Tumor microenvironment: bone marrow-mesenchymal stem cells as key players.

BBACAN-87929; No. of pages: 15; 4C: 4, 6, 10, 11 Biochimica et Biophysica Acta xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbacan

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Pedro Barcellos-de-Souza a,b,⁎, Valentina Gori c, Franco Bambi c, Paola Chiarugi a b c

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Department of Experimental and Clinical Biomedical Sciences, University of Florence, Tuscany Tumor Institute and Center for Research, Transfer and High Education DenoTHE, Florence, Italy CAPES Foundation, Ministry of Education of Brazil, Brasília, DF, Brazil Azienda Ospedaliero Universitaria Meyer, Florence, Italy

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Article history: Received 23 June 2013 Received in revised form 15 October 2013 Accepted 18 October 2013 Available online xxxx

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Tumor progression is a multistep phenomenon in which tumor-associated stromal cells perform an intricate cross-talk with tumor cells, supplying appropriate signals that may promote tumor aggressiveness. Among several cell types that constitute the tumor stroma, the discovery that bone marrow-derived mesenchymal stem cells (BM-MSC) have a strong tropism for tumors has achieved notoriety in recent years. Not only are the BMMSC recruited, but they can also engraft at tumor sites and transdifferentiate into cells such as activated fibroblasts, perivascular cells and macrophages, which will perform a key role in tumor progression. Whether the BM-MSC and their derived cells promote or suppress the tumor progression is a controversial issue. Recently, it has been proposed that proinflammatory stimuli can be decisive in driving BM-MSC polarization into cells with either tumor-supportive or tumor-repressive phenotypes (MSC1/MSC2). These considerations are extremely important both to an understanding of tumor biology and to the putative use of BM-MSC as “magic bullets” against tumors. In this review, we discuss the role of BM-MSC in many steps in tumor progression, focusing on the factors that attract BM-MSC to tumors, BM-MSC differentiation ability, the role of BM-MSC in tumor support or inhibition, the immunomodulation promoted by BM-MSC and metastatic niche formation by these cells. © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

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Keywords: Bone marrow-derived mesenchymal stem cells Tumor progression Tumor tropism Evasion from tumor site Cancer-associated fibroblasts Metastatic niche

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Origin and characterization of BM-MSC . . . . . . . . . . . . . . Recruitment of BM-MSC to tumor sites . . . . . . . . . . . . . . Fate of BM-MSC in tumor microenvironments . . . . . . . . . . . 3.1. BM-MSC transdifferentiation into CAF-like cells . . . . . . . 3.2. Acquisition of a vascular-like phenotype by BM-MSC . . . . 3.3. Hematopoietic transdifferentiation by BM-MSC . . . . . . . 4. The role of BM-MSC in tumor growth . . . . . . . . . . . . . . . 4.1. Tumor progression evidence . . . . . . . . . . . . . . . 4.1.1. Role of BM-MSC-derived CAF in tumor progression . 4.1.2. BM-MSC-induced tumor neovascularization . . . . 4.2. Anti-tumor progression evidence . . . . . . . . . . . . . 5. BM-MSC and evasion from tumor sites . . . . . . . . . . . . . . 5.1. BM-MSC modulation of innate immunity . . . . . . . . . . 5.2. Adaptive immunity modulation by BM-MSC . . . . . . . . 6. BM-MSC and the metastatic niche . . . . . . . . . . . . . . . . 7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Tumor microenvironment: Bone marrow-mesenchymal stem cells as key players☆

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64 ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: Department of Experimental and Clinical Biomedical Sciences, University of Florence, Viale Morgagni 50-50134, Florence, Italy. Tel.: +39 055 2751247; fax: +39 055 2751201. E-mail address: [email protected] (P. Barcellos-de-Souza). 0304-419X/$ – see front matter © 2013 The Authors. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbcan.2013.10.004

Please cite this article as: P. Barcellos-de-Souza, et al., Tumor microenvironment: Bone marrow-mesenchymal stem cells as key players, Biochim. Biophys. Acta (2013), http://dx.doi.org/10.1016/j.bbcan.2013.10.004

It has been described that BM-MSC show a strong tropism towards injured tissues because the intravenous delivery of BM-MSC results in their migration to specific sites of injury [9]. Moreover, endogenous BM-MSC are mobilized in response to inflammation or injury, thus increasing their numbers in the bloodstream and targeting specific tissues via active mechanisms [10]. Tumors are often considered to be “wounds that never heal” because they continuously produce inflammatory mediators that maintain the

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Aminopeptidase N Integrin β1 chain Hyaluronan receptor Integrin α1-5 chain ICAM-1 Lymphocyte function-associated antigen 3 Integrin β3 chain Transferrin receptor Ecto-5′-nucleotidase Thy-1 Endoglin; TGFβRIII VCAM-1 PDGFR-β MCAM (MUC18) Raph blood group ALCAM p75; NGFR

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23

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CD13 [150,151] CD29 [150] CD44 [150,151] CD49 [152] CD54 [153,154] CD58 [154] CD61 [155] CD71 [153] CD73 [6] CD90 [6] CD105 [6] CD106 [151,154] CD140b [155,156] CD146 [154,156,157] CD151 [158] CD166 [153,154] CD271 [156,159] HLA-ABC [154] Stro-1 [160]

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tumor microenvironment and assist with tumor progression [11]. In the last 10 years, it has been acknowledged that BM-MSC target to and engraft at sites of tumor development, which is a complex response that involves an intricate multistep process. The BM-MSC must be able to sense the signaling molecules released from the distant altered tissues, to move from their niche in the bone marrow into the circulation, and then to migrate into the target tissue. Once incorporated into the tumor microenvironment, the BM-MSC may differentiate into functional cells that participate in microenvironment homeostasis. It is likely that the selective tropism of BM-MSC to both tumors and sites of injury is due to the upregulation of similar inflammatory mediators in both circumstances. Many reports describe that BM-MSC can home and engraft to different types of solid tumors, including breast [12–15], lung [16,17], pancreatic [18], colon [19,20], ovarian [21–23] and prostate carcinomas [24,25], melanoma [26], glioma [27,28], Kaposi's sarcoma [29], and osteosarcoma [30] among other primary and metastatic tumors. The attraction of BM-MSC to tumor sites has been experimentally characterized by the injection by different routes of ex vivo-expanded BM-MSC and the subsequent cell-tracking utilizing a variety of labeling probes based on fluorescence, bioluminescence, positron emission, and magnetic resonance [31]. To support the biological role of endogenous BM-MSC migration to tumors, reports in which the bone marrow of sub-lethally irradiated SCID mice is repopulated with labeled bone marrow cells have concluded that these cells can efficiently home to the stroma of distally established tumors [32,33]. This idea highlights the concept that bone marrow-derived cells are significant components of the tumor stroma [21,32,34–36]. Due to their tropism to inflammatory sites, the chemotactic responses of BM-MSC are generally considered to resemble those of immune cells. Consistent with this, inflammatory cytokines are strongly involved in modulating the mobilization of the BM-MSC in the bone marrow niche and the further trafficking and homing of those cells to tumor sites. BM-MSC express a set of chemokine receptors, such as CCR1, -2, -4, -7 to -10, CXCR1, -2, -4 to -6, and CXC3 [37–40] and migrate in vitro in response to a number of molecules, e.g., CCL1 to -5, -7, -17, -19, -21 to -23, -25, -27, -28, CXCL-5, -8, -12, -13, -16 and CX3CL-1 (for details, see [41]). In addition to the chemokine receptors, it has also been described that BM-MSC express cell surface markers and receptors associated with migration such as growth hormone receptors, adhesion molecules and Toll-like receptors (TLR) [41]. These molecular mechanisms are similar to the leukocyte migratory apparatus, which

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Recent years have witnessed numerous publications about mesenchymal stem cells (MSC). This is mainly due to the characteristics of these cells, particularly their ability to proliferate ex vivo, their potential for multilineage differentiation and their immunomodulatory properties. These distinctive features, together with an outstanding tropism for tumor sites, have highlighted MSC as putative genetically engineered vehicles that specifically deliver therapeutic agents into tumors. Nonetheless, an important role for both endogenous and exogenous MSC in tumor progression has been described. In addition to their tumor-homing ability, these cells can engraft and differentiate into tumor-associated stromal cells, ultimately leading to different consequences. MSC are widely distributed in a variety of adult tissues such as the umbilical cord, Wharton's Jelly, bone marrow, adipose tissue, peripheral blood and the lungs. In such tissues, MSC are either constantly present or their pool is replenished by the migration of bone marrow-derived MSC (BM-MSC) [1,2]. It has recently been demonstrated that MSC are also present in the placenta and fetal tissues [1]. After an ex vivo expansion, the MSC isolated from diverse tissues have similar properties and minor differences, which may be due to their microenvironment of origin. Nevertheless, they can exert different effects depending on their biological contexts [3]. The bone marrow is the most extensively studied source of MSC. Despite an invasive surgical procedure needed for BM-MSC recovery, a relatively low cell yield (0.001%–0.01%) is obtained, which is inversely correlated with the age of the donor [4]. Despite many limitations, the BM-MSC population is the gold standard for MSC clinical applications and in vitro experiments [5]. There is still a wide perception in the literature that MSC, particularly BM-MSC, represent a phenotypically heterogeneous population of cells; therefore, great confusion still exists among cell biologists regarding the identity of human BM-MSC. The International Society for Cell Therapy has proposed the following criteria for human BM-MSC characterization: (a) the adherence to plastic in standard culture conditions; (b) the expression of surface molecules such as CD73, CD90, and CD105 in the absence of CD34, CD45, HLA-DR, CD14 or CD11b, CD79a, or CD19, as assessed by fluorescence-activated cell sorter analysis; and (c) the capacity to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro [6]. It has been shown that BM-MSC do not express endothelial markers such as CD31, CD34 and von Willebrand Factor (vWF). They express a large number of adhesion molecules (e.g., CD44 and integrins), some stromal cell markers (e.g., Src Homology-2, -3 and -4) and some cytokine receptors (e.g., Interleukin (IL)-1R and the tumor necrosis factor (TNF)-αR) [7]. Table 1 summarizes the currently used markers for BM-MSC characterization. Recent studies have suggested that MSC are capable of differentiating into endodermal and ectodermal lineages, including the lungs, retinal pigment, skin, sebaceous duct cells, renal tubular cells, neural cells, hepatocytes, and pancreatic islets [8]. Furthermore, in vitro functional assays are needed to demonstrate the multilineage differentiation of expanded cells, and a demonstration of pluripotency is necessary to discriminate between bona fide MSC and their precursors.

Table 1 t1:1 Markers expressed by BM-MSC. The principal markers that are used for the standard char- t1:2 acterization of BM-MSC are listed in the table. t1:3

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After reaching the tumor site, BM-MSC engraft into the signalreleasing microenvironment and differentiate into functional stromal cells [31]. It is well established that BM-MSC can give rise to mesenchymal lineages such as chondrocytes, adipocytes and osteocytes [6]. They can also differentiate into myofibroblast-like cells, allowing the formation of connective tissue components and, ultimately, tissue repair [9,53]. Finally, the role of BM-MSC in the tumor stroma has become increasingly important because their ability to differentiate into carcinoma-associated fibroblasts (CAF) and other tumor-associated cells such as endothelial and pericyte-like cells, as well as the newly recognized macrophage-like cells [31,54] has been described.

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It has been shown that engrafted bone marrow cells can develop into α-smooth muscle actin (SMA)-expressing myofibroblasts, usually recognized as CAF, in the tumor microenvironment [32,33]. In particular, BM-MSC co-cultured in vitro with cancer cells can be activated and may have a CAF-like phenotype. The markers used to define the CAF-like phenotype are divided into four groups: 1) the fibroblast activation markers, which include fibroblast specific protein (FSP, also named S100A4) and fibroblast activation protein (FAP); 2) the aggressiveness markers thrombospondin-1, tenascin-C and stromelysin; 3) the pro-vasculogenesis markers (desmin-1, α-SMA and VEGF-AA); and 4) the growth factors that support tumor growth and inflammation (EGF, HGF, IL-6, bFGF) [21,34–36].

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conditioning provides BM-MSC with higher migratory ability and increases the expression of c-Met, the major receptor for the HGF/scatter factor [48]. Moreover, the BM-MSC enhanced migration under conditions of hypoxia can be due to the increased expression of 3BP2/ SH3BP2 (Abl SH3-binding protein 2), an adaptor/scaffold protein that regulates leukocyte differentiation and motility, via HIF-mediated transcription. These data reinforce the resemblance between leukocyte and BM-MSC migratory machineries [47]. Furthermore, a hypoxic environment results in the generation of reactive oxygen species in the surrounding cells, which can increase genetic damage, leading to cell necrosis and tissue damage and consequently to inflammation [49]. Thus, hypoxia can maintain and amplify the inflammatory process in tumors, resulting in the production of chemokines and proteases that both attract immune cells and recruit BM-MSC. In solid tumors, cell death via the necrotic pathway can be a recurrent event, as malignant cells often show apoptosis resistance that is useful for enduring the impaired oxygen and nutrient supplies and the constant attack by immune cells. Therefore, the debris generated by cell necrosis is recognized by the immune cells through damageassociated molecular patterns (DAMP), thereby promoting inflammation and priming the adaptive immune responses. DAMP are a heterogeneous group of endogenous molecules, which include hyaluronan fragments, heat shock proteins, the S100 protein family, amyloid-β fibrils, uric acid, cytokines (IL-1α and IL-33) and the DNA-binding molecule high-mobility group box (HMGB)-1. Recent studies have linked DAMP with the chemoattraction of BM-MSC and have identified HMGB-1 as a crucial DAMP mediator [50,51]. When oxidized, HMGB-1 loses many of the functions typical of native HMGB-1, such as subcellular localization, interaction with DNA, and proinflammatory activities [52]. Interestingly, oxidized DAMP showed a significantly reduced induction of BM-MSC migration [51]. Although the direct oxidation of DAMP due to hypoxia has not been clearly reported, under the severe hypoxic conditions of a tumor microenvironment, it is likely that the DAMP are protected from oxidation and thereby they may act as important chemoattractants for BM-MSC.

