al., 2000 ). It is well documented that TAM aid tumour progression by promoting angiogenesis, invasion and metastasis (reviewed in Seyfried, 2001 ; Lewis and Pollard, 2006 ). However, fusion events between tumour cells and tissue macrophage/microglia would also accelerate tumour progression since resulting daughter cells would inherit the invasive/migratory potential of microglia and unlimited proliferative potential of the tumour cell. Hence, a microglial/glioma cell hybrid could rapidly acquire an invasive phenotype in the absence of novel mutations.

The macrophage-like characteristics and invasive properties of the murine VM-M2 and VM-M3 brain tumours are more similar to human GBM than to most previously described experimental mouse brain tumour models ( Shelton et al., 2010b ). If fusion hybridization of neoplastic cells with macrophages/microglia could be the origin of invasive cells within human GBM, why are these cell types rarely found in rodent brain tumour transplant models? Most macrophage/microglia seen in chemically induced brain tumours are derived from resident microglia and TAM ( Ecsedy et al., 1998 ). We suggest that the mouse or the rat brain would respond to a tumour implant as if it were an acute infection. This would involve invasion of TAM and activation of local microglia. This is not what usually occurs in the development of a human GBM, where neoplastic transformation is a protracted process. For example, GBM can arise many years after head wound trauma ( Gruss et al., 1993 ; Sabel et al., 1999 ). It is therefore possible that conditions for macrophage/microglia fusogenicity are greater in the microenvironment of human brain than in the microenvironment of the rodent brain during malignant transformation.

GLIOMA CELL EXPRESSION OF MYELOID ANTIGENS

Macrophages and microglia express unique antigens that can help identify these cells in various tissues ( Roggendorf et al., 1996 ; Guillemin and Brew, 2004 ). A list of some of these antigens is shown in Table 1 . Numerous studies have reported high numbers of cells in GBM that express antigens seen only in macrophages/microglia ( Rossi et al., 1987 ; Roggendorf et al., 1996 ; Leung et al., 1997 ; Badie and Schartner, 2000 ; Graeber et al., 2002 ). As mentioned above, most cells within GBM that express macrophage antigens are generally considered host-infiltrating TAM, and are not considered part of the neoplastic tumour cell population ( Roggendorf et al., 1996 ; Seyfried, 2001 ). However, several reports show that macrophage/microglial antigens are expressed on neoplastic cells within GBM ( Figure 3 ) ( Rossi et al., 1987 ; Leenstra et al., 1995 ; Graeber et al., 2002 ; Deininger et al., 2003 ; Strojnik et al., 2006 ). These findings in GBM are consistent with findings reported for other human cancer types including breast, lung, skin, ovarian, pancreatic, rectal and renal ( Rossi et al., 1987 ; Leenstra et al., 1995 ; Deininger et al., 2003 ; Matyja et al., 2003 ; Strojnik et al., 2006 , 2009 ; Colman et al., 2010 ) and reviewed in Huysentruyt and Seyfried (2010) . The expression of macrophage antigens in neoplastic cell subpopulations within GBM would support our hypothesis that malignant microglia are a component of human GBM.

Deininger et al. (2003) utilized double labelling experiments to demonstrate that neoplastic cells located in regions of infiltrative glioma growth were positive for both GFAP and the macrophage marker CD14. It is currently thought that TAM, localized at the leading edge of the tumour, facilitate local tumour cell invasion ( Lewis and Pollard, 2006 ). However, the invading cells were CD14+/GFAP+ suggesting that these cells were part of the invading tumour cell population and not TAM. It is possible that these cells arise through fusion of tumour cells with macrophages/microglia, thus representing a neoplastic hybrid-cell population. In contrast to the Deininger et al.'s study ( Deininger et al., 2003 ), most studies of macrophages in brain tumours have not used double labelling to distinguish neoplastic cells from non-neoplastic cells ( Rossi et al., 1987 ; Shinonaga et al., 1988 ; Morimura et al., 1990 ; van Landeghem et al., 2009 ). Consequently, it is often difficult to determine with certainty the origin of all cell types that express macrophage/microglia antigens in GBM. However, the human glioblastoma cell line U138 expressed the macrophage marker Ki-M1P (CD68) in an in vitro environment where there were no contaminating TAMs ( Figure 3A ) ( Paulus et al., 1992 ). This finding is similar to what we showed for the murine VM-M2 and VM-M3 cell lines ( Huysentruyt et al., 2008 ). A few investigators have entertained the possibility that some macrophage/microglial-type cells in GBM might be part of the neoplastic cell population. Several previous studies show, however, that subpopulations of neoplastic macrophage-like cells are present in most human cancers (reviewed in Huysentruyt and Seyfried, 2010 ). It would therefore be important to consider the possibility that some GBM cell subpopulations, previously identified as TAM, are in fact neoplastic cells.

Like GBM, PXA (pleomorphic xanthoastrocytoma) contains cells of multiple morphologies ( Matyja et al., 2003 ). As in GBM, MNGC are also a characteristic cell type in PXA ( Matyja et al., 2003 ). In an attempt to further characterize this cell subtype, Matyja et al. (2003) evaluated the co-expression of glial and macrophage markers in the various tumour cell populations in eight cases of PXA. The authors identified a population of neoplastic cells that expressed GFAP and either HLA-class II or CD68 within the cytoplasm ( Matyja et al., 2003 ). Many of these neoplastic cells were large and lipid-laden suggesting a mesenchymal origin. According to our hypothesis, neoplastic microglia/macrophages could give rise to PXA following fusion hybridizations.

