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

    Leanne C. Huysentruyt1

    , Zeynep Akgoc2

    , and Thomas N. Seyfried*2

    1 Department of Medicine, Hematology and Oncology, University of California, SanFrancisco, California, USA2 Department of Biology, Boston College, Chestnut Hill, Massachusetts, USA

    Short running title: Neoplastic macrophages/microglia in GBM

    *Corresponding author:Thomas N. SeyfriedBiology Department, Boston College, Chestnut Hill, MA 02467Phone: 1-617-552-3563Fax: 1-617-552-2011Email: [email protected]

    Keywords: Macrophage, microglia, glioma, glioblastoma multiforme, fusion,phagocytosis

    Abbreviations:CNS central nervous systemGBM glioblastoma multiformeMDSC myeloid derived suppressor cellMNGC multinucleated giant cellPXA pleomorphic xanthoastrocytomaRTG retrogradeTAM tumor associated macrophageMDSC myeloid derived suppressor cell

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    Abstract

    Most malignant brain tumors contain various numbers of cells with characteristics ofactivated or dysmorphic macrophages/microglia. These cells are generally consideredpart of the tumor stroma and are often described as tumor-associated macrophages(TAM). These types of cells are thought to either enhance or inhibit brain tumorprogression. Recent evidence indicates that neoplastic cells with macrophagecharacteristics are found in numerous metastatic cancers of non-CNS origin. Evidenceis presented here suggesting that sub-populations of cells within human gliomas,specifically glioblastoma multiforme (GBM), are neoplastic macrophages/microglia.

    These cells are thought to arise following mitochondrial damage in fusion hybridsbetween neoplastic stem cells and macrophages/microglia.

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    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 mosthighly invasive and metastatic cells within the tumor. It is not clear if similar kinds ofcells are part of the malignant cell population in human glioblastoma multiforme (GBM).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 about 10% of patientssurviving 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 invasivenature of this tumor. GBM invades throughout the brain and often produces multi-centric secondary legions at sites distant from the primary tumor (Laws et al., 1993;Rubinstein, 1972; Scherer, 1940). Complete surgical resection of GBM is extremelyrare. Radiation therapy, which enhances the necrotic microenvironment, often results infurther tissue damage and more aggressive tumors (Kargiotis et al., 2010; Lakka andRao, 2008; Seyfried et al., 2011; Seyfried et al., 2010). Thus, effective therapeuticoptions are desperately needed for GBM patients.

    GBM is classified as either primary or secondary. Primary GBM arises de novo withoutany prior evidence of a low-grade tumor, whereas secondary GBM arises frommalignant progression of a lower grade glioma (Ohgaki and Kleihues, 2009). A definingcharacteristic of GBM is the secondary structures of Scherer which include diffuse

    parenchymal invasion, perivascular growth, subpial surface growth and invasion alongwhite matter tracks (Scherer, 1940; Shelton et al., 2010). While systemic metastasis ofGBM is uncommon due to early patient death, GBM can be extremely metastaticespecially if the neoplastic cells gain access to extraneural sites (Frank et al., 2009;Gotway et al., 2011; Hoffman and Duffner, 1985; Ng et al., 2005; Rubinstein, 1972;

    Taha et al., 2005; Zhen et al., 2010). These findings indicate that GBM is not onlyhighly invasive within the central nervous system (CNS), but can also invade outside theCNS.

    GBM generally contain multiple morphologically diverse cell types that express neural,glial and myeloid markers (Huysentruyt et al., 2008; Rubinstein, 1972; Seyfried, 2001;Wood and Morantz, 1979; Yuan et al., 2004). In fact, mesenchymal cells withcharacteristics of tumor associated macrophages (TAMs) and/or microglia can compriseup to 70% of some GBM (Morantz et al., 1979). It has been difficult to determine with

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    GBM may arise from resident or infiltrating myeloid cells of the tumor stroma that thenbecome neoplastic during disease progression (Huysentruyt and Seyfried, 2010).