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suggests that BM-MSC migration occurs in a leukocyte-similar fashion. This similarity to leukocyte motility is also reported in a study that demonstrates the coordinated rolling and adhesion of BM-MSC on endothelial cells [42]. Although the signals released by tumors as well as the molecular mechanisms involved in the mobilization of BM-MSC are currently poorly understood, a number of studies using in vivo and in vitro approaches have identified some key players in the tumor microenvironment scenario, which range from cytokines to growth factors. Despite some conflicting results, which are probably the consequence of different experimental conditions and/or variability in the BM-MSC heterogeneity, a consistent panel of molecules involved in the promotion of BM-MSC migration towards different types of tumor is currently being identified (Fig. 1). Accordingly, inflammatory cytokines and chemokines released by tumors, including IL-6, CXCL8/IL-8, CCL2/MCP-1, CXCL1, CXCL2, CXCL12/SDF-1, transforming growth factor (TGF)-β and Neurotropin3, acknowledged to recruit leukocytes to sites of injury, have been implicated in regulating BM-MSC mobilization and migration to tumors (Fig. 1). Growth factors such as epidermal growth factor (EGF), hepatocyte growth factor (HGF), bFGF and platelet derived-growth factor (PDGF)-BB have been shown to induce BM-MSC migration, and the effects of these factors can be stronger when they are combined [41]. Recently, the post-transcriptional regulation of SDF-1 mRNA expression by the microRNA (miR) pair 126/126* was identified as a critical mechanism in the BM-MSC recruitment by breast cancer cells. The tissue sample analyses demonstrated that breast cancer patients, with a highly methylated T-2 promoter in the Egfl7 gene, express low levels of miR-126. As the SDF-1 mRNA is a target for the miR126/126* complex, this epigenetic modification allows high expression of SDF-1, which is responsible for the attraction of BM-MSC [15]. In both inflammatory and tumor sites, the activation of extracellular matrix (ECM) proteases, which degrade the ECM and allow cell migration into these locations, is a common event. BM-MSC migrate more efficiently to high aggressive tumors of different origins that show a higher expression of the urokinase plasminogen activator (uPA) and uPA receptor than to less aggressive tumors, evidencing uPA as a chemoattractant molecule. This mechanism may also involve the participation of high levels of proinflammatory cytokines, such as IL-6, IL-8 and MCP-1, which may be correlated with the uPA and soluble uPA receptor expression [16]. Other inflammatory cytokines (TGF-β, TNF-α, IL-1β) are able to increase metalloproteinase (MMP) activity in BMMSC, which is crucial for promoting cell migration [43]. In line with this, MMP-1 activity mediates BM-MSC tropism towards human glioma cells through the cleavage of the protease-activated receptor expressed on the BM-MSC surface [44]. Another similarity between inflammatory and tumor sites is the prompt angiogenic response that commonly occurs. Vascular endothelial growth factor (VEGF)-AA and IL-8 are involved in the recruitment of endothelial cells and in the coordination of an angiogenic process. Of note, these molecules also promote tumor homing for BM-MSC (Fig. 1). Certainly, these findings underscore the importance of the angiogenic scenario in the recruitment of BM-MSC towards tumor sites. As a tumor grows, its blood supply can be insufficient, thereby creating regions of low oxygen concentration that are characterized by variable hypoxia. A decrease in oxygen availability is frequent occurrence within tumor microenvironment, thus promoting the activation of hypoxia-inducible factors (HIF), a family of transcription factors that induce the transcription of several genes, such as VEGF, TNF-α and proinflammatory cytokines, which in turn are able to activate nuclear factor (NF)-κB [45]. NF-κB is a prominent proinflammatory factor that has a prominent role in inducing the transcription of a large number of chemokines and growth factors, including CCL2, -3, -5, CXCL2 and -8 that are implicated in BM-MSC migration. Indeed, BM-MSC migrate in vitro towards the hypoxic environment, mainly through the MT1MMP-1 axis modulated by HIF-1 [46,47]. In addition, hypoxic pre-

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Fig. 1. Principal mediators identified in the BM-MSC tropism to tumors. BM-MSC recruitment to several tumors with different etiologies has been reported in the last 10 years. The main molecules involved in this phenomenon are described above.

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The understanding of the importance BM-MSC in a tumor microenvironment is further extended by reports showing their transdifferentiation into endothelial-like cells, which suggests that they maintain their progenitor cell potential and, consequently, participate in functional vasculature structures. Hence, BM-MSC incorporated into tumor sites can transdifferentiate into cells with pro-vascular characteristics, e.g., endothelial cells and pericytes, although conflicting results still raise doubts about their true ability to adopt a classical endothelial phenotype. As mentioned above, the work by Kidd et al. is in agreement with other reports in which MSC isolated from adipose tissue were described to have undergone endothelial differentiation [55,56]. In regard to BMMSC, different investigations have reached different conclusions. It has been reported that murine bone marrow-derived cells expressing CD31+, an endothelial cell surface marker, were present within and around the cancer in a xenograft model of pancreatic cancer [32]. However, those data do not elucidate whether there was transdifferentiation of the BM-MSC or the engraftment of other bone marrow cells, such as multipotent progenitor cells. Annabi and collaborators reported that murine BM-MSC, which were formerly CD31−, contributed to the formation of highly vascularized tumors in vivo, and ultimately those cells differentiated into CD31+ cells [46]. In vitro results have shown that human BM-MSC cultured in the presence of VEGF could be differentiated into endothelial-like cells, expressing the endothelial-specific markers VEGFR-1, -2 and vWF, although they remained negative for CD31 or CD34 [57]. Moreover, culturing BM-MSC in endothelial cell growth supplements, and exposing those cells to shear force and extracellular matrix stimuli provided both phenotypic and functional endothelial-like characteristics to BM-MSC [58]. In models of melanoma and lung adenocarcinoma, after tumor engraftment, some BM-MSC were closely integrated into the tumor vasculature and expressed CD31+ but not α-SMA, which is often used as a pericyte marker, suggesting a direct support of the tumor vasculature, similar to an endothelial phenotype [17]. A different biological scenario, however, can lead to the prevalence of a pericyte-like differentiation rather than an endothelial-like one. Recent reports have identified that BM-MSC do not differentiate into endothelial cells in vitro; however, reactivity to endothelin-1 in vivo, which is a similarity with perivascular cell precursors, has been reported [18,59]. Rat BM-MSC integrated into tumor vessel walls expressed pericyte markers such as α-SMA, NG2, PDGFR-β, but not endothelial cell markers. The pericyte marker expression profile and the perivascular location of the engrafted BM-MSC indicate that these cells act as pericytes within tumors [60]. Human BM-MSC treated with glioblastoma conditioned medium express CD 151, desmin, α-SMA, nestin, and NG2, whereas no expression of the vWF or myosin could be detected, indicating a differentiation into pericyte-like cells, instead of endothelial or smooth muscle cells. From a morphologic point of view, the BM-MSC capillary-like networks substantially differ from human

Recently, as already demonstrated for adipose tissue-MSC [63], it has been identified that BM-MSC can undergo hematopoietic differentiation and achieve macrophage-like phenotypic and functional aspects [54]. After either direct or indirect co-culture with nasopharyngeal carcinoma and lung adenocarcinoma cells, BM-MSC expressed hematopoietic markers such as CD11b, CD34 and CD45. Moreover, BM-MSC conditioned by co-culturing with these cancer cells expressed the mature monocyte/macrophage markers CD68, CSF1R, and MRC1. On the contrary, they had diminished expression of the pluripotent stem cell markers OCT3/4, SOX2, and Nanog. The acquisition of a macrophagelike phenotype is reinforced by a morphology alteration to roundshaped cells, the expression of CXCL5, G-CSF, GM-CSF, CXCL1, IL-7, CCL7, CXCL6, and CCL10 and enhanced macrophage effector functions such as a phagocytic capability and nitric oxide production. The BMMSC hematopoietic transdifferentiation is dependent on a decreased transient influx of Ca2+ [54]. Although interesting, this new role for tumor engrafted BM-MSC needs further investigations into (a) the molecular signals that promote the ability of BM-MSC to transdifferentiate into hematopoietic cells; and (b) the role of the macrophage-like BM-MSC in tumor progression. Although BM-MSC plasticity has been well characterized, more studies are needed to elucidate the role of a diverse biological background in influencing the behavior of the BM-MSC. As BM-MSC have been shown to give rise to many tissues, migrating BM-MSC may represent a source of pluripotent cells that are constantly available for tumor stroma formation and for promoting the developing cancer-related inflammatory environment (Fig. 2). 4. The role of BM-MSC in tumor growth

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In the tumor microenvironment, the BM-MSC, and their subsequently differentiated cells with distinct phenotypes, interact with the tumor cells and with the other stromal cells via a number of signaling molecules, resulting in a complex crosstalk. The factors that are produced by the BM-MSC directly, or that are produced by others cells in the tumor microenvironment after BM-MSC contact, can range from mitogens, ECM proteins, and angiogenic and inflammatory mediators and are therefore potential regulators of tumor growth and spread. The outcome of this intricate signaling network is not yet clear because BMMSC have been shown to have both stimulating and inhibiting effects on tumor progression (Fig. 3). It is conceivable that the biological context, i.e., the tumor histotypes or their local microenvironments may be critical to setting the balance between the pro- and anti-survival effects elicited by the BM-MSC.

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During the past decade, it has been acknowledged that tumor progression is driven not only by alterations in the cancer cells but also by contributions of the cancer-associated stroma [64]. To evaluate the BM-MSC ability to induce tumor growth in vivo, assays that utilize co-

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endothelial cells because the tube network formed by the pericyte cells without an endothelial lining has finer branched networks, reflecting the early participation of pericytes in the angiogenic sprouting [59,61]. In fact, it must be considered that the BM-MSC exhibit a remarkable similarity to pericytes, not only because these two cell types are of a mesenchymal origin and have a resemblance in their protein marker profiles but also because they can perform analogous functions in supporting a tumor vascular network. Furthermore, as BM-MSC can be a source of pericytes, the latter can also acquire an MSC phenotype [62]. Thus, BM-MSC transdifferentiation into pericytes seems to be a plausible and recurrent event in the tumor microenvironment.

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Recent reports have shed new light on the importance of the CAFlike derived-BM-MSC in the tumor microenvironment. The exact percentage of cells involved in this phenomenon is highly dependent on the experimental conditions and the model used; in a model of pancreatic cancer, it has been documented that ~20% of the CAF originated from BM-MSC [33], while in an ovarian cancer study, a significant 60– 70% of the FAP+ and FSP+ CAF were reported to have been derived from the bone marrow [36]. In fact, the origins of the stroma-recruited MSC were reported by Kidd et al., and it was proposed that the majority of the pericytes (neuron/glial antigen (NG)2+ and α-SMA+) and endothelial cells (CD31+) are recruited from the tumor adjacent tissue, such as local adipose tissue, whereas the FAP+ and FSP+ CAF-like cells, involved in extracellular matrix remodeling, are attracted from the host bone marrow populations, such as BM-MSC [36].

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Fig. 2. BM-MSC transdifferentiation ability and the role of their derived phenotypes in tumor progression. The BM-MSC potential to transdifferentiate into several cells types is recognized in many reports. The tumor microenvironment is acknowledged to drive the BM-MSC into activated fibroblast-like or perivascular-like differentiated cells.

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the proliferation of osteosarcoma cells [30]. In keeping with a key role for BM-MSC in the formation of distant metastases, BM-MSC injected intravenously into nude mice bearing orthotopic osteosarcomas led to increased metastatic nodules, and CCL5 has been proposed to play the main role [30]. The proliferation of the Saos-2 osteosarcoma can also be induced by IL-6, secreted by BM-MSC through the activation of the STAT3 transcription factor. The IL-6/STAT3 signaling pathway gives rise to a survival signal in Saos-2 cells, thereby increasing cell migration and invasion and promoting the metastasis of osteosarcoma [71]. Additionally, the IL-6 secretion by BM-MSC increases the number of colorectal tumorinitiating cells and promotes tumor formation [69]. BM-MSC also produce TGF-β1 , acknowledged as a tumor inducing cytokine for prostate cancer cells [24]. LL-37 (leucine, leucine-37), a proinflammatory cytokine with chemoattractive properties for immune cells that also has a pro-

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injection of these cells with different types of tumors in immunecompromised animals have been performed by several groups. BMMSC co-injected with cancer cells increased tumor growth in models of B16 melanoma [17,65], colon cancer [20,66], breast cancer [12,67], as well as in osteosarcoma [68], ovarian [21], colorectal [69,70], lung [17], gastric [34] and prostate carcinomas [24,25]. Signaling molecules secreted by BM-MSC have been acknowledged to promote tumor progression. Karnoub and collaborators investigated the tumor onset, growth kinetics and metastasis formation of four different human mammary carcinoma cell lines co-injected subcutaneously with BM-MSC. Although the BM-MSC only increased the primary tumor growth of one cell type (MCF7/Ras), they enhanced the metastatic potential of all the cancer cell lines. CCL5/RANTES, secreted by BMMSC and acting primarily through Akt signaling, was identified as a key factor in allowing metastatic colonization [12]. In addition, CCL5 produced by BM-MSC is also important in mobilizing and increasing

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Motility Proliferation Transdifferentiation into CAF-like cells Invasion EMT Metastasis Apoptosis Inhibition Increase tumor-initiating cells Angiogenesis Transdifferentiation into perivascular-like cells Immunosuppression CCL5, TGF-β, IL-6, IL-10, VEGF, MMP, SDF-1, Neuregulin1

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Fig. 3. Positive or negative role of BM-MSC in tumor progression. BM-MSC can support tumor progression by modulating several processes elicited by tumor cells or the tumor-associated stroma. Nonetheless, reports have also proposed an antitumorigenic function for BM-MSC.


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4.1.1. Role of BM-MSC-derived CAF in tumor progression In agreement with their role as tumor stroma-associated cells, BMMSC conditioned in vitro for several days with tumor derived medium can acquire a CAF-like phenotype and can start producing SDF-1, thereby sustaining tumor growth both in vitro and in vivo [35]. SDF-1 secretion is considered a critical feature of the CAF in several tumors and one of the factors which supports tumor progression [85]. On the other hand, non-conditioned BM-MSC expressed undetectable levels of the CAF markers and did not support tumor growth [35]. The importance of resident CAF in supporting tumor progression and the preparation of a metastatic niche is well acknowledged for prostate and breast cancers [86]. The CAF engage in a biunivocal interplay with cancer cells, described as the efferent and the afferent pathways. In the efferent branch of the loop, the cancer cells cause the activation of the bystander CAF, enhancing their reactivity in terms of contractility,

change of secreted ECM proteins and soluble factors [87]. The CAF undergo activation in response to cancer derived soluble factors such as IL-6, TGF-β or CCL2 [88]. To complete the diabolic circuitry through the afferent pathway, the activated CAF affect the malignancy of the cancer cells, mainly by eliciting EMT in the primary tumor cells, as well as by enhancing tumor growth and metastatic spread [88]. Of note, the signaling elicited by the resident CAF in cancer cells undergoing EMT is similar to the proinflammatory cues that involve cyclooxygenase-2, NF-κB and HIF-1 [89]. Once activated, the CAF become active players in the cancer malignancy in several other ways. First, they can cooperate in the recruitment and organization of endothelial precursor cells, thereby facilitating de novo vessel formation and tumor growth [90,91]. Second, they can participate in the orchestration of an inflammatory response, thereby enhancing the recruitment of monocytes to the primary tumor, as well as their polarization towards the M2 tumor-promoting phenotype [92]. Third, the CAF infiltrating aggressive cancers undergo expression/activation of carbonic anhydrase IX, leading to an extracellular pH decrease and favoring extracellular protease activation, thereby driving the EMT of the cancer cells [93]. Finally, the tumor-educated CAF are active modulators of tumor metabolism, behaving as tumor cell feeders and allowing the cancer cells to regain a respiratory phenotype. Indeed, upon contact with bystander CAF, the tumor cells induce a clear Warburg metabolism to their associated CAF, which enhances glucose uptake, aerobic glycolysis and lactate secretion through the increased expression of monocarboxylate transporter (MCT)-4. In turn, the cancer cells begin to exploit this lactate, uploaded through the specific import lactate carrier, MCT-1. The cancer cells use the CAF-derived lactate by both respiring it in their reactivated mitochondrial respiration, as well as by driving it into anabolic pathways to obtain proteins and biomass to facilitate tumor growth [94]. This reconverted metabolism has been called reverse Warburg metabolism [94,95]. The role of the CAF as feeders of cancer cells has been expanded to the secretion of ketone bodies, which are useful for maintaining a high lipid synthesis rate during glucose deprivation [96]. In addition, CAF have also been shown to undergo autophagy to fuel cancer cells and sustain their growth [97]. Of course, all these metabolic adaptations can foster the metabolic pathways of the tumor cells, thereby providing them with lactate and allowing the adaptation of the tumor cells to a low glucose environment [94]. Again, the role of CAF in supporting the metabolism of cancer cells is driven by oxidative stress and the activation of HIF-1, which is a key regulator of glucose addiction and catabolic/autophagic processes [94]. Hence, the metabolic reprogramming of cancer cells to a reverse Warburg phenotype allows the tumors that are highly infiltrated by a reactive stroma to allocate a Warburg metabolism to the stromal fibroblasts and to use their byproducts to survive and grow in a glucose-free ischemic milieu. The complexity of the mutual cooperation between the stromal fibroblasts and cancer cells is further underscored by some reports that again describe a mandatory interplay among these cells, but in the opposite metabolic direction. Indeed, immunohistochemistry data suggest that in some tumor types, the cancer cells express lactate dehydrogenase (LDH)-5 and MCT-4, which is essentially linked to anaerobic metabolism, while the stromal cells express high levels of LDH-1 and MCT-1, suggesting that they use aerobic metabolism [98,99]. This idea has also been confirmed in a breast cancer model, where BM-MSC are able to upload the lactate expelled from the tumor cells and use it as a source of energy [100]. Whatever the direction of the metabolic interplay, the stromal and cancer cells exchange lactate and energy-rich compounds in a mutually favorable relationship that favors cancer malignancy. Of course, these data have important pharmacological implications, as they include infiltrated CAF, either resident or recruited by bone marrow-derived cells, as key regulators of metabolic adaptive strategies engaged by the cancer cells to face the hostile tumor environment, thereby adding a further level of complexity to the system. In line with the role of engrafted BM-MSC in a tumor microenvironment, CXCL16 expression by prostatic carcinoma cells, besides

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angiogenic role, is found upregulated in ovarian, breast and lung tumors [72–74]. Coffelt and collaborators described a role for LL-37 in recruiting BM-MSC to tumor sites [23]. Furthermore, LL-37-stimulated BM-MSC actively secrete proinflammatory cytokines, including IL-6, IL-10, CCL5, VEGF, as well as MMP-2. Thereby, LL-37 facilitates ovarian tumor progression through the support of the tumor engrafted BM-MSC, which direct the pro-angiogenic, metastatic and immunosuppressive responses [23]. Once the migrating BM-MSC have reached the tumor site, they can face a developing environment that is inhabited by inflammatory cells. Despite a lack of exploration of the influence of macrophages on BMMSC behavior, it has been suggested that BM-MSC respond to macrophage-derived IL-8, CCL2 and CCL5 and, in turn, acquire a motile phenotype, characterized by the secretion of IL-6, CCL5 and CXCL10 [75]. These cytokines participate in both the maintenance of a proinflammatory state and in tumor growth. Nevertheless, further investigations are necessary to clarify the relationship between BM-MSC and cancer-associated macrophages. Epithelial cells form a tissue sheet through the binding of adhesion molecules, most prominently epithelial cadherin (E-Cad). Thus, the disaggregation of both normal and tumor epithelial cells is characterized by a decreased expression of E-Cad. Moreover, E-Cad downregulation is also a key event in the activation of a post-transcriptional program termed epithelial-to-mesenchymal transition (EMT), in which the cells gradually lose their epithelial characteristics and acquire a more motile and invasive mesenchymal-like phenotype, which is associated with metastatic features [76,77]. Notably, the composition of the microenvironment, including oxygen tension, growth factors, and cytokines, has recently been suggested to modulate the EMT process in cancer cells [77,78]. Breast cancer cells, upon contact with BM-MSC, increase their expression of EMT specific markers, allowing a loss of the cellular adherens-junctions and of polarization, thereby suggesting that BMMSC can induce EMT [79,80]. Lysyl oxidase has been determined to be necessary for such an interaction. Indeed, BM-MSC contact induces de novo expression of lysyl oxidase and is a key event in enhancing the lung and bone metastasis of poorly metastatic cells [81]. BM-MSC have also elicited EMT in hepatocellular carcinoma cells, leading to their metastatic progression [82]. Emphasizing the role of a proinflammatory context to elicit BM-MSC pro-tumor actions, the prometastatic effect of BM-MSC could also be reproduced with the supernatant of BM-MSC pretreated with interferon (IFN)-γ and TNF-α. Moreover, treatment of hepatocellular carcinoma cells with this supernatant leads to EMT, mainly due to the secretion of TGF-β by BM-MSC stimulated with those proinflammatory cytokines. BM-MSC also enhance the metastatic ability of ovarian and colon carcinoma cells [20,83]. Of note, circulating BM-MSC are not only able to migrate towards the orthotopic primary colon carcinoma, but they are also recruited by liver metastatic colonies. In both primary and metastatic tumors, PDGF-BB signaling plays a critical role in the interaction between BM-MSC and tumor cells [84].

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Depending on the experimental setting, BM-MSC have also been acknowledged to lead anti-tumorigenic responses. Khakoo et al. described intrinsic BM-MSC antineoplastic properties through Akt inactivation in Kaposi's sarcoma cells [29]. In SCID mice bearing disseminated nonHodgkin's lymphoma xenografts, the BM-MSC exhibited antitumor activity and, although they increased the release of pro-angiogenic cytokines and induced the migration of endothelial cells, they also promoted endothelial cell apoptosis [109]. Even though studies underline BM-MSC assistance in engineering long-term functional vasculature, either by transdifferentiation into endothelial/pericyte phenotypes or by the stimulation of provasculogenesis paracrine factors [46,59–61], a putative antiangiogenic role for these cells has been proposed. This outcome can occur due to BM-MSC migration towards the capillaries and their intercalation between those endothelial cells. This would increase the production of ROS by the endothelial cells, thereby leading to their apoptosis and subsequent capillary degeneration [110]. BM-MSC inhibit the in vivo tumor growth and in vitro proliferation, migration, and invasion of lung adenocarcinoma cells through the secretion of oncostatin-M, a differentiation-promoting cytokine, that exerts antiproliferative effects against several types of cancers [78]. The inhibition of glioma cell line tumorigenesis has also been attributed to BM-MSC action because the co-injection of BM-MSC with the glioma cells resulted in a significant reduction in the tumor volume and vascular density. In particular, the effects observed in the co-culture model have to be related to impaired tumor angiogenesis by virtue of reduced secretion of PDGF-BB and IL-1β [111] (Fig. 3). To better understand the controversy concerning the role of BMMSC on tumor progression, it must be considered that the complex and variable crosstalk within the tumor microenvironment modulates the BM-MSC effects. It is likely that hypoxia, the composition of the extracellular matrix, the extracellular acidity as well as the inflammatory component of the stroma are all crucial players in determining the outcome of these BM-MSC effects. When BM-MSC have been engrafted into the primary tumor site, the balance between the pro- or anti-tumor effects is determined by the tumor microenvironmental changes. In a non-adverse milieu, BM-MSC can actually repair tumor “wounds” and can have antitumor effects. Conversely, in case of adverse and sustained inflammatory conditions, recruited BM-MSC behave contrasting healing of the tumor, and concurring to sustain malignancy of the neoplasia.

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the tumor neovasculature, subsequently acquiring a pericyte-like phenotype and acting as pericytes in the tumor stroma [60]. A decreased oxygen concentration, a common state in growing solid tumors, acquires an important role in promoting angiogenesis because hypoxia can augment the expression of several angiogenic factors, including the VEGFs, angiopoietin-2, PDGF-BB, placenta growth factor, TGF-β, IL-8, and HGF [105,106]. In keeping with this, BM-MSC also respond to hypoxia, increasing their secretion of VEGF-AA, thereby supporting the idea for a key role for BM-MSC during de novo angiogenesis [107]. Moreover, hypoxia was also important in stimulating TGF-β secretion from BM-MSC, which is important in breast tumor vascularization [19]. Inflammation also interacts with the BM-MSC induced vascularization as colon cancer growth was accelerated when the BM-MSC were pre-stimulated with the inflammatory cytokines IFN-γ and TNF-α. The BM-MSC pre-stimulated by both inflammatory factors expressed higher levels of VEGF via the HIF-1 signaling pathway and then had enhanced tumor angiogenesis, finally leading to colon cancer growth in mice [108].

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attracting BM-MSC, is also able to induce their transdifferentiation into CAF-like cells, which secrete high levels of SDF-1. Thus, the SDF-1 produced by these CAF-like cells, originated from BM-MSC, is able to induce EMT in prostate tumor cells, consequently promoting metastasis [25]. In addition to prostate and breast cancers, similar cross-talk between CAF and cancer cells has been reported in other tumor histotypes. Skov3 ovarian cancer cells secrete IL-6, VEGF-AA and TGF-β1 which affect the recruitment and differentiation of BM-MSC. The BM-MSC establish a reciprocal connection with the Skov-3 cells and upregulate the secretion of these cytokines by the tumor cells. Indeed, the tumor-engrafted BM-MSC subsequently differentiate into CAF-like cells and contribute to tumor microvascularization, stromal networks, and the production of tumor-stimulating paracrine factors [21]. Aligned with the importance of the inflammatory component of BMMSC homing to tumor sites, CAF that originated from BM-MSC are recruited to inflammation-induced gastric cancers in a TGF-β and SDF1α-dependent manner [34]. Consequently, the BM-MSC-derived CAF show DNA-hypomethylation, which is a feature of CAF, and they express IL-6 and other factors that contribute to a stem cell niche, such as Wnt5α and BMP4, ultimately promoting tumor growth [34]. In a primary hepatocellular carcinoma, the engrafted BM-MSC show increased expression of FSP, a CAF marker. The BM-MSC-derived CAF increase tumor proliferation and invasion, mainly through the FSPdependent up-regulation of miR155 expression. The upregulation of miR155 has been correlated with the negative regulation of suppressor of cytokine signaling 1, thereby activating STAT3 signaling. In turn, STAT3 promotes MMP-9 expression, thus increasing tumor invasiveness [101]. In colorectal cancers, Neuregulin1 protein is secreted by BMMSC, which activates the human epidermal growth factor receptor (HER) 2/3, a family of proteins implicated in tumor progression [102]. The sustained HER2/3 activation leads to PI3K/Akt signaling, consequently increasing the survival, invasion and metastatic spread of colorectal cancer [70]. Similarly to colorectal cancer cells, cancerassociated MSC demonstrate higher expression of transmembrane Neuregulin1, a feature significantly associated with advanced cancer stages, invasion depth and a decreased 5-year progression-free survival [70]. MMPs secreted by MSC-derived CAF play a pivotal role in inducing tumor pro-invasive characteristics. In a murine model of skin carcinoma, the BM-MSC engrafted at tumor sites have turned into pericytelike cells or α-SMA expressing CAF, and the latter were exclusively responsible for producing MMPs in the tumor stroma, thereby affecting stromal reactivity [103]. Although several factors that are present in the tumor stroma can putatively drive the BM-MSC transdifferentiation towards CAF, a main role for TGF-β1 was proposed. Indeed, the inhibition of the TGF-β1/ SMAD pathway blocks the BM-MSC differentiation into CAF-like cells, and the inhibition of this signaling suppressed BM-MSC pro-tumor effect [104]. 4.1.2. BM-MSC-induced tumor neovascularization The pro-tumor behavior of BM-MSC may be due to their combined effect on tumor proliferation, tumor dissemination, and vessel distribution [21,23,66]. In certain studies, the BM-MSC-elicited tumor progression was not primarily due to the promotion of tumor cell growth or an increased number of BM-MSC, but rather to the secretion of angiogenic factors [17]. Several pro-angiogenic factors, including VEGF-AA, IL-6, TGF-β, IL-8, leukemia inhibitory factor, macrophage-colony stimulating factor and macrophage inflammatory protein-2 are produced by BM-MSC, mainly in response to treatment with tumor cell conditioned medium [17,21]. Aside from producing factors that trigger and stimulate angiogenesis, the transdifferentiation into endothelial and/or pericytes-like cells endows BM-MSC with a structural role in tumor neo-angiogenesis. In experimental glioma models, BM-MSC can efficiently integrate into

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5. BM-MSC and evasion from tumor sites

5.2. Adaptive immunity modulation by BM-MSC

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The growth of a tumor mass and the subsequent tumor cell evasion from the primary neoplastic site into the bloodstream relies on the impairment of the immune surveillance of the tumor microenvironment. The concept that BM-MSC are potent suppressors of many immune cells has been widely spread in the last decade. More recently, several authors have suggested that the BM-MSC could perform an intricate cross-talk with many effectors of the immune system, rather than a simple immune-suppression. BM-MSC are considered to be immune-privileged cells because they express MHC class I molecules, whereas they have low expression of MHC class II and co-stimulatory molecules on their cell surface. However, they still have a fascinating relationship with immune cells through direct cell contact or the secretion of soluble factors [112].

Several studies have investigated the interaction between BMMSC and T cells, the main effectors of adaptive immunity. Briefly, BM-MSC have been classified as T cell suppressors for both CD4+ T helper cells and CD8+ cytotoxic T lymphocytes. It has been described that BM-MSC can induce T cell anergy, which renders them functionally inactive [122,123], inhibits their proliferation [124] and elicits their apoptosis [125]. Moreover, BM-MSC inhibit proinflammatory cytokine production, including that of IFN-γ and TNF-α and suppress naïve and memory T cell responses to their cognate antigens [123,126]. In addition, BM-MSC also impair proliferation and partially affect the cytokine production and cytotoxic activity of natural killer T cells and γδ lymphocytes, two subsets of T cells that link innate and adaptive immunity [127]. Interestingly, BM-MSC can induce a pro-Th2 response in disorders that are associated with the dominance of Th1 cells [128] or modulate the Th1 response in Th2prevalent scenarios [129], consequently restoring the balance between these two T helper subtypes. Therefore, it has been proposed that BM-MSC sense the immunological environment and respond to it accordingly [112]. Moreover, in accordance with the complex crosstalk between BM-MSC and inflammatory and immune cells, the full T cell-suppressive capacity of human BM-MSC requires monocytes that produce IL-1β. The IL-1β, in turn, enhances the BM-MSC-mediated secretion of TGF-β, which is involved in lymphocyte suppression [130]. BM-MSC immunomodulatory effects are also supported by the upregulation of regulatory T (TReg) cells, an immune-suppressive population that is able to inhibit the production of the proinflammatory cytokines IL-17 and IL-22 by Th17 cells and to reprogram differentiated Th17 cells into IL-10+ FOXP3+ TReg cells [131]. The recent literature emphasizes the significance of the proinflammatory microenvironment on shaping the immunomodulatory repertoire of human BM-MSC. Their plasticity endows them with the capacity to adapt to external conditions by switching suppressive mechanisms [120]. Some studies have also investigated the relationship between BMMSC and dendritic cells (DC), the main type of antigen-presenting cells that modulate the adaptive immunity. BM-MSC impair DC differentiation and maturation because DC matured with TNF-α and cultured in the presence of BM-MSC failed to upregulate the maturation marker CD86 and did not increase the cell surface expression of MHC class II. Hence, BM-MSC promote a phenotype that leads to T cell anergy and TReg cell induction [132]. The immunosuppressive role of BM-MSC also extends to their effects on B-cells because when co-cultured with BM-MSC, B-cell proliferation and differentiation are impaired, resulting in decreased immunoglobulin production and impaired chemotaxis [133]. Recently, in a clear parallel with macrophage polarization, it has been proposed that BM-MSC could polarize into two different types of cells with distinct phenotypes, depending on the triggered TLR signaling. Waterman and collaborators suggested this model based on observations that TLR4-primed BM-MSC mostly secrete proinflammatory mediators, while TLR3-primed BM-MSC usually express immunosuppressive ones. Thus we can identify proinflammatory and immunocompetent MSC1 and immunosuppressive MSC2 phenotypes [134]. Accordingly, the in vitro co-culture of MSC1 with several cancer cell lines diminished tumor growth, whereas an MSC2 co-culture had opposite effects. Likewise, the treatment of tumors established in immune competent models with MSC1 resulted in attenuated tumor growth and metastasis while an MSC2-treatment led to tumor growth and spread promotion [135]. These data spotlight the importance of the activation state/polarization of the BM-MSC for their role in the microenvironment community and the further development of pro- or antitumor actions (Fig. 4). It is widely accepted that BM-MSC need to be activated by neighboring cells/factors to exert their immunomodulatory properties. In this

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5.1. BM-MSC modulation of innate immunity

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After migrating into the tissues, or in response to an inflammatory burst, monocytes can be differentiated into macrophages, which are key players in inflammatory responses and tissue regeneration. Macrophages are phenotypically differentiated into classically activated type macrophages (M1), which initiate and sustain proinflammatory actions, or alternatively polarized macrophages (M2), which are further classified into subtypes M2a, 2b or 2c according to their stimulation and subsequent cytokine and surface molecule expression. M2 macrophages are recognized to have immunoregulatory functions that are involved in tissue repair and ongoing chronic inflammation [113]. Thus, M1 macrophages assist in the Th1 responses guided by cytokines such as IL-12 and IL-23, whereas M2 macrophages express low levels of IL-12 but high levels of IL-10. In the tumor microenvironment, cancerassociated macrophages (CAM) represent the major inflammatory component, and clinical observations indicate that the CAM mainly show an M2 phenotype polarization [114]. Thus, M2-CAM influence multiple steps in tumor development including growth, invasion and metastasis of tumor cells. The CAM participate in a reciprocal interplay with CAF and the tumor cells that eventually increases tumor malignancy and favors tumor metastasis [92]. The chemokines produced by BM-MSC, including CCL3, CCL12 and CXCL12, attract and mobilize monocytes and macrophages into inflammatory sites [115]. Macrophages co-cultivated with BM-MSC show high levels of IL-10 production and phagocytic activity, and low levels of TNF-α and IFN-γ production as well as MHC class II expression, leading to polarization towards an M2-like macrophage phenotype [116,117]. The inflammatory component plays an important role in the modulation of the BM-MSC/macrophage crosstalk because BM-MSC stimulation by proinflammatory cytokines, such as IFN-γ, TNF-α, lipopolysaccharide (LPS) [118,119], or activated T cells [120] supports macrophage polarization towards an M2-like phenotype, through the expression of indoleamine 2,3-dioxygenase enzymes in BM-MSC. Neutrophils, another leukocyte cell type, are also recruited to inflamed tissues containing BM-MSC, as IFN-γ-activated BM-MSC stimulated with a TLR4 agonist secrete the neutrophil chemoattractants IL-1, IL-6 and CCL5 [121]. Taken as a whole, it is postulated that the BM-MSC/macrophage interactions in homeostatic conditions usually direct wound healing effects by the promotion of a regulatory M2-like phenotype. This crosstalk is regulated in feedback loops to prevent excessive inflammatory responses and to support tissue regeneration [112]. In a tumor microenvironment situation, where a constant and sustained proinflammatory scenario is common, this modulation is impaired and the BM-MSC-induced macrophage polarization into an M2type could allow tumor progression. This tumor progression could be due to an increase in the pro-angiogenic and immunosuppressive factors produced by M2-macrophages.

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Tumor metastasis is a multistep process. First, it requires adjacent stromal invasion and subsequent intravasation into the bloodstream by the tumor cells. Next, the cells must survive in the circulation, adhere to the endothelium, extravasate, invade the matrix and grow in the target organ. In all these stages, the tumor cells must escape from recognition and avoid destruction by the immune system. Finally, tumor cells have to adapt to several other circumstances such as oxidative stress, surviving anoikis, and the interaction with different ECM components at the metastatic site [138]. As already discussed, BM-MSC and their engrafted differentiated cells with distinct phenotypes have been involved in promoting metastasis in several tumor models. In addition to affecting invasion, tumor

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growth and evasion from a primary neoplastic site, the biological responses elicited by BM-MSC may influence in several ways the further steps of the metastatic progression, particularly the formation of the metastatic niche. According to Paget's hypothesis, the sites where metastases develop are not randomly chosen; on the contrary, the tumor cell (“the seed”) has a specific affinity for the microenvironment of certain organs (“the soil”). Therefore, tumor growth in the metastatic niche is dependent on the proper growth signals from the environment where the malignant cells anchor [139]. Interestingly, clinical and experimental observations often indicate that metastases take place at sites of injury [140]. It is hypothesized that signals indicating a loss of differentiated cells can induce the self-renewal of cancer stem cells (CSC), a small subpopulation of tumor cells with an infinite proliferative potential. CSC are proposed to give rise to new tumors and, therefore, be the cause of relapse and metastasis formation [141]. CSC can also start a regenerative wound-healing process, which is thought to activate the inflammatory processes that culminate in delivering signals required to initiate metastatic tumor growth [142]. Consequently, as the BM-MSC preferentially migrate to tumors and sites of tissue injury, it is likely that they may prepare the sites for subsequent colonization, analogously to those CSC effects. This perspective could explain the evidence that BM-MSC behave in a pro-metastatic manner [140]. The role of BM-MSC in metastatic niche formation can also be extended to the regulation of CSC features. When co-cultured with breast cancer cells, the BM-MSC promoted an increase in the CSC aldehyde dehydrogenase 1-positive population [67]. This co-culture also enhanced mammosphere-forming capacities, which is consistent with the acquisition of CSC traits [81]. In addition, tumor-secreted IL-1 induces prostaglandin E(2) secretion by BM-MSC. Prostaglandin E(2), in an autocrine manner, provokes the expression of a group of cytokines which, in turn, activate β-catenin signaling in the tumor cell, triggering EMT and creating a CSC niche [143]. Moreover, Giannoni and collaborators observed that the CAF-mediated EMT in prostate carcinoma cells can increase CSC features [88]. This might suggest a role for BM-MSCderived CAF in the CSC niche formation. Bone marrow is a target organ in many metastases. BM-MSC have a role in bone marrow metastatic niche formation, as they can interact with disseminated tumor cells into the marrow space, and modify

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scenario, an inflammatory environment seems to be necessary to promote their effect and some inflammation-related molecules such as TNF-α, IFN-γ and IL-1β might be implicated [136]. Thus, proinflammatory mediators activate, or employing a definition used by immunologists, “license” BM-MSC to be immunosuppressive or to improve their suppressive actions. The proinflammatory background is usually found at tumor sites as a result of cancer-related inflammation exerted by stromal and immune cells. The immunomodulation exerted by BMMSC would be a result of the context produced by immune cellderived signals and not a stereotypical immunosuppressive response [108,112,121]. Experimental evidence indicates that the BM-MSC immuneregulatory properties support the development of tumors as evaluated in allogeneic immunocompetent mice after the co-injection of BMMSC and B16 melanoma cells locally or at distant sites [65]. Further data suggests that the BM-MSC in the tumor inflammatory microenvironment may be endowed with immunosuppressive functions, which will help tumor cells to escape from the immune surveillance [137]. In conclusion, BM-MSC, through a complex crosstalk with several elements, inhibit many effector functions of immune cells, thereby promoting an immunosuppressive state in the tumor microenvironment. This scenario allows tumor cells and their associated stroma to overcome the immune surveillance, ultimately leading to tumor evasion from primary neoplastic sites and enhanced metastasis formation.

840 841

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Fig. 4. Principal aspects of BM-MSC polarization towards MSC1 or MSC2 phenotypes. The suggested MSC1 and MSC2 polarization phenotypes and their main characteristics are listed above.


876 877 878 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914


936 937 938 939 940 941 942 943 944 945 946 947 948 949 950 951

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In the past decade, several groups have investigated the role of BMMSC on tumor progression. The initial studies demonstrated a remarkable homing to tumor sites and a putative immune-privilege from BM-MSC, which have promoted enthusiasm because these cells can be used as suitable carriers to deliver anti-tumor agents to a tumor microenvironment. Because of their features BM-MSC have the potential to serve as useful vehicles for many anti-tumor agents, especially for those having a short systemic half-life and excessive toxicity. In addition, therapeutic BM-MSC may be able to reach poorly accessible body sites. Based on gene modification techniques, researchers are transferring the killing activity of immune cells to BM-MSC, creating the socalled mesenkillers [149]. Nonetheless, subsequent data have identified BM-MSC as pro-active tumor stroma-associated cells; hence, they have been implicated in promoting almost all the features recognized as hallmarks of cancer [64], including cell survival, angiogenesis, invasion, evasion of immune system and metastasis. Moreover, significant cell plasticity has been identified, which allows engrafted-BM-MSC to acquire a range of phenotypes that is determined by the complex tumor microenvironment. Aside from the classic bone, cartilage and fat transdifferentiation ability, certain scenarios can induce myofibroblast, endothelial, pericyte or macrophage-like cell types. The cancer-related inflammatory

P

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964

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7. Conclusions

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EMT induction [79–82,143], despite recent work suggesting that these cells can also inhibit EMT or even promote MET. Conditioned medium from BM-MSC treated with bFGF and EGF increases HGF secretion and inhibits TGF-β-induced EMT in renal tubular epithelial cells [148]. In lung adenocarcinoma cells, the treatment with conditioned medium from BM-MSC elicits a clear MET, mainly by the enhancement of epithelial markers and suppression of EMT markers and the Nanog stemness factor [78]. Therefore, depending on the context, the induction of MET by BM-MSC may be a double-edge sword because it could diminish metastatic progression, if it takes place in the primary site; however, it could also favor the metastatic exit from dormancy and colony growth, if it occurs within the metastatic niche (Fig. 5).

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their proliferative and morphogenetic organization patterns [144]. Moreover, BM-MSC can also facilitate breast cancer cell migration across bone marrow endothelial cells, assisting tumor cell entry into the bone marrow. Of note, low invasive breast cancer cells seem to have a preferential tropism to bone marrow compared with highly invasive cells [145]. Therefore, it is likely that cancer subpopulations with a special tropism towards bone marrow at an initial tumorigenesis could be responsible for cancer recurrence and the resistance to therapy [145]. Interestingly, metastatic cells can bring with them certain stromal components, including activated CAF, during their circulation. This heterotypic association between the metastatic tumors and the CAF provides higher viability and growth advantages to the metastatic cells [146]. Indeed the CAF induce EMT, an epigenetic motogenic program that is correlated with resistance to anoikis, which is useful when traveling cancer cells lack adhesion in the bloodstream, and to the acquisition of stem-like traits, that are effective in regenerating tumors in the metastatic niche. As BM-MSC can engraft at tumor sites and transdifferentiate into CAF, it is tempting to assume a role for the CAF-like BM-MSC in promoting metastatic progression. Furthermore, a recent report shows that BM-MSC can spontaneously form hybrids with breast cancer cells. These hybrids showed predominantly mesenchymal morphological characteristics, mixed gene expression profiles, and increased DNA ploidy, which gave them an increased metastatic ability. Both in culture and in xenografts, the hybrids subsequently underwent DNA ploidy reduction and a morphological reversal to breast carcinoma-like morphological characteristics, while maintaining a mixed breast cancer–mesenchymal expression profile [147]. As has been argued before, EMT is considered to be critical to endowing carcinomas with metastatic features because it enhances cell motility by changing cell–cell interactions and adhesive characteristics and augments cell matrix degradation, resulting in a more invasive cell type. However, to establish and stabilize distant metastases, it is suggested that cancer cells revert to an epithelial phenotype and incorporate into remote tissues through the execution of mesenchymal-toepithelial transition (MET), the post-transcriptional reprogram that is the opposite of EMT. Several studies have noted a role for BM-MSC in

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11

Fig. 5. BM-MSC association with tumor cells during circulation and metastatic niche formation. As it has been acknowledged for CAF, so we suggest that tumor educated-BM-MSC can (1) be transported with tumor cells during their circulation, (2) help tumor transendothelial migration, and (3) contribute to the formation of metastasis in the appropriate niche. Recently, it has been described that BM-MSC can form a putative hybrid with tumor cells [147]. Although this needs to be supported by more experimental data, we speculate that (4) the hybrid fusion possibly supports tumor survival in the bloodstream, inhibiting anoikis, assisting (5) transendothelial migration and (3) the development of a metastatic niche, where a reversion to carcinoma-like morphological characteristics through MET takes place.


954 955 956 957 958 959 960 961 962 963

965 966 967 968 969 970 971 972 973 974 975 976 977 978 979 980 981 982 983 984 985 986

12

987 988


999 1001 1000

Abbreviations bFGF Basic Fibroblast Growth Factor

1002

BM-MSC CAF CAM CSC DAMP DC E-Cad ECM EGF EMT FAP FSP HGF HIF HMGB IFN IL LDH LPS MCP MCT MET MHC miR MMP MSC NG PDGF NF-κB SDF SMA TGF TNF TLR TReg UCB uPA VEGF vWF

1015 1016 1017 1018 1019 1020 1021 1022 1023 1024 1025 1026 1027 1028 1029 1030 1031 1032 1033 1034 1035 1036 1037 1038 1039 1040

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Acknowledgements

The authors would like to thank the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq — Ministry of Education, Brazil), the Italian Association for Cancer Research (AIRC), and the Istituto Toscano Tumori 1046 and FESR-PorCreo 2012 for financial support. We would also like to 1047 express our gratitude to Dr. Andrea Morandi for kindly revising this 1048 manuscript. 1044 1045

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Berry, S. McGee, E. Lee, H. Sun, J. Wang, T. Jin, H. Zhang, J. Dai, P.H. Krebsbach, E.T. Keller, K.J. Pienta,

T

1009 1010

C

1007 1008

E

1005 1006

bone marrow-derived MSC cancer-associated fibroblasts cancer-associated macrophages cancer stem cells damage-associated molecular patterns dendritic cells epithelial cadherin extracellular matrix epidermal growth factor epithelial-to-mesenchymal transition fibroblast activation protein fibroblast specific protein hepatocyte growth factor hypoxia-inducible factors high-mobility group box interferon Interleukin lactate dehydrogenase lipopolysaccharide Monocyte Chemoattractant Protein monocarboxylate transporter mesenchymal-to-epithelial transition major histocompatibility complex microRNA Metalloproteinases mesenchymal stem cells neuron/glial antigen Platelet-Derived Growth Factor Nuclear Factor-κB Stromal-Derived Factor smooth muscle actin transforming growth factor tumor necrosis factor Toll-like receptors Regulatory T cells Umbilical Cord Blood urokinase plasminogen activator vascular endothelial growth factor von Willebrand Factor.

R

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R

996 997

O

994 995

C

993

N

991 992

U

989 990

1049

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component plays a major role in directing BM-MSC actions in the tumor stroma, not only in the recruitment and induction of subsequent transdifferentiation but also in the favoring of MSC polarization towards a pro- or anti-tumor supportive type (MSC1/2). Likewise, inflammation has been proposed to promote BM-MSC licensing to exert immune-suppressive effects, impairing immune surveillance in the tumor microenvironment and enhancing tumor progression. To date, the understanding of BM-MSC biology is far from complete and strongly needs further work that will lead to the development of more rational and selective therapies in advance of their safe use in patients.


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Tumor-associated macrophages as major players in the tumor microenvironment.

Tumor-associated stromal cells as key contributors to the tumor microenvironment.

Mesenchymal stem cells in the tumor microenvironment.

Bone Microenvironment, Stem Cells, and Bone Tissue Regeneration.

The SOX transcription factors as key players in pluripotent stem cells.

Bone mesenchymal stem cells differentiate into myofibroblasts in the tumor microenvironment.

OsteoMacs: Key players around bone biomaterials.

The bone microenvironment - Multiple players involved in cancer progression.

B cells and the humoral response in melanoma: The overlooked players of the tumor microenvironment.

Mimicking biophysical stimuli within bone tumor microenvironment.

Cancer stem cells and the tumor microenvironment: interplay in tumor heterogeneity.

Tertiary Lymphoid Structure-Associated B Cells are Key Players in Anti-Tumor Immunity.

Glioma Stem Cells: Signaling, Microenvironment, and Therapy.

Nanotechnology, and Application.

RhoGTPases as key players in mammalian cell adaptation to microgravity.

B-lymphocytes as key players in chemical-induced asthma.

Platelets and their microparticles as key players in pathophysiological responses.

ROS as key players in plant stress signalling.

Interplay between Tumor Microenvironment and Cancer Cells.

Glial cells as key players in schizophrenia pathology: recent insights and concepts of therapy.

The microenvironment reprograms circuits in tumor cells.

NK cells in the tumor microenvironment.

Immunoregulatory effects of bone marrow-derived mesenchymal stem cells in the nasal polyp microenvironment.

Hepatocyte Growth Factor, a Key Tumor-Promoting Factor in the Tumor Microenvironment.