Expression of macrophage markers has been reported in high-grade gliomas including GBM ( Rossi et al., 1987 ; Leenstra et al., 1995 ; Strojnik et al., 2006 ). Leenstra et al. (1995) examined macrophage characteristics in six malignant glioma cell lines. All the cell lines examined were classified as malignant gliomas based upon DNA flow cytometry and GFAP expression. Interestingly, all six glioma lines co-expressed macrophage markers and GFAP. In contrast, macrophage antigens were not found in cultured astrocytes isolated from healthy brain tissue, suggesting that the macrophage markers expressed by malignant astrocytes were not artefacts of an in vitro culture environment ( Leenstra et al., 1995 ). In a separate study, immunohistochemical analysis revealed that 40% of grade III astrocytomas and GBM tumour specimens contained neoplastic cells with macrophage markers ( Rossi et al., 1987 ). The U87 GBM model has also been shown to express both CD68 ( Figure 3B ) and glia markers when grown either in vitro or in vivo ( Strojnik et al., 2006 ). Strojnik et al. (2009) evaluated the prognostic significance of CD68 expression in malignant human gliomas. It was clear form their study that the presence of high levels of CD68-positive tumour cells was predictive of reduced survival. CD68 was expressed by both microglia and tumour cells ( Strojnik et al., 2009 ). The macrophage/microglia marker, Iba1, is also expressed by the VM-M2 mouse GBM tumour cells ( Figure 3C ) ( Huysentruyt et al., 2008 ). Taken together, these findings support our hypothesis and indicate that some neoplastic GBM cells could be of macrophage/microglial origin.

NUMEROUS HUMAN CANCERS EXPRESS MACROPHAGE PROPERTIES

Evidence presented above suggests that subpopulations of the invasive GBM tumour cells include neoplastic cells with multiple characteristics of macrophages/microglia. Recent studies show that macrophage properties and antigens are expressed in the neoplastic cells of numerous human metastatic cancers including bladder, brain, breast, carcinoma of unknown primary, endometrial, fibrosarcoma, gall bladder, liver, lung, lymphoma, melanoma, multiple myeloma, ovarian, pancreatic, rectal, rhabdomyosarcoma and renal cancers (reviewed in Huysentruyt and Seyfried, 2010 ). Commonly, these macrophage characteristics are observed in the invasive and metastatic cell populations, further highlighting the possibility that the highly-invasive tumour cells may be of mesenchymal origin. Indeed, these observations led to our recent prediction that the metastatic cells in most human cancers are of myeloid origin ( Huysentruyt and Seyfried, 2010 ). As microglia are mesenchymal cells of myeloid origin, we suggest that neoplastic microglia can represent an invasive cell population in GBM. This shared property among numerous invasive/metastatic tumours could have broad therapeutic implications. For example, any therapy that is able to reduce invasion and metastasis of one tumour type would likely be effective in treating tumours with macrophage characteristics.

POSSIBLE MECHANISMS

How could resident microglia or TAM become part of the neoplastic cell population in GBM? We recently reviewed evidence indicating that all cancer, regardless of tissue or cellular origin, is primarily a disease of impaired cellular energy metabolism ( Seyfried and Shelton, 2010 ). Otto Warburg first proposed that all types of cancers arise from irreversible damage to cellular respiration ( Warburg, 1931 , 1956 ). Persistent injury to oxidative phosphorylation will require compensatory non-oxidative energy metabolism to maintain viability ( Seyfried and Shelton, 2010 ; Seyfried et al., 2010 ; Shelton et al., 2010b ). The mitochondrial stress response or RTG (retrograde) signalling from the mitochondria to the nucleus is required to up-regulate the oncogenes needed to sustain glycolysis and non-oxidative mitochondrial energy production ( Seyfried and Shelton, 2010 ; Seyfried et al., 2010 ). However, persistent RTG activation leads to eventual nuclear genomic instability and other recognized hallmarks of cancer ( Butow and Avadhani, 2004 ; Singh et al., 2005 ; Seyfried and Shelton, 2010 ). Mitochondria energy production is often damaged as a consequence of mutagens, hypoxia, inflammation, ROS or inherited mutations ( Kiebish et al., 2008 ). We recently showed that mitochondria isolated from murine gliomas contain numerous lipid defects supporting previous findings that respiratory energy production is dysfunctional in tumour mitochondria ( Kiebish et al., 2008 , 2009 ; Ordys et al., 2010 ; Seyfried and Shelton, 2010 ). Macrophages/microglia home to mitochondria-damaging environments in response to inflammation, infection, wound repair and tumorigenesis ( Giulian et al., 1989 ; Chettibi, 1999 ; Bingle et al., 2002 ; Graeber et al., 2002 ; Lewis and Murdoch, 2005 ; Martin and Leibovich, 2005 ; Lewis and Pollard, 2006 ). Inflammation and hypoxia in the tumour's microenvironment can damage macrophage mitrochondria ( Frost et al., 2005 ; Navarro and Boveris, 2005 ). It is therefore possible that mitochondria could become dysfunctional when macrophages/microglia respond to various chronic tissue injuries, resulting in a persistent RTG response with eventual malignant transformation ( Seyfried and Shelton, 2010 ). Hence, we suggest that some invasive GBM cells could arise from resident tissue microglia or TAM that have suffered mitochondrial damage during tumour initiation or progression.

Focal hypoxia combined with persistent inflammation and stem cell proliferation could produce macrophage–microglia or macrophage–stem cell fusions. Respiratory damage could either proceed or follow fusion events thus initiating the path to frank neoplasia. Additionally, damaged mitochondria could also be passed from one cell to another via cytoplasmic inheritance during cellular-fusion events ( Seyfried and Shelton, 2010 ). Although normal mitochondria are known to suppress tumorigenesis in fusion hybrids ( Seyfried and Shelton, 2010 ), persistent inflammation in the tumour microenvironment could contribute to continued mitochondrial damage in the fused hybrids. This would enhance the Warburg effect, unbridled proliferation and genomic instability. As microglia naturally embody the ability to migrate throughout the brain ( Graeber and Streit, 1990 ; Amat et al., 1996 ), we suggest that transformed cells of macrophage/microglia origin would possess invasive potential.

Currently, the cause of macrophage and tumour cell hybridization is unknown. Various stages of the macrophage response to tumour development could increase the probability for cell-fusion events. The host immune system treats tumours as unhealed wounds ( Dvorak, 1986 ; Seyfried, 2001 ; Bissell and Hines, 2011 ). Macrophages home to hypoxic tumour areas in the process of wound healing. The failure of macrophages to completely digest apoptotic cells could result in macrophage×tumour hybrid cells ( Pawelek, 2000 ). It is therefore possible that macrophages fuse with tumour cells in an attempt to ‘heal’ the tumour, as macrophages are known to fuse with non-myeloid cells during tissue repair. Radiation is commonly used as a primary therapy for most brain tumours. However, radiation therapy enhances the incidence of fusion hybrid formation and could result in a more aggressive and invasive tumours ( Shabo et al., 2009 ; Seyfried et al., 2011 ). This could account in part for the exacerbating effects of radiation therapy on GBM progression ( Seyfried et al., 2010 ).

The macrophage fusion hybrid hypothesis could potentially explain many of the distinguishing characteristics seen in invasive and metastatic GBM tumour cells. These macrophage-reprogramming strategies may account for some of the cellular heterogeneity seen in human GBM since macrophage×CNS cell hybrids would likely retain the histology of the CNS fusion partner. Tumour hybrids could also account for the cancer cell aneuploidy and chromosomal abnormalities often seen in tumour cells ( Steinhaus, 1981 ; Pawelek, 2000 ). Further studies will be needed to determine the extent to which fusion hybridization might contribute to the invasive properties seen in GBM.

CONCLUDING REMARKS

GBM is a highly complex and lethal tumour. Unfortunately, the current therapies available to GBM patients are largely ineffective and often have a negative impact on the patient's quality of life. In this commentary, we aim to highlight the possibility that the most aggressive and invasive cells in human GBMs are neoplastic macrophages/microglia. While further experimentation is needed to confirm the gene expression and immunohistochemical data reviewed here, we believe that novel therapeutic approaches could be developed targeting the macrophage characteristics of invasive GBM cells. It would be of particular interest to determine if the CD68+ cell population of human GBM has neoplastic properties.

ACKNOWLEDGEMENTS

We thank Dr Annette Persson for providing Figure 1(b) and for invaluable discussions. We also thank Dr Werner Paulus for providing the high-resolution image for Figure 3(a) .

This work was supported, in part, by the National Institutes of Health [grant numbers HD -39722, NS - 55195 and CA -102135], a grant from the American Institute of Cancer Research and the Boston College Expense Fund .

REFERENCES

Ackman JB Siddiqi F Walikonis RS LoTurco JJ Fusion of microglia with pyramidal neurons after retroviral infection. J Neurosci 2006 26 11413 11422 17079670 10.1523/JNEUROSCI.3340-06.2006 PMC6674527

Alvarez-Dolado M Pardal R Garcia-Verdugo JM Fike JR Lee HO Pfeffer K Lois C Morrison SJ Alvarez-Buylla A Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 2003 425 968 973 14555960 10.1038/nature02069

Amat JA Ishiguro H Nakamura K Norton WT Phenotypic diversity and kinetics of proliferating microglia and astrocytes following cortical stab wounds. Glia 1996 16 368 382 8721677 10.1002/(SICI)1098-1136(199604)16:4<368::AID-GLIA9>3.0.CO;2-W

Badie B Schartner JM Flow cytometric characterization of tumor-associated macrophages in experimental gliomas. Neurosurgery 2000 46 957 961 10764271 10.1097/00006123-200004000-00035

Banaei-Bouchareb L Peuchmaur M Czernichow P Polak M A transient microenvironment loaded mainly with macrophages in the early developing human pancreas. J Endocrinol 2006 188 467 480 16522727 10.1677/joe.1.06225

Bingle L Brown NJ Lewis CE The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol 2002 196 254 265 11857487 10.1002/path.1027

Bissell MJ Hines WC Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression. Nat Med 2011 17 320 329 21383745 10.1038/nm.2328 PMC3569482

Biswas SK Sica A Lewis CE Plasticity of macrophage function during tumor progression: regulation by distinct molecular mechanisms. J Immunol 2008 180 2011 2017 18250403 10.4049/jimmunol.180.4.2011

Bjerknes R Bjerkvig R Laerum OD Phagocytic capacity of normal and malignant rat glial cells in culture. J Natl Cancer Inst 1987 78 279 288 3468291

Bottazzi B Polentarutti N Acero R Balsari A Boraschi D Ghezzi P Salmona M Mantovani A Regulation of the macrophage content of neoplasms by chemoattractants. Science 1983 220 210 212 6828888 10.1126/science.6828888

Burke B Lewis CE The Macrophage. 2002 New York Oxford University Press

Butow RA Avadhani NG Mitochondrial signaling: the retrograde response. Mol Cell 2004 14 1 15 15068799 10.1016/s1097-2765(04)00179-0

Camargo FD Chambers SM Goodell MA Stem cell plasticity: from transdifferentiation to macrophage fusion. Cell Prolif 2004a 37 55 65 14871237 10.1111/j.1365-2184.2004.00300.x PMC6496757

Camargo FD Finegold M Goodell MA Hematopoietic myelomonocytic cells are the major source of hepatocyte fusion partners. J Clin Invest 2004b 113 1266 1270 15124017 10.1172/JCI21301 PMC398434

Chakraborty A Lazova R Davies S Backvall H Ponten F Brash D Pawelek J Donor DNA in a renal cell carcinoma metastasis from a bone marrow transplant recipient. Bone Marrow Transplant 2004 34 183 186 15195072 10.1038/sj.bmt.1704547

Chakraborty AK de Freitas Sousa J Espreafico EM Pawelek JM Human monocyte x mouse melanoma fusion hybrids express human gene. Gene 2001 275 103 106 11574157 10.1016/s0378-1119(01)00647-3

Chakraborty AK Sodi S Rachkovsky M Kolesnikova N Platt JT Bolognia JL Pawelek JM A spontaneous murine melanoma lung metastasis comprised of host×tumor hybrids. Cancer Res 2000 60 2512 2519 10811133

Chang GH Barbaro NM Pieper RO Phosphatidylserine-dependent phagocytosis of apoptotic glioma cells by normal human microglia, astrocytes, glioma cells. Neuro Oncol 2000 2 174 183 11302338 10.1093/neuonc/2.3.174 PMC1920494

Chettibi S MWJ Ferguson Snyderman J Wound repair: an overview. In Inflammation: Basic Principals and Clinical Correlates 1999 New York Lippincott Williams and Wilkins 865 881

Colman H Zhang L Sulman EP McDonald JM Shooshtari NL Rivera A Popoff S Nutt CL Louis DN Cairncross JG Gilbert MR Phillips HS Mehta MP Chakravarti A Pelloski CE Bhat K Feuerstein BG Jenkins RB Aldape K A multigene predictor of outcome in glioblastoma. Neuro Oncol 2010 12 49 57 20150367 10.1093/neuonc/nop007 PMC2940562

Coopman PJ Do MT Thompson EW Mueller SC Phagocytosis of cross-linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity. Clin Cancer Res 1998 4 507 515 9516943

Deininger MH Meyermann R Schluesener HJ Expression and release of CD14 in astrocytic brain tumors. Acta Neuropathol (Berl) 2003 106 271 277 12835948 10.1007/s00401-003-0727-9

Deininger MH Seid K Engel S Meyermann R Schluesener HJ Allograft inflammatory factor-1 defines a distinct subset of infiltrating macrophages/microglial cells in rat and human gliomas. Acta Neuropathol (Berl) 2000 100 673 680 11078219 10.1007/s004010000233

Duelli D Lazebnik Y Cell fusion: a hidden enemy? Cancer Cell 2003 3 445 448 12781362 10.1016/s1535-6108(03)00114-4

Duffy PE Astrocytes: Normal, Reactive, and Neoplastic. 1983 New York Raven Press 224

Dvorak HF Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 1986 315 1650 1659 3537791 10.1056/NEJM198612253152606

Ecsedy JA Yohe HC Bergeron AJ Seyfried TN Tumor-infiltrating macrophages contribute to the glycosphinglipid composition of brain tumors. J Lipid Res 1998 39 2218 2227 9799808

Fais S Cannibalism: a way to feed on metastatic tumors. Cancer Lett 2007 258 155 164 17977647 10.1016/j.canlet.2007.09.014

Fan X Salford LG Widegren B Glioma stem cells: evidence and limitation. Semin Cancer Biol 2007 17 214 218 16750389 10.1016/j.semcancer.2006.04.002

Frank S Kuhn SA Brodhun M Mueller U Romeike B Kosmehl H Regenbrecht CR Ewald C Reichart R Kalff R Metastatic glioblastoma cells use common pathways via blood and lymphatic vessels. Neurol Neurochir Pol 2009 43 183 190 19484696

Fraser H Astrocytomas in an inbred mouse strain. J Pathol 1971 103 266 270 5563880 10.1002/path.1711030410

Fraser H Brain tumours in mice, with particular reference to astrocytoma. Food Chem Toxicol 1986 24 105 111 3957158 10.1016/0278-6915(86)90344-3

Frost MT Wang Q Moncada S Singer M Hypoxia accelerates nitric oxide-dependent inhibition of mitochondrial complex I in activated macrophages. Am J Physiol Regul Integr Comp Physiol 2005 288 R394 R400 15486095 10.1152/ajpregu.00504.2004

Gabrilovich DI Nagaraj S Myeloid-derived suppressor cells as regulators of the immune system. Nat Rev Immunol 2009 9 162 174 19197294 10.1038/nri2506 PMC2828349

Ghoneum M Gollapudi S Phagocytosis of Candida albicans by metastatic and non metastatic human breast cancer cell lines in vitro . Cancer Detect Prev 2004 28 17 26 15041073 10.1016/j.cdp.2003.10.001

Ghoneum M Hamilton J Brown J Gollapudi S Human squamous cell carcinoma of the tongue and colon undergoes apoptosis upon phagocytosis of Saccharomyces cerevisiae , the baker's yeast, in vitro . Anticancer Res 2005 25 981 989 15868937

Ghoneum M Matsuura M Braga M Gollapudi S S. cerevisiae induces apoptosis in human metastatic breast cancer cells by altering intracellular Ca 2+ and the ratio of Bax and Bcl-2. Int J Oncol 2008 33 533 539 18695883

Ghoneum M Wang L Agrawal S Gollapudi S Yeast therapy for the treatment of breast cancer: a nude mice model study. In Vivo 2007 21 251 258 17436573

Giulian D Chen J Ingeman JE George JK Noponen M The role of mononuclear phagocytes in wound healing after traumatic injury to adult mammalian brain. J Neurosci 1989 9 4416 4429 2480402 10.1523/JNEUROSCI.09-12-04416.1989 PMC6569632

Gordon S Gallin J Snyderman R Development and distribution of mononuclear phagocytes: revelance to inflammation. In Inflammation: Basic Principles and Clinical Correlates 1999 New York Lippincott Williams and Wilkins 35 48

Gordon S Alternative activation of macrophages. Nat Rev Immunol 2003 3 23 35 12511873 10.1038/nri978

Gotway MB Conomos PJ Bremner RM Pleural metastatic disease from glioblastoma multiforme. J Thorac Imaging 2011 26 W54 W58 20634762 10.1097/RTI.0b013e3181d449d6

Graeber MB Scheithauer BW Kreutzberg GW Microglia in brain tumors. Glia 2002 40 252 259 12379912 10.1002/glia.10147

Graeber MB Streit WJ Microglia: immune network in the CNS. Brain Pathol 1990 1 2 5 1669689 10.1111/j.1750-3639.1990.tb00630.x

Gruss P Spohr A Leiber A Tasler J Menzl H Hofstadter F The traumatic origin of a glioblastoma. Zentralbl Neurochir 1993 54 186 189 8128788

Guillemin GJ Brew BJ Microglia, macrophages, perivascular macrophages, and pericytes: a review of function and identification. J Leukoc Biol 2004 75 388 397 14612429 10.1189/jlb.0303114

Han SJ Yang I Otero JJ Ahn BJ Tihan BJ McDermott MW Berger MS Chang SM Parsa AT Secondary gliosarcoma after diagnosis of glioblastoma: clinical experience with 30 consecutive patients. J Neurosurg 2010 112 990 996 19817543 10.3171/2009.9.JNS09931

Handerson T Camp R Harigopal M Rimm D Pawelek J Beta1,6-branched oligosaccharides are increased in lymph node metastases and predict poor outcome in breast carcinoma. Clin Cancer Res 2005 11 2969 2973 15837749 10.1158/1078-0432.CCR-04-2211

Hoffman HJ Duffner PK Extraneural metastases of central nervous system tumors. Cancer 1985 56 1778 1782 4027909 10.1002/1097-0142(19851001)56:7+<1778::aid-cncr2820561309>3.0.co;2-i

Homma T Fukushima T Vaccarella S Yonekawa Y Di Patre PL Franceschi S Ohgaki H Correlation among pathology, genotype, and patient outcomes in glioblastoma. J Neuropathol Exp Neurol 2006 65 846 854 16957578 10.1097/01.jnen.0000235118.75182.94

Huysentruyt LC Mukherjee P Banerjee D Shelton LM Seyfried TN Metastatic cancer cells with macrophage properties: evidence from a new murine tumor model. Int J Cancer 2008 123 73 84 18398829 10.1002/ijc.23492

Huysentruyt LC Seyfried TN Perspectives on the mesenchymal origin of metastatic cancer. Cancer Metastasis Rev 2010 29 695 707 20839033 10.1007/s10555-010-9254-z PMC2962789

Huysentruyt LC Shelton LM Seyfried TN Influence of methotrexate and cisplatin on tumor progression and survival in the VM mouse model of systemic metastatic cancer. Int J Cancer 2010 126 65 72 19536778 10.1002/ijc.24649

Kargiotis O Geka A Rao JS Kyritsis AP Effects of irradiation on tumor cell survival, invasion and angiogenesis. J Neurooncol 2010 100 323 338 20449629 10.1007/s11060-010-0199-4

Kiebish MA Han X Cheng H Chuang JH Seyfried TN Cardiolipin and electron transport chain abnormalities in mouse brain tumor mitochondria: lipidomic evidence supporting the Warburg theory of cancer. J Lipid Res 2008 49 2545 2556 18703489 10.1194/jlr.M800319-JLR200 PMC2582368

Kiebish MA Han X Cheng H Seyfried TN In vitro growth environment produces lipidomic and electron transport chain abnormalities in mitochondria from non-tumorigenic astrocytes and brain tumours. ASN NEURO 2009 1 e00011 19570033 10.1042/AN20090011 PMC2695587

Krex D Klink B Hartmann C von Deimling A Pietsch T Simon M Sabel M Steinbach JP Heese O Reifenberger G Weller M Schackert G Long-term survival with glioblastoma multiforme. Brain 2007 130 2596 2606 17785346 10.1093/brain/awm204

Lakka SS Rao JS Antiangiogenic therapy in brain tumors. Expert Rev Neurother 2008 8 1457 1473 18928341 10.1586/14737175.8.10.1457 PMC2656359

Laws Jr ER Goldberg WJ Bernstein JJ Migration of human malignant astrocytoma cells in the mammalian brain: Scherer revisited. Int J Dev Neurosci 1993 11 691 697 8116480 10.1016/0736-5748(93)90056-j

Leek RD Lewis CE Whitehouse R Greenall M Clarke J Harris AL Association of macrophage infiltration with angiogenesis and prognosis in invasive breast carcinoma. Cancer Res 1996 56 4625 4629 8840975

Leenstra S Das PK Troost D de Boer OJ Bosch DA Human malignant astrocytes express macrophage phenotype. J. Neuroimmunol 1995 56 17 25 7822478 10.1016/0165-5728(94)00128-b

Leung SY Wong MP Chung LP Chan AS Yuen ST Monocyte chemoattractant protein-1 expression and macrophage infiltration in gliomas. Acta Neuropathol (Berl) 1997 93 518 527 9144591 10.1007/s004010050647

Lewis C Murdoch C Macrophage responses to hypoxia: implications for tumor progression and anti-cancer therapies. Am J Pathol 2005 167 627 635 16127144 10.1016/S0002-9440(10)62038-X PMC1698733

Lewis CE Pollard JW Distinct role of macrophages in different tumor microenvironments. Cancer Res 2006 66 605 612 16423985 10.1158/0008-5472.CAN-05-4005

Mallat M Marin-Teva JL Cheret C Phagocytosis in the developing CNS: more than clearing the corpses. Curr Opin Neurobiol 2005 15 101 107 15721751 10.1016/j.conb.2005.01.006

Mantovani A Sica A Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr Opin Immunol 2010 22 231 237 20144856 10.1016/j.coi.2010.01.009

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 23 549 555 12401408 10.1016/s1471-4906(02)02302-5

Martin P Leibovich SJ Inflammatory cells during wound repair: the good, the bad and the ugly. Trends Cell Biol 2005 15 599 607 16202600 10.1016/j.tcb.2005.09.002

Matyja E Kroh H Taraszewska A Naganska E Zabek M Marchel A Expression of macrophage/histiocytic antigens in pleomorphic xanthoastrocytomas. Folia Neuropathol 2003 41 89 95 12899201

Morantz RA Wood GW Foster M Clark M Gollahon K Macrophages in experimental and human brain tumors. Part 2: studies of the macrophage content of human brain tumors. J Neurosurg 1979 50 305 311 422981 10.3171/jns.1979.50.3.0305

Morimura T Neuchrist C Kitz K Budka H Scheiner O Kraft D Lassmann H Monocyte subpopulations in human gliomas: expression of Fc and complement receptors and correlation with tumor proliferation. Acta Neuropathol (Berl) 1990 80 287 294 2399810 10.1007/BF00294647

Munzarova M Kovarik J Is cancer a macrophage-mediated autoaggressive disease? Lancet 1987 1 952 954 2882343 10.1016/s0140-6736(87)90295-9

Murdoch C Giannoudis A Lewis CE Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood 2004 104 2224 2234 15231578 10.1182/blood-2004-03-1109

Navarro A Boveris A Hypoxia exacerbates macrophage mitochondrial damage in endotoxic shock. Am J Physiol Regul Integr Comp Physiol 2005 288 R354 R355 15637172 10.1152/ajpregu.00726.2004

Ng WH Yeo TT Kaye AH Spinal and extracranial metastatic dissemination of malignant glioma. J Clin Neurosci 2005 12 379 382 15925766 10.1016/j.jocn.2004.11.004

Nitta T Okumura K Sato K Lysosomal enzymic activity of astroglial cells. Pathobiology 1992 60 42 44 1543550 10.1159/000163695

Ohgaki H Kleihues P Epidemiology and etiology of gliomas. Acta Neuropathol (Berl) 2005 109 93 108 15685439 10.1007/s00401-005-0991-y

Ohgaki H Kleihues P Genetic alterations and signaling pathways in the evolution of gliomas. Cancer Sci 2009 100 2235 2241 19737147 10.1111/j.1349-7006.2009.01308.x PMC11159448

Ojalvo LS King W Cox D Pollard JW High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors. Am J Pathol 2009 174 1048 1064 19218341 10.2353/ajpath.2009.080676 PMC2665764

Ojalvo LS Whittaker CA Condeelis JS Pollard JW Gene expression analysis of macrophages that facilitate tumor invasion supports a role for Wnt-signaling in mediating their activity in primary mammary tumors. J Immunol 2010 184 702 712 20018620 10.4049/jimmunol.0902360 PMC3226722

Ordys BB Launay S Deighton RF McCulloch J Whittle IR The role of mitochondria in glioma pathophysiology. Mol Neurobiol 2010 42 64 75 20414816 10.1007/s12035-010-8133-5

Ostrand-Rosenberg S Myeloid-derived suppressor cells: more mechanisms for inhibiting antitumor immunity. Cancer Immunol Immunother 2010 59 1593 1600 20414655 10.1007/s00262-010-0855-8 PMC3706261

Paulus W Roggendorf W Kirchner T Ki-M1P as a marker for microglia and brain macrophages in routinely processed human tissues. Acta Neuropathol (Berl) 1992 84 538 544 1462766 10.1007/BF00304473

Pawelek JM Tumour cell hybridization and metastasis revisited. Melanoma Res 2000 10 507 514 11198471 10.1097/00008390-200012000-00001

Pawelek JM Tumour-cell fusion as a source of myeloid traits in cancer. Lancet Oncol 2005 6 988 993 16321767 10.1016/S1470-2045(05)70466-6

Pawelek JM Chakraborty AK Fusion of tumour cells with bone marrow-derived cells: a unifying explanation for metastasis. Nat Rev Cancer 2008 8 377 386 18385683 10.1038/nrc2371

Persson A Englund E The glioma cell edge-winning by engulfing the enemy? Med Hypotheses 2009 73 336 337 19423236 10.1016/j.mehy.2009.03.042

Persson A Englund E The glioma cell lead winning by eating? In 9th European Congress of Neuropathology 2008 Athens, Greece Clinical Neuropathology 194

Pollard JW Macrophages define the invasive microenvironment in breast cancer. J Leukoc Biol 2008 84 623 630 18467655 10.1189/jlb.1107762 PMC2516896

Powell AE Anderson EC Davies PS Silk AD Pelz C Impey S Wong MH Fusion between intestinal epithelial cells and macrophages in a cancer context results in nuclear reprogramming. Cancer Res 2011 71 1497 1505 21303980 10.1158/0008-5472.CAN-10-3223 PMC3079548

Preusser M de Ribaupierre S Wohrer A Erridge SC Hegi M Weller M Stupp R Current concepts and management of glioblastoma. Ann Neurol 2011 70 9 21 21786296 10.1002/ana.22425

Qian BZ Pollard JW Macrophage diversity enhances tumor progression and metastasis. Cell 2010 141 39 51 20371344 10.1016/j.cell.2010.03.014 PMC4994190

Rachkovsky M Pawelek J Acquired melanocyte stimulating hormone-inducible chemotaxis following macrophage fusion with Cloudman S91 melanoma cells. Cell Growth Differ 1999 10 517 524 10437919

Rachkovsky M Sodi S Chakraborty A Avissar Y Bolognia J McNiff JM Platt J Bermudes D Pawelek J Melanoma×macrophage hybrids with enhanced metastatic potential. Clin Exp Metastasis 1998 16 299 312 9626809 10.1023/a:1006557228604

Raychaudhuri B Rayman P Ireland J Ko J Rini B Borden EC Garcia J Vogelbaum MA Finke J Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma. Neuro Oncol 2011 13 591 599 21636707 10.1093/neuonc/nor042 PMC3107102

Ricci-Vitiani L Pallini R Biffoni M Todaro M Invernici G Cenci T Maira G Parati EA Stassi G Larocca LM De Maria R Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature 2010 468 824 828 21102434 10.1038/nature09557

Roggendorf W Strupp S Paulus W Distribution and characterization of microglia/macrophages in human brain tumors. Acta Neuropathol (Berl) 1996 92 288 293 8870831 10.1007/s004010050520

Rossi ML Hughes JT Esiri MM Coakham HB Brownell DB Immunohistological study of mononuclear cell infiltrate in malignant gliomas. Acta Neuropathol (Berl) 1987 74 269 277 3314311 10.1007/BF00688191

Rubinstein LJ Tumors of the central nervous system. 1972 Washington, DC Armed Forces Institute of Pathology 400

Sabel M Felsberg J Messing-Junger M Neuen-Jacob E Piek J Glioblastoma multiforme at the site of metal splinter injury: a coincidence? Case report. J Neurosurg 1999 91 1041 1044 10.3171/jns.1999.91.6.1041 10584854

Scherer H The forms of growth in gliomas and their practical significance. Brain 1940 63 1 34

Seyfried TN Perspectives on brain tumor formation involving macrophages, glia, and neural stem cells. Perspect Biol Med 2001 44 263 282 11370160 10.1353/pbm.2001.0035

Seyfried TN Kiebish MA Marsh J Shelton LM Huysentruyt LC Mukherjee P Metabolic management of brain cancer. Biochim Biophys Acta 2011 1807 577 594 20804725 10.1016/j.bbabio.2010.08.009

Seyfried TN Shelton LM Cancer as a metabolic disease. Nutr Metab (Lond) 2010 7 7 20181022 10.1186/1743-7075-7-7 PMC2845135

Seyfried TN Shelton LM Mukherjee P Does the existing standard of care increase glioblastoma energy metabolism? Lancet Oncol 2010 11 811 813 20634134 10.1016/S1470-2045(10)70166-2

Shabo I Olsson H Sun XF Svanvik J Expression of the macrophage antigen CD163 in rectal cancer cells is associated with early local recurrence and reduced survival time. Int J Cancer 2009 125 1826 1831 19582880 10.1002/ijc.24506

Shelton LM Huysentruyt LM Mukherjee P Seyfried TN Calorie restriction as an anti-invasive therapy for malignant brain cancer in the VM mouse. ASN NEURO 2:e00038 2010a 10.1042/AN20100002 PMC2908744 20664705

Shelton LM Mukherjee P Huysentruyt LC Urits I Rosenberg JA Seyfried TN A novel pre-clinical in vivo mouse model for malignant brain tumor growth and invasion. J Neurooncol 2010b 99 165 176 20069342 10.1007/s11060-010-0115-y

Shinonaga M Chang CC Suzuki N Sato M Kuwabara T Immunohistological evaluation of macrophage infiltrates in brain tumors. Correlation with peritumoral edema. J Neurosurg 1988 68 259 265 3276837 10.3171/jns.1988.68.2.0259

Sica A Saccani A Mantovani A Tumor-associated macrophages: a molecular perspective. Int Immunopharmacol 2002 2 1045 1054 12349942 10.1016/s1567-5769(02)00064-4

Sica A Schioppa T Mantovani A Allavena P Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer 2006 42 717 727 16520032 10.1016/j.ejca.2006.01.003

Singh KK Kulawiec M Still I Desouki MM Geradts J Matsui S Inter-genomic cross talk between mitochondria and the nucleus plays an important role in tumorigenesis. Gene 2005 354 140 146 15979824 10.1016/j.gene.2005.03.027

Steinhaus J Ueber carcinom-einschlusse. Virchows Arch 1981 126 533 535

Stossel T Snyderman J Mechanical responses of white blood cells. In Inflammation: Basic Principals and Clinical Correlates 1999 New York Lippincott Williams and Wilkins 661 679

Strojnik T Kavalar R Lah TT Experimental model and immunohistochemical analyses of U87 human glioblastoma cell xenografts in immunosuppressed rat brains. Anticancer Res 2006 26 2887 2900 16886610

Strojnik T Kavalar R Zajc I Diamandis EP Oikonomopoulou K Lah TT Prognostic impact of CD68 and kallikrein 6 in human glioma. Anticancer Res 2009 29 3269 3279 19661345

Stupp R Hegi ME Mason WP van den Bent MJ Taphoorn MJ Janzer RC Ludwin SK Allgeier A Fisher B Belanger K Hau P Brandes AA Gijtenbeek J Marosi C Vecht CJ Mokhtari K Wesseling P Villa S Eisenhauer E Gorlia T Weller M Lacombe D Cairncross JG Mirimanoff RO Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised phase III study: 5-year analysis of the EORTC-NCIC trial. Lancet Oncol 2009 10 459 466 19269895 10.1016/S1470-2045(09)70025-7

Taha M Ahmad A Wharton S Jellinek D Extra-cranial metastasis of glioblastoma multiforme presenting as acute parotitis. Br J Neurosurg 2005 19 348 351 16455543 10.1080/02688690500305506

Takeuchi H Kubota T Kitai R Nakagawa R Hashimoto N CD98 immunoreactivity in multinucleated giant cells of glioblastomas: an immunohistochemical double labeling study. Neuropathology 2008 28 127 131 18021193 10.1111/j.1440-1789.2007.00859.x

Talmadge JE Donkor M Scholar E Inflammatory cell infiltration of tumors: Jekyll or Hyde. Cancer Metastasis Rev 2007 3–4 373 400 10.1007/s10555-007-9072-0 17717638

Tso CL Shintaku P Chen J Liu Q Liu J Chen Z Yoshimoto K Mischel PS Cloughesy TF Liau LM Nelson SF Primary glioblastomas express mesenchymal stem-like properties. Mol Cancer Res 2006 4 607 619 16966431 10.1158/1541-7786.MCR-06-0005

van Landeghem FK Maier-Hauff K Jordan A Hoffmann KT Gneveckow U Scholz R Thiesen B Bruck W von Deimling A Post-mortem studies in glioblastoma patients treated with thermotherapy using magnetic nanoparticles. Biomaterials 2009 30 52 57 18848723 10.1016/j.biomaterials.2008.09.044

Vignery A Osteoclasts and giant cells: macrophage–macrophage fusion mechanism. Int J Exp Pathol 2000 81 291 304 11168677 10.1111/j.1365-2613.2000.00164.x PMC2517739

Vignery A Macrophage fusion: are somatic and cancer cells possible partners? Trends Cell Biol 2005 15 188 193 15817374 10.1016/j.tcb.2005.02.008

Vural G Hagmar B Walaas L Extracranial metastasis of glioblastoma multiforme diagnosed by fine-needle aspiration: a report of two cases and a review of the literature. Diagn Cytopathol 1996 15 60 65 8807254 10.1002/(SICI)1097-0339(199607)15:1<60::AID-DC12>3.0.CO;2-A

Warburg O The Metabolism of Tumours 1931 New York 327

Warburg O On the origin of cancer cells. Science 1956 123 309 314 13298683 10.1126/science.123.3191.309

Watters JJ Schartner JM Badie B Microglia function in brain tumors. J Neurosci Res 2005 81 447 455 15959903 10.1002/jnr.20485

Waziri A Glioblastoma-derived mechanisms of systemic immunosuppression. Neurosurg Clin N Am 2010 21 31 42 19944964 10.1016/j.nec.2009.08.005

Wood GW Morantz RA Immunohistologic evaluation of the lymphoreticular infiltrate of human central nervous system tumors. J Natl Cancer Inst 1979 62 485 491 216840 10.1093/jnci/62.3.485

Yang L DeBusk LM Fukuda K Fingleton B Green-Jarvis B Shyr Y Matrisian LM Carbone DP Lin Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 2004 6 409 421 15488763 10.1016/j.ccr.2004.08.031

Yates AJ An overview of principles for classifying brain tumors. Mol-Chem-Neuropathol 1992 17 103 120 1329796 10.1007/BF03159986

Yilmaz Y Lazova R Qumsiyeh M Cooper D Pawelek J Donor Y chromosome in renal carcinoma cells of a female BMT recipient: visualization of putative BMT-tumor hybrids by FISH. Bone Marrow Transplant 2005 35 1021 1024 15778726 10.1038/sj.bmt.1704939

Youness E Barlogie B Ahearn M Trujillo JM Tumor cell phagocytosis. Its occurrence in a patient with medulloblastoma. Arch Pathol Lab Med 1980 104 651 653 6893661

Yuan X Curtin J Xiong Y Liu G Waschsmann-Hogiu S Farkas DL Black KL Yu JS Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 2004 23 9392 9400 15558011 10.1038/sj.onc.1208311

Zagzag D Esencay M Mendez O Yee H Smirnova I Huang Y Chiriboga L Lukyanov E Liu M Newcomb EW Hypoxia- and vascular endothelial growth factor-induced stromal cell-derived factor-1alpha/CXCR4 expression in glioblastomas: one plausible explanation of Scherer's structures. Am J Pathol 2008 173 545 560 18599607 10.2353/ajpath.2008.071197 PMC2475791

Zhen L Yufeng C Zhenyu S Lei X Multiple extracranial metastases from secondary glioblastoma multiforme: a case report and review of the literature. J Neurooncol 2010 97 451 457 19898745 10.1007/s11060-009-0044-9

Zimmer C Weissleder R Poss K Bogdanova A Wright SC Jr Enochs WS MR imaging of phagocytosis in experimental gliomas. Radiology 1995 197 533 538 7480707 10.1148/radiology.197.2.7480707