Vitronectin as a molecular player of the tumor microenvironment in neuroblastoma

The composition, morphology and organization of the extracellular matrix (ECM) is key to both healthy and pathological environments. In healthy tissue, the ECM regulates development and homeostasis, whereas in tumors it displays mechanical properties such as stiffness that confer malignant characteristics to cell behaviour including proliferation, cell-death resistance, angiogenesis, invasion and metastasis [1‐3]. The interaction between tumor cells and their surrounding elements is the first step in the development of metastasis, since cell movement requires firm cell-ECM adhesions to break down, as well as molecules to guide the migration. Different cancer-promoting biological pathways of interest for further exploration are cell-cell or cell-ECM adhesions, proteases and chemokines [4]. In addition, recent studies show that cancer invasion and metastasis are driven by physical and chemical interactions between tumor cells and the ECM that translate into a stiff neoplastic ECM and soft or deregulated tumor cells, which in turn lead to a more favorable microenvironment for cancer dissemination [5‐8].

Vitronectin (VN) is an adhesive glycoprotein that acts as a link between cells and the ECM through several ligands such as: integrins, plasminogen activator inhibitor-1(PAI-1) and urokinase plasminogen activator receptor (uPAR). VN is present in plasma as a monomeric or dimeric structure (folded or native form) and in the ECM of several tissues as a multimeric formation (unfolded or active form) [9, 10]. It is mainly synthesized by hepatocytes in the liver, although it has also been found in smaller amounts in extrahepatic tissues such as: brain, lung, kidney and vascular wall of adrenal gland [11‐13]. It has also been observed that some tumor cells secrete VN as well as tumor-infiltrating T-lymphocytes (TIL) which bind to VN through TIL uPAR expression [14, 15]. The biological functions of VN, derived from its domains which bind several ligands in its activated form, are: preservation of vascular homeostasis (thrombosis and fibrinolysis), control of the innate immune system, facilitating cell adhesion and participation in migration in tissue repair and regeneration [16]. VN has a role in the provisional matrix of tumors, where it can promote cell adhesion and matrix degradation by binding to integrins, PAI-1 and uPAR [17]. In fact, in several human neoplasms, VN is associated with tumor invasion, metastasis and angiogenesis [18‐20].

Neuroblastoma (NB) originates from the neural crest in the Sympathetic Nervous System and is one of the most common pediatric solid tumors [21]. Although several clinical, biological and genetic markers define the risk of progression in NB patients [22], the mechanisms that control communication between tumor cells and the ECM, and can influence aggressiveness are not yet clear. Our group has already described the aggressive pattern of a stiff ECM defined as: ECM with cross-linked and disorganized reticulin fiber networks, scant amount of collagen type I fibers and glycosaminoglycans and large and abundant irregularly-shaped and high blood vessels, associated with a poor outcome in NB patients [23‐26]. Hence, to better comprehend this tumor cell-ECM communication we searched for targets within the ECM elements. Previous studies in NB have noted VN expression in ganglion cells that could suggest a differentiation role for this molecule [12] and its α_vβ₃ integrin receptor, which is highly expressed in high-risk NB [27].

In this study, we used digital image analysis to examine the immunohistochemical expression of VN in NB to better understand the mechanical signals between neuroblasts and the ECM and their influence on tumor growth, differentiation and dissemination. In addition, we have done in vitro and in vivo experