Tumor-Associated Macrophages (TAMs) Form an Interconnected Cellular Supportive Network in Anaplastic Thyroid Carcinoma

Background A relationship between the increased density of tumor-associated macrophages (TAMs) and decreased survival was recently reported in thyroid cancer patients. Among these tumors, anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumors in humans. TAMs (type M2) have been recognized as promoting tumor growth. The purpose of our study was to analyze with immunohistochemistry the presence of TAMs in a series of 27 ATC. Methodology/Principal Findings Several macrophages markers such as NADPH oxidase complex NOX2-p22phox, CD163 and CD 68 were used. Immunostainings showed that TAMs represent more than 50% of nucleated cells in all ATCs. Moreover, these markers allowed the identification of elongated thin ramified cytoplasmic extensions, bestowing a “microglia-like” appearance on these cells which we termed “Ramified TAMs” (RTAMs). In contrast, cancer cells were totally negative. Cellular stroma was highly simplified since apart from cancer cells and blood vessels, RTAMs were the only other cellular component. RTAMs were evenly distributed and intermingled with cancer cells, and were in direct contact with other RTAMs via their ramifications. Moreover, RTAMs displayed strong immunostaining for connexin Cx43. Long chains of interconnected RTAMs arose from perivascular clusters and were dispersed within the tumor parenchyma. When expressed, the glucose transporter Glut1 was found in RTAMs and blood vessels, but rarely in cancer cells. Conclusion ATCs display a very dense network of interconnected RTAMs in direct contact with intermingled cancer cells. To our knowledge this is the first time that such a network is described in a malignant tumor. This network was found in all our studied cases and appeared specific to ATC, since it was not found in differentiated thyroid cancers specimens. Taken together, these results suggest that RTAMs network is directly related to the aggressiveness of the disease via metabolic and trophic functions which remain to be determined.


Introduction
Anaplastic thyroid carcinoma (ATC) represents less than 5% of all thyroid cancers and is one of the most aggressive malignancies in humans [1][2][3]. Despite multimodality therapeutic approaches, ATC still carries a dismal prognosis and new treatment, based on a better knowledge of the pathogenesis and progression of this cancer, are therefore required.
Anaplastic carcinoma arises from thyroid follicular cells and is characterized by atypical cells with large, bizarre nuclei with numerous atypical mitotic figures that resemble polymorphic mesenchymal sarcoma. There is no thyroid or epithelial differentiation. This explains why the macrophage component, termed ''tumor-associated macrophages'', (TAMs), which is closely mixed with cancer cells, was only recently recognized [4,5]. Among TAMs, M1 macrophages which display ''classic'' activation are separated from ''alternative'' M2 macrophages [6][7][8][9][10]. It is well established that the main function of M1 is phagocytosis in response to bacterial stimuli and/or Th1 cytokines while the main function of M2 is immunosuppression and trophic activity in response to Th2 cytokines (e.g. IL10, TGFbeta) [11]. In thyroid cancers, an increased density of TAMs was associated with decreased survival, reflecting their M2 nature [5].
In the present study, we report on a very dense and diffuse intratumor infiltration of ATC by TAMs. These TAMs organize themselves in an interconnected network in close contact with cancer cells and blood vessels.

Clinical and Histological data
Twenty-seven patients who were treated for ATC at Institut Gustave-Roussy from 1998 to 2007 were identified. All these patients died within 1 year after diagnosis. Routine histological sections stained with hematoxylin and eosin were reviewed to confirm the diagnosis and corresponding paraffin-embedded tissue blocks were obtained for immunohistochemical studies. The protocol was approved by Institutional Review Board at the Institut Gustave-Roussy and all patients gave their written informed consent.
-NOX2 is often referred to as the phagocyte NADPH oxidase and is still widely considered to have a limited, essentially phagocyte-specific tissue expression [12]. -P22phox forms a heterodimer with NOX2 that is located at the cell surface membrane [12]. -CD163 is expressed on most subpopulations of mature tissue macrophages [13] and is linked to anti-inflammatory macrophage functions [14]. -CD68 has long been used as a specific macrophage marker [15], but is currently no longer considered a specific marker for macrophages but rather an antigen indicative of lysosomes [16]. -Cx43 (connexin 43) is expressed by several types of cells including macrophages [17,18] and is one of 21 members of the human connexin family. It is the most widely distributed gap junction isoform and enables the intercellular passage of numerous small molecules ranging from ions to much larger metabolites [18][19][20]. Subcellular Cx43 immunostaining correlates with protein synthesis in endoplasmic reticulum, the golgi apparatus, cytoplasmic transport in small vesicles and finally on cell surface membrane [21,22]. -Glut-1 is a glucose transporter that mediates cellular glucose uptake. It is inducible in monocytes and macrophages [23]. -CD34 is a marker of vascular endothelial cells [24]. -Alpha-Smooth Muscle Actin (Alpha-SMA) is present in ''activated fibroblasts'' or myofibroblasts [25] -CD3 recognizes lymphocyte T cells [26]. -Ki67 nuclear expression is associated with cell proliferation [27].
The origin, characteristics and conditions of use of these antibodies are summarized in table 1.

Determination of percentage of TAMs among nucleated cells
TAMs were counted by surface unit of histological section on digitalized photos with the same format and at the same original magnification (6100) with the help of ImageJ software (National Center for Biotechnology Information). For each case, four microscopic fields were digitalized; two with NOX2 immunostaining and two with CD163 immunostaining. The number and percentage of stained and unstained nucleated cells were calculated. To avoid any overestimation of the number of TAMs which could have been due to extended cytoplasmic ramifications, we counted only cells with a visible nucleus (Figs. 1-3, S1). TAMs in  the cellular cycle by double immunostaining with Ki67  and P22phox antibodies See Text S1.

Transmission electron microscopy
Three cases of ATC were obtained for electron microscopy study (See Text S1).

Clinical data
Gender, age at the time of the diagnosis and survival of each patient are summarized in table 2.

NOX2, CD163 and CD68 immunostainings
TAMs displayed strong staining for NADPH oxidase NOX2, CD163 and CD 68 which clearly contrasted with cancer cells that were entirely negative for each of these markers. All ATC cases displayed a high density of TAMs that were diffusely dispersed within the tumor (Fig. 1). Furthermore TAMs and cancer cells could also be distinguished on morphological grounds. TAMs nuclei were small, regular and ovoid, whereas cancer cell nuclei were 5 to 10fold larger, irregular and dystrophic (Figs. 1, 2A and 3). At the subcellular level, the strong staining obtained with all these markers, allowed us to study their cytoplasmic extensions. Three types of subcellular stainings were observed: a/ diffuse cytoplasmic staining ; b/ granular staining with small vesicles located in the cell body and/ or in the cytoplasmic ramifications; c/ plasma membrane staining (Figs. 1,3 and S2). All these three types of staining were found in all the tumors according to the observed area.

P22phox immunostaining
p22phox is required for the formation of a functionally active NADPH oxidase. Strong p22phox staining, similar to NOX2 staining, was found in all ATCs (Fig. 1D). However, in some areas of few cases, there was strong NOX2 positivity contrasting with weak p22phox staining. All cancer cells were negative for p22phox.

Number and percentage of TAMs
In the 27 ATC studied, the mean proportion of TAMs was 57% of the total number of cells with a visible nucleus ( Table 2). In fact, this percentage is likely to be a wide underestimation of the real percentage of TAMs since TAM nuclei were 5 to 10-fold smaller than cancer cell nuclei and the probability of cutting these nuclei on a 2D histological section is 5 to 10 times smaller than that of cutting cancer cell nuclei (Fig. 3).
Shape and subcellular NOX2, CD163 and CD68 staining of TAMs Strong NOX2, CD163 and CD 68 stainings of TAMs allowed us to describe the following characteristics that were not visible with the usual hematoxylin and eosin staining: 1/ TAMs displayed very elongated cytoplasmic processes (up to 200 microns apart from the cell body); 2/ staining in these processes was often irregular with a moniliform pattern; 3/ these processes could divided in several more or less thinner branches; 4/ at their extremity, they often exhibited a small cytoplasmic 'button shaped'' enlargement; 5/ these extremities were in close contact with other TAMs, cancer cells and blood vessels; 6/ TAMs displayed no or very few intracytoplasmic phagocytic materials (Figs. 1, 2A, 3 and S2). Electron microscopy revealed cytoplasmic extensions of TAMs and close association with cancer cells (Fig. 4). We propose to call these TAMs ''Ramified TAMs'' (RTAMs). These features are in contrast to those of ''classic'' macrophages which present a more or less regular amoeboid round shape without major cytoplasmic extensions (Fig. 2B). Moreover this type of macrophages often presents intracellular phagocytized material.

RTAM network
On NOX2, P22phox, CD163 and CD68 stainings, most TAMs displayed direct contact with other TAMs, forming networks of connected cells which could be tracked at least partially on 2D histological sections. These cellular networks connected TAMs to each other, enfolding dispersed isolated cancer cells and often in direct contact with them (Figs. 1, 3 and 4).

Connexin 43 (Cx43) immunostaining
In normal thyroid tissue, Cx43 staining was localized to characteristic punctuate structures at sites of cell-cell apposition (Fig. 5A). Cx43 positive staining was observed in all ATCs, in the majority of RTAMs and blood vessels and in a lower percentage of   6). Unlike in normal thyroid tissue, the staining was localized in the cytoplasm, the golgi area, small vesicles and/or characteristic intercellular punctuate structures suggesting a synthesis of CX43 and a more rapid turnover of the protein in ATC than in normal differentiated thyroid tissue [21]. Long chains of connected Cx43-positive RTAMs could be recognized and tracked within the tumor (Fig. 6). Moreover, it was possible to detect the Cx43 connection between positive blood vessels and RTAMs (Fig. 5D). At electron microscopy intercellular junctions consistent with gap junctions were found between RTAMs and between RTAMs and cancer cells (Fig. 7).

Blood vessels
In well differentiated thyroid carcinoma immunostaining of blood vessels by CD34 displayed vasculature in close association with epithelial cancer cells. Almost each cancer cell is in direct contact with a blood vessel (Fig. 8B). In contrast, in ATC, vasculature was much more disorganized and anaplastic cancer cells could be localized in avascular areas at a distance of more than 150 microns from blood vessels (Fig. 8A). RTAMs were often located in clusters around blood vessels separating these vessels from cancer cells (Fig. 9), and long connected chains of RTAMs were infiltrating the avascular tumor tissue (Fig. 10).

GLUT1 immunostaining
GLUT1 was positive in red blood cells and vessels. In some cases and in some areas, RTAMs were positive for GLUT1 whereas the great majority of cancer cells were negative for GLUT1 (Fig. 11).

Single Ki67 immunostaining
In the majority of tumors, the proportion of Ki67-positive nuclei was around 50% (Table 2).

Double Ki67-p22phox immunostaining
This staining allowed recognizing four types of cells on the same section: Ki672 P22phox2 and Ki67+ P22phox2 cancer cells; Ki672 P22phox+ and Ki67+ and P22phox+ TAMs (Figs. 12 and S3). Each type of cells was counted with the help of the ImageJ software in the same way as for determining the whole percentage of TAMs (see Table 2). It appears that the mean proliferative rate of RTAMs was estimated to be 57% and the mean proliferative rate of cancer cells was estimated to be 45%.

Alpha-SMA immunostaining
The staining was restricted to pericytes around blood vessels. All the other cells were negative. (Data not shown).

CD3 immunostaining
This staining demonstrated an absence or very rare presence of CD3+ T lymphocytes (less than 0, 01%) (Data not shown).

F-FDG PET/CT
All the patients suffering of ATC and studied at the institute Gustave-Roussy showed high to very intense FDG uptake. [28] (Fig  S4).

Discussion
Nowadays, TAMs are classified in several subcategories according to different phenotypes that are dependent upon the type of tumors and microenvironments [29,30]. In this study, we used NOX2, P22phox, CD163, CD68 and CD34 immunostainings to reveal and to characterize TAMs in ATC. With the use of these well recognized markers, it appeared that ATC tissue harbored only three types of cells: NOX2+ P22phox+ CD163+ CD68+ CD342 macrophages; NOX22 P22phox2 CD1632 CD682 CD342 cancer cells and NOX22 CD1632 CD682 CD34+ blood vessel endothelium. Other cell types, such as lymphoid cells, fibroblasts and/or myofibroblasts were absent or very rare. Moreover ATC cancer cells were totally negative for all of these markers. This study confirms the presence of a high number of TAMs in most ATC that represent the majority of intra-tumor nucleated cells. Furthermore immunohistochemistry allowed us to accurately describe the morphology and the organization of TAMs.
Then we studied the ramified morphology of these TAMs and CX43 expression: TAMs in ATC displayed long and thin cytoplasmic processes which could extend up to 150 microns from the cell body. More often, these elongated processes were irregular and moniliform containing small stained vesicles, and were usually divided into several branches conferring a ''microglia-like'' appearance ( Figs. 2A,3,4,S2). We propose to call them ''Ramified TAMs'' or ''RTAMs''. RTAMs were in direct contact with other RTAMs, with cancer cells and with blood vessel endothelium. We suggest that these ramifications correspond to specific functions of type M2 TAMs and contrast to the absence of ramifications and the amoeboid shape of classic type M1 macrophages (Fig. 2B). Our hypothesis is that these cytoplasmic extensions of RTAMs may permit ''cross talk'' and molecular transfers between RTAMs and cancer cells. This ''symbiotic'' feature has been considered as a requirement for tumor aggressiveness [31].
Although differently expressed, CX43 was observed in the three cell types and at their interfaces. The great majority of RTAMs displayed CX43 positivity in the cytosol, in small vesicles either located in the cell body or along cytoplasmic processes and at appositional membrane areas with characteristic ''punctuate'' gap junction appearances, corresponding to the different phases of synthesis and processing of the protein [21].
As CX43 allows the intercellular passage of numerous small molecules ranging from ions to larger metabolites, these findings support our hypothesis that gap junctions between TAMs allow an efficient metabolic support by coordinating intercellular signaling and connections to cancer and endothelial cells.  We have schematically represented TAMs network on figure 13. A biological network structure is much more robust and resistant to diverse types of aggression or dysfunction than a non-interconnected structure [32][33][34]. This could give to ATC a definitive advantage on the neighboring non-tumor tissues The vasculature could be considered the basis for the unique physiology of solid tumors in comparison with normal tissue [35][36][37]. In well-differentiated thyroid cancer, tumor cells appear in close contact with blood vessels like thyrocytes in normal thyroid tissue. In contrast, many cancer cells in ATC were located at a distance of more than 150 microns from blood vessels (Fig. 8A). This distance is considered to be the maximum distance of oxygen diffusion in tissues from blood vessels [38,39]. However, these cancer cells, in direct contact with RTAMs did not show any sign of necrosis or apoptosis. Moreover, RTAMs were strikingly clustered around blood vessels and from these cell clusters, long chains of connected RTAMs extended to avascular areas, as attempting to establish contact with distant cancer cells ( Fig. 9 and  10).
Several mechanisms other than tumor vasculature can provide the tumor with oxygen and nutrients [40]. Monocytes/macro-  phages may have the capacity to mimick blood vessels [41], in the in vitro formation of cord-like structures or branched cell columns in matrigel under angiogenic conditions [42][43][44] and in the in vivo transformation of monocytes into blood vessels [45,46]. Besides morphological features, lineage markers were found to be coexpressed by both endothelial cells and monocytes [42] [47,48]. However, in our study RTAMs and blood vessels were constantly clearly distinct since RTAMs were CD163+, CD342 and blood vessels CD1632, CD34+. Glod et al. underlined [46] that ''the continued expression of myeloid proteins implies that the cells do not trans-differentiate into mature endothelial cells, but rather take on specific endothelial characteristics while remaining inherently monocytic cells.'' Therefore, it is tempting to speculate that the interconnected RTAM network plays a vessel-like role in supplying nutrients from the blood to cancer cells.
Staining of Ki67 showed that the majority of cells were in a proliferative state and the double immunostaining for Ki67 and P22phox showed that the proliferation rates of RTAMs and cancer cells were roughly similar ( Table 2). Thus these rapidly growing tumors display apparently the same RTAMs/cancer cells ratio during their evolution in accordance with the ''symbiotic'' hypothesis.
GLUT1 protein has been recognized as the main isoform of glucose transporters in malignant tumors [49]. Stimulation of glycolysis is an activation signal of macrophages [50]. In ATC, the fact that the majority of cancer cells were negative for GLUT1 and that only blood vessels and a subset of RTAMs were positive, supports the notion that glucose uptake from the blood takes place mainly in RTAMs and not or to a far lesser degree in cancer cells. Indeed GLUT1 has also been recognized as the main transporter for the glucose analogue 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) in positron-emission tomography (PET) [49,51] and ATC displays an intense uptake of 18 FDG [28,52] (Fig S4). Furthermore Kubota et al. [53] showed that 18 FDG uptake was 2-4 times higher in tumor-associated macrophages tissues than in tumor cells.
These data suggest that the high uptake of FDG in ATC could be mostly related to the high number of RTAMs and that RTAMs network is related to glucose metabolism in the tumor.
TAMs displayed strong staining for NOX2 and its partner p22phox. NOX2 is the catalytic subunit of NADPH oxidase which produces reactive oxygen species (O 2 2 and H 2 0 2 ) in activated macrophages in order to kill bacteria. Thus, this proinflammatory activity appears to be related more to ''classic'' M1 macrophages than to that of anti-inflammatory M2 macrophages. However, recent studies have revealed other functions of the NADPH oxidase systems [12]. In this context, macrophages in rats could highly potentiate the invasive capacity of hepatoma tumor cells, both in vitro and in vivo [54,55] and this was inhibited by superoxide dismutase and catalase, This indicates a role for superoxide (O 2 -) and H 2 O 2 , both of which are generated by NADPH oxydase. Moreover, these studies argued in favor of direct contact between macrophages and tumor cells. Furthermore, the immunosuppressive properties of NOX2 and H 2 O 2 have been demonstrated [56,57] and reactive oxygen species (ROS) generated by NADPH oxidase are necessary for invadopodia and podosome formation which facilitates invasive behavior [58][59][60]. ROS can also modulate cell proliferation and the continuous generation of H2O2 is required for these mitogenic effects [61].
In ATC, cancer cells were negative for both NOX2 and p22phox. p22phox is also the functional partner of other NOX isoforms such as NOX1, NOX3 and NOX4 [12], and its lack of expression indicates that ATC cancer cells do not express these NADPH oxidase systems and consequently do not produce superoxide and H 2 O 2 on their own. ROS within cells act as secondary messengers in intracellular signaling cascades which induce and maintain the survival and oncogenic phenotype of cancer cells [62,63]. Numerous types of cancer cells display innate H 2 O 2 overproduction, and this has been correlated with increased malignant potential [56][57][58] [64,65]. Given the absence of the H 2 O 2 generator in ATC cancer cells and the symbiotic function of RTAMs, it may be hypothesized that H 2 O 2 produced by RTAMs could fuel cancer cells through interconnecting mechanisms such as Cx43. Moreover, beyond its role as a gap junction on the plasma membrane, Cx43 induces resistance to H 2 O 2 -mediated apoptosis, thus conferring an advantage for tumor growth [66].
Although essentially descriptive, our study demonstrates the presence in ATC of a dense and diffuse interconnected TAMs  network. To our knowledge this is the first time that such a network is described in a malignant tumor. This network found in all of our studied ATC cases appears related to the anaplastic nature of the proliferation since it was not present in more differentiated thyroid carcinomas and implies that the cancer cells were isolated by TAMs ramifications. We proposed some hypotheses concerning its possible functions given the aggressiveness of the disease and its need in a great amount of energy. Further studies using experimental models are now warranted to investigate the role of such interconnected macrophage network. Finally, TAMs were recently manipulated in vivo and in situ with pharmacological agents or cytokines to induce cytotoxic activity against cancer cells [67][68][69]. Given its dismal prognosis, ATC appears to be a candidate for such therapeutic approaches.

Supporting Information
Text S1 Supplemental material and methods.