    Our hypothesis comes from information on the VM mouse model of GBM which consistsof highly invasive tumors (Shelton et al., 2010). The VM tumors arise spontaneously inthe brains of inbred VM mice (Fraser, 1971; Fraser, 1986; Shelton et al., 2010). Theneoplastic cells in the invasive VM brain tumors express multiple properties ofmacrophages and microglial cells and the VM-M3 and VM-M2 tumors have beenestablished as a model system for human GBM (Huysentruyt et al., 2008; Huysentruytand Seyfried, 2010; Huysentruyt et al., 2009; Shelton et al., 2010). We recently

    reviewed evidence showing that many invasive and metastatic human cancers alsoexpress multiple properties of mesenchymal myeloid cells (Huysentruyt and Seyfried,2010). If many non-neural metastatic tumors might arise from mesenchymal myeloidtype cells, what about malignant tumors of the CNS? The goal of this commentary is tohighlight similarities between myeloid cells and the invasive cell populations of GBM,and to discuss possible mechanisms by which neoplastic cells could express theseproperties.

    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 (Huysentruytand Seyfried, 2010; Munzarova and Kovarik, 1987; Pawelek, 2000; Pawelek andChakraborty, 2008; Rachkovsky et al., 1998; Vignery, 2005). Although large numbersof cells with macrophage and myeloid properties are found in most malignant cancers

    including GBM, these cells are generally considered part of the tumor stroma and notpart of the neoplastic population (Mantovani et al., 2002; Seyfried, 2001). These cellsare often referred to as tumor-associated macrophages (TAM), and are thought tofacilitate tumor development and malignant progression (Bingle et al., 2002; Lewis and

    Pollard, 2006; Pollard, 2008; Seyfried, 2001; Talmadge et al., 2007). The tumor stromais a complex microenvironment that is generally comprises malignant tumor cells andhost infiltrating cells that include immune and epithelial cells (Bissell and Hines, 2011).The most commonly accepted view is that tumor-derived chemoattractants signal

    monocytes to extravasate out of the bloodstream and infiltrate the tumors mass(Bottazzi et al., 1983; Murdoch et al., 2004). Once in the tumor microenvironment,enhane inflammation and angiogenesis establish the pre-metastatic niche, thuscontributing to tumor progression (Bingle et al., 2002; Lewis and Pollard, 2006; Pollard,2008; Seyfried, 2001; Talmadge et al., 2007). However, little consideration has beengiven to the possibility that microglial or TAM subsets might actually be part of the

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    (Burke and Lewis, 2002; Gordon, 1999; Huysentruyt et al., 2008; Stossel, 1999).Macrophages fluctuate between two activation states depending upon the

    microenvironment. Macrophages can become classically activated (M1 activation) inresponse to pathogens and pro-inflammatory molecules resulting in generation of nitricoxide and reactive oxygen species (Biswas et al., 2008; Mantovani and Sica, 2010;Qian and Pollard, 2010; Sica et al., 2002; Sica et al., 2006). In contrast, macrophagescan become alternatively activated (M2 activation) in response to anti-inflammatorymolecules such as IL-4 and IL-10 and are the major wound-healing cell (Biswas et al.,2008; Gordon, 2003). Interestingly, the two macrophage subpopulations play separateroles in tumorigenic processes (Biswas et al., 2008). M1 macrophages, located at sites

    of chronic inflammation, contribute to neoplastic transformation through the release ofcytotoxic and DNA damaging pro-inflammatory molecules (Biswas et al., 2008).Macrophages with an M2 phenotype contribute to angiogenesis and metastasis withinthe established tumor (Biswas et al., 2008; Huysentruyt and Seyfried, 2010; Mantovaniand Sica, 2010; Mantovani et al., 2002; Sica et al., 2006). However recent evidencesuggests that TAMs may be a more complex subtype that can share features of bothactivation states (Ojalvo et al., 2009; Ojalvo et al., 2010; Qian and Pollard, 2010).

    Ineffective antitumor immunity resulting from reduced T cell function is a hallmark ofmany cancers including GBM (Raychaudhuri et al., 2011; Waziri, 2010). Recently it hasbeen suggested that another myeloid cell type, myeloid-derived suppressor cells(MDSCs), are partially responsible for GBM-related immune suppression((Raychaudhuri et al., 2011)). MDSCs are a heterogeneous population of immaturemyeloid cells that accumulate in tumor bearing hosts where they suppress T cellfunction resulting in ineffective anti-tumor immunity (Gabrilovich and Nagaraj, 2009;Ostrand-Rosenberg, 2010; Raychaudhuri et al., 2011). This cell type accumulates inthe blood, lymph nodes and spleen in patients with solid tumors and has been reportedto represent approximately 5% of the total tumor mass in various experimental tumormodel systems (Yang et al., 2004). Raychaudhuri et aldemonstrated that patients withGBM have increased MDSC counts in their blood when compared to healthy controlsand these cells likely promote T cell immune suppression in this patient population(Raychaudhuri et al., 2011). While the phenotype and function of MDSCs is believed to7be distinct from those of TAMs, MDSCs can differentiate into TAMs within the tumor

    microenvironment thus further contributing to tumor progression (reviewed in(Gabrilovich and Nagaraj, 2009)). To our knowledge, it has not been demonstrated ifMDSCs contribute to GBM tumor mass.

    Phagocytosis: a defining behavior of macrophages and invasive cells

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    through the extracellular matrix and into surrounding tissues (Bjerknes et al., 1987;Coopman et al., 1998).

    The phagocytic/cannibalistic behavior of tumor cells was first described over a centuryago 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 microorganisms use cannibalism as a feedingmechanism, cellular cannibalism is an exclusive property of malignant tumor cells (Fais,2007). Both human and murine cancers have been shown to phagocytose tumor cells,

    erythrocytes, leukocytes, platelets, apoptotic cells, and extracellular particles (reviewedin (Huysentruyt and Seyfried, 2010)). Interestingly, phagocytic tumor cells are observedin malignant gliomas, especially in GBM (Figure 1) (Bjerknes et al., 1987; Chang et al.,2000; Nitta et al., 1992; Persson and Englund, 2009; van Landeghem et al., 2009;Youness et al., 1980; Zimmer et al., 1995).

    We previously identified two spontaneous brain tumors (VM-M2 and VM-M3) in theinbred VM mouse strain that are highly invasive in brain (Huysentruyt et al., 2008;

    Shelton et al., 2010). These VM brain tumors express multiple properties ofmacrophages including phagocytic activity and are a model for human GBM(Huysentruyt et al., 2008; Shelton et al., 2010). The phagocytic phenotype in thesecells 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 thephagocytic activity of four rat glioma cell lines with varying degrees of in vivoinvasiveness, Bjerknes et aldemonstrated that the invasive glioma cells phagocytosedbacteria, red blood cells, zymosan particles and glia cell fragments (Bjerknes et al.,1987). Furthermore, the two most invasive rat glioma cell lines displayed the highestlevel of phagocytosis. The authors noted that the phagocytic behavior correlated withthe amount of rat brain destruction during tumor cell invasion, suggesting thatphagocytic activity may be connected to the excretion of lysosomal enzymes (Bjerkneset al., 1987). Lysosomal enzyme secretion and phagocytosis are macrophage-specificbehaviors. Invasive glioma cell lines that display macrophage/microglia-like behaviorssuggest a myeloid origin. Chang et alalso demonstrated that the glioma cell lines U87,

    U251, and SF268 exhibit phagocytic behavior against apoptotic glioma cells (Chang etal., 2000). These findings further demonstrate that invasive glioma cells can exhibitmacrophage-like phagocytic behavior.

    Although astrocytes have been described as semi-professional phagocytes, theirphagocytic ability is limited. It could be suggested that the phagocytic behavior

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    GBM cell population (Persson and Englund, 2009). These findings support thehypothesis that neoplastic macrophages/microglia can exist within GBM.

    In addition to GBM, other invasive CNS tumors contain neoplastic phagocytic cells. Forexample, phagocytic cells were observed in the bone marrow of a patient a highlyinvasive and metastatic medulloblastoma, which involved invasion and metastasis tobrainstem, bone marrow, lymph nodes and spinal cord (Youness et al., 1980). Afterextensive characterization of the phagocytic cells via histopatholgy, electron microscopyand cytogenics, the authors stated that the phagocytic cells were unquestionablymetastatic medulloblastoma cells. Interestingly, this tumor metastasized outside the

    CNS after excision of the primary tumor from the right cerebellar region (Youness et al.,1980). While extracranial metastasis of CNS tumors is uncommon, invasive CNStumors can metastasize throughout the body if they gain access to extraneural sites(Hoffman and Duffner, 1985; Taha et al., 2005; Vural et al., 1996). In fact, severalreports show that GBM can be highly metastatic (Frank et al., 2009; Gotway et al.,2011; Hoffman and Duffner, 1985; Ng et al., 2005; Rubinstein, 1972; Taha et al., 2005;

    Zhen et al., 2010). It therefore appears that his case of metastatic medulloblastomaexhibited the macrophage/microglia characteristics seen in some GBM.

    While it might be difficult to demonstrate a myeloid origin of invasive GBM, the evidencereviewed here suggests that subpopulations of neoplastic GBM cells display thephagocytic behavior of macrophages/microglia. As microglia are the residentmacrophages of the brain, we propose that subpopulations of the malignant GBM cellscould arise from microglia/macrophages (Morantz et al., 1979; Seyfried, 2001). HumanGBM contain mixtures of numerous neoplastic cell types, many of which have

    mesenchymal properties, do not express GFAP, and are of unknown origin (Duffy,1983; Han et al., 2010; Ohgaki and Kleihues, 2009). Indeed, the original nineteenthcentury observations of Virchow (1863/1865) described glioblastomas as gliosarcomasof mesenchymal origin (Scherer, 1940; Zagzag et al., 2008). While numerousmesenchymal cells are frequently seen in GBM, the specific classification of all tumorcell types within human GBM remains ambiguous at best (Fan et al., 2007; Tso et al.,2006; Yates, 1992). According to our hypothesis, some of these neoplasticmesenchymal cells could arise from transformed macrophages/microglia.

    Novel therapeutic approaches to GBM management become possible if the phagocyticbehavior can be targeted. Gollapudi and colleagues demonstrated that tumor cellsundergo apoptosis after feeding tumor cells anti-tumor agents (Ghoneum andGollapudi, 2004; Ghoneum et al., 2005; Ghoneum et al., 2008; Ghoneum et al., 2007).They showed that various tumor cells, grown either in vitro or in vivo, underwent a

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    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 transformedmacrophages/microglia as well as non-neoplastic TAM.

    Fusogenicity

    Fusogenicity involves the merging of two distinct plasma membranes and is a definingcharacteristic of macrophages and microglia (Duelli and Lazebnik, 2003; Huysentruytand Seyfried, 2010; Vignery, 2000). Cell fusion is a highly regulated process that is

    essential for fertilization, skeletal muscle and placental formation (Duelli and Lazebnik,2003). Outside of normal developmental processes, fusion events are normallyrestricted to cells of myeloid origin (reviewed in (Duelli and Lazebnik, 2003)).Macrophages often fuse during osteoclast formation and wound healing in orderincrease cell volume to facilitate engulfment of large extracellular materials (Vignery,2000; Vignery, 2005). Macrophages also undergo heterotypic fusions with numerouscell types. It is currently believed that macrophage heterotypic fusion is a mechanismby which macrophages can heal a defective cell or tissue (Camargo et al., 2004;

    Camargo et al., 2004; Powell et al., 2011; Vignery, 2000; Vignery, 2005). There is alsoconsiderable evidence demonstrating that macrophages fuse with tumor cells (reviewedin (Huysentruyt and Seyfried, 2010)). These properties would also be expected inmicroglia since microglia are the resident macrophages of the CNS.

    Nearly a century ago, Aichel first suggested that fusions between somatic cells andleukocytes could result in aneuploidy and malignant hybrids (reviewed in (Rachkovskyet al., 1998)). Sixty years later, Mekler and Warner proposed that fusions betweenmyeloid cells and tumor cells would produce daughter cells endowed with the invasiveproperties of the myeloid cell as well as the unlimited proliferative potential of the tumorcell (reviewed in (Rachkovsky et al., 1998)). The fusion hybrid hypothesis has emergedas a credible alternative to the epithelial mesenchymal transition for the origin ofinvasive and metastatic cancers (Huysentruyt and Seyfried, 2010; Pawelek andChakraborty, 2008). Indeed, Pawelek and co-workers showed that fusions betweennon-metastatic cells and macrophages result in a cells with the ability to invade and

    metastasize (Chakraborty et al., 2004; Chakraborty et al., 2001; Chakraborty et al.,2000; Handerson et al., 2005; Pawelek, 2000; Pawelek, 2005; Pawelek andChakraborty, 2008; Rachkovsky and Pawelek, 1999; Rachkovsky et al., 1998; Yilmaz etal., 2005). Additionally, they provided evidence demonstrating that these fusion eventsoccur spontaneously both in vitro and in vivo in a variety of animal models and in humancancers (Chakraborty et al., 2004; Chakraborty et al., 2000; Rachkovsky et al., 1998;

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    GBM is composed of neoplastic cells with features that vary from small round cells tomultinucleated giant cells (MNGCs) (Homma et al., 2006). MNGCs arise from the

    fusion of two or more macrophages (Vignery, 2000; Vignery, 2005). Recent reportssuggest that MNGCs in GBM could arise through fusions of peripheral macrophages ormicroglia with glioma cells (Alvarez-Dolado et al., 2003). A recent study demonstratedthat CD98, a protein that regulates monocyte fusion events, was expressed on thesurface of MNGCs in 15/16 GBMs (Figure 2B) (Takeuchi et al., 2008). These findingssuggest that MNGC formation in GBM is similar to what occurs during normalmonocyte/macrophage fusion events (Takeuchi et al., 2008). Microglia and bonemarrow derived cells can fuse with CNS cells resulting in MNGCs (Ackman et al., 2006;

    Alvarez-Dolado et al., 2003). The MNGCs in GBM often express GFAP, a proteinexpressed on CNS stem cell and astrocytes, suggesting that this cell populationcontains macrophage/microglia and CNS stem cell/astrocyte hybrids (Takeuchi et al.,2008). Patient prognosis is generally worse in when GBMs contain large numbers ofMNGCs and other macrophage-like cells when compared to GBMs containing fewer ofthese cells (Deininger et al., 2003). This situation is similar to that seen in non-CNStumors in that prognosis is generally worse for malignant tumors with higher than lowernumbers of macrophages (Homma et al., 2006; Huysentruyt and Seyfried, 2010; Leek

    et al., 1996; Shabo et al., 2009).

    The large size and unique phenotype of MNGCs make them easily detectable in braintumor biopsy specimens (Figure 1B) (Rubinstein, 1972). However, it can be difficult todetect fusion events among tumor cells and myeloid cells with subsequent nuclearfusions. Utilizing a double immunostaining technique, Deiningeret alidentified a subsetof glioma cells that were positive for both GFAP and allograft inflammatory factor 1(AIF1), a microglial marker also referred to as Iba1 (Deininger et al., 2000).GFAP+/AIF1+ cells were localized near areas of tumor growth and were indicative ofspontaneous in vivo fusion events (Deininger et al., 2000). It is well documented thatTAM aid tumor progression by promoting angiogenesis, invasion and metastasis(reviewed in (Lewis and Pollard, 2006; Seyfried, 2001)). However, fusion eventsbetween tumor cells and tissue macrophage/microglia would also accelerate tumorprogression since resulting daughter cells would inherit the invasive/migratory potentialof microglia and unlimited proliferative potential of the tumor cell. Hence, a

    microglial/glioma cell hybrid could rapidly acquire an invasive phenotype in the absenceof novel mutations.

    The macrophage-like characteristics and invasive properties of the murine VM-M2 andVM-M3 brain tumors are more similar to human GBM than to most previously describedexperimental mouse brain tumor models (Shelton et al., 2010). If fusion hybridization of

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    and Seyfried, 2010)). It would therefore be important to consider the possibility thatsome GBM cell subpopulations, previously identified as TAM, are in fact neoplastic

    cells.

    Like GBM, pleomorphic xanthoastrocytoma (PXA) contains cells of multiplemorphologies (Matyja et al., 2003). As in GBM, MNGC are also a characteristic celltype in PXA (Matyja et al., 2003). In an attempt to further characterize this cell sub-type, Matyja et al., evaluated the co-expression of glial and macrophage markers in thevarious tumor cell populations in eight cases of PXA (Matyja et al., 2003). The authorsidentified 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 werelarge 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 includingGBM (Leenstra et al., 1995; Rossi et al., 1987; Strojnik et al., 2006). Leenstra et alexamined macrophage characteristics in six malignant glioma cell lines (Leenstra et al.,1995). All the cell lines examined were classified as malignant gliomas based upon

    DNA flow cytometry and by GFAP expression. Interestingly, all six glioma lines co-expressed macrophage markers and GFAP. In contrast, macrophage antigens werenot found in cultured astrocytes isolated from healthy brain tissue suggesting that themacrophage markers expressed by malignant astrocytes were not artifacts of an in vitroculture environment (Leenstra et al., 1995). In a separate study, immunohistochemicalanalysis revealed that 40% of grade III astrocytomas and GBM tumor specimenscontained neoplastic cells with macrophage markers (Rossi et al., 1987). The U87GBM model has also been shown to express both CD68 (Figure 3B) and glia markerswhen grown either in vitro orin vivo (Strojnik et al., 2006). Strojnik et alevaluated theprognostic significance of CD68 expression in malignant human gliomas (Strojnik et al.,2009). It was clear form their study that the presence of high levels of CD68-positivetumor cells was predictive of reduced survival. CD68 was expressed by both microgliaand tumor cells (Strojnik et al., 2009). The macrophage/microglia marker, Iba-1, is alsoexpressed by the VM-M2 mouse GBM tumor 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 tumorcells include neoplastic cells with multiple characteristics of macrophages/microglia.

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    tumor cells may be of mesenchymal origin. Indeed, these observations led to ourrecent prediction that the metastatic cells in most human cancers are of myeloid origin

    (Huysentruyt and Seyfried, 2010). As microglia are mesenchymal cells of myeloidorigin, we suggest that neoplastic microglia can represent an invasive cell population inGBM. This shared property amongst numerous invasive/metastatic tumors could havebroad therapeutic implications. For example, any therapy that is able to reduce invasionand metastasis of one tumor type would likely be effective in treating tumors withmacrophage characteristics.

    Possible mechanisms

    How could resident microglia or TAM become part of the neoplastic cell population inGBM? We recently reviewed evidence indicating that all cancer, regardless of tissue orcellular origin, is primarily a disease of impaired cellular energy metabolism (Seyfriedand Shelton, 2010). Otto Warburg first proposed that all cancers arise from irreversibledamage to cellular respiration (Warburg, 1931; Warburg, 1956). Persistent injury tooxidative phosphorylation will require compensatory non-oxidative energy metabolism tomaintain viability (Seyfried and Shelton, 2010; Seyfried et al., 2010; Shelton et al.,

    2010). The mitochondrial stress response or retrograde signaling (RTG) from themitochondria to the nucleus is required to upregulate the oncogenes needed to sustainglycolysis and non-oxidative mitochondrial energy production (Seyfried and Shelton,2010; Seyfried et al., 2010). However, persistent RTG activation leads to eventualnuclear genomic instability and other recognized hallmarks of cancer (Butow and

    Avadhani, 2004; Seyfried and Shelton, 2010; Singh et al., 2005). Mitochondria energyproduction is often damaged as a consequence of mutagens, hypoxia, inflammation,reactive oxygen species or inherited mutations (Kiebish et al., 2008). We recentlyshowed that mitochondria isolated from murine gliomas contain numerous mitochondriallipid defects supporting previous findings that respiratory energy production isdysfunctional in tumor mitochondria (Kiebish et al., 2008; Kiebish et al., 2009; Ordys etal., 2010; Seyfried and Shelton, 2010). Macrophages/microglia hone to mitochondria-damaging environments in response to inflammation, infection, would repair, andtumorigenesis (Bingle et al., 2002; Chettibi, 1999; Giulian et al., 1989; Graeber et al.,2002; Lewis and Murdoch, 2005; Lewis and Pollard, 2006; Martin and Leibovich, 2005).

    Inflammation and hypoxia in the tumor microenvironment can damage macrophagemitrochondria (Frost et al., 2005; Navarro and Boveris, 2005). It is therefore possiblethat mitochondria could become dysfunctional when macrophages/microglia respond tovarious chronic tissue injuries, resulting in a persistent RTG response with eventualmalignant transformation (Seyfried and Shelton, 2010). Hence, we suggest that someinvasive GBM cells could arise from resident tissue microglia or TAM that have suffered

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    Although normal mitochondria are known to suppress tumorigenesis in fusion hybrids(Seyfried and Shelton, 2010), persistent inflammation in the tumor microenvironment

    could contribute to continued mitochondrial damage in the fused hybrids. This wouldenhance the Warburg effect, unbridled proliferation, and genomic instability. Asmicroglia naturally embody the ability to migrate throughout the brain (Amat et al., 1996;Graeber and Streit, 1990), we suggest that transformed cells of macrophage/microgliaorigin would possess invasive potential.

    Currently, the cause of macrophage and tumor cell hybridization is unknown. Variousstages of the macrophage response to tumor development could increase the

    probability for cell fusion events. The host immune system treats tumors as unhealedwounds (Bissell and Hines, 2011; Dvorak, 1986; Seyfried, 2001). Macrophages hometo hypoxic tumor areas in the process of wound healing. The failure of macrophages tocompletely digest apoptotic cells could result in macrophage x tumor hybrid cells(Pawelek, 2000). It is therefore possible that macrophages fuse with tumors cells in anattempt to heal the tumor, as macrophages are known to fuse with non-myeloid cellsduring tissue repair. Radiation is commonly used as a primary therapy for most braintumors. However, radiation therapy enhances the incidence of fusion hybrid formation

    and could result in a more aggressive and invasive tumors (Seyfried et al., 2011; Shaboet al., 2009). This could account in part for the exacerbating effects of radiation therapyon GBM progression (Seyfried et al., 2010).

    The macrophage fusion hybrid hypothesis could potentially explain many of thedistinguishing characteristics seen in invasive and metastatic GBM tumor cells. Thesemacrophage-reprogramming strategies may account for some of the cellularheterogeneity seen in human GBM since macrophage x CNS cell hybrids would likelyretain the histology of the CNS fusion partner. Tumor hybrids could also account for thecancer cell aneuploidy and chromosomal abnormalities often seen in tumor cells(Pawelek, 2000; Steinhaus, 1981). Further studies will be needed to determine theextent to which fusion hybridization might contribute to the invasive properties seen inGBM

    Concluding remarks

    Glioblastoma multiforme is a highly complex and lethal tumor. Unfortunately, thecurrent therapies available to GBM patients are largely ineffective and often have anegative impact on the patients quality of life. In this commentary, we aim to highlightthe possibility that the most aggressive and invasive cells in human GBMs areneoplastic macrophages/microglia. While further experimentation is needed to confirmthe gene expression and immunohistochemical data reviewed here, we believe that

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    This work was supported in part from NIH grants (HD-39722, NS- 55195, and CA-102135), a grant from the American Institute of Cancer Research, and the Boston

    College Expense Fund.

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    Figure legends

    Figure 1. Phagocytic behavior of malignant glioma cells. A) Phagocytic behavior of theVM-M2 and macrophage RAW 264.7 cell lines was assessed from merging (Merge) thefluorescence (Fl) images and the differential interference contrast images after feedingthe cells florescent beads. The image indicated that the malignant VM-M2 cells werephagocytic. B) Multinucleated giant cell (MNGC) in a human GBM containing engulfedcell debris (arrow). Reproduced with permissions: panel A (Huysentruyt et al., 2008)

    panel B (Persson, 2008).

    Figure 2. Fusogenic properties of malignant glioma cells. A) The fusion hybridhypothesis suggests that a macrophage x tumor cell hybrid will express genetic andfunctional traits of both parental cells. B) A bi-nucleated cell in human GBM expressingthe macrophage fusion protein CD98 (arrows). C) Human pleomorphicxanthoastrocytoma (PXA) demonstrating double immunoreactivity of the glial markerGFAP (brown) and macrophage CD68 (red). Reproduced with permissions: panel B(Takeuchi et al., 2008) panel C (Matyja et al., 2003).

    Figure 3. Malignant glioma cells express macrophage-specific antigens. A) Expressionof the macrophage marker CD68 (Ki-M1P) in the the human GBM cell line U138 (X475).B) CD68 expression in most tumor cells of a human GBM (X400). C) Expression of themacrophage/microglia marker Iba-1 in tumor cells of the VM-M2 tumor, a mouse modelof human GBM (X630). Reproduced with permission A (Paulus et al., 1992) B (Strojniket al., 2009) C (Huysentruyt et al., 2008).

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    Table 1. Malignant glioma cell expression of myeloid antigens

    Myeloid Antigen Reference

    CD98 (Takeuchi et al., 2008) AIF-1 (Iba-1) (Deininger et al., 2000;

    Huysentruyt et al.,2008)

    CD14 (Deininger et al., 2003)HLA-class II (Matyja et al., 2003;

    Rossi et al., 1987)CD68 (Huysentruyt et al.,

    2008; Leenstra et al.,1995; Matyja et al.,2003; Strojnik et al.,2006; Strojnik et al.,

    2009)CD11b (Huysentruyt et al.,

    2008; Leenstra et al.,1995)

    HAM56 (Leenstra et al., 1995)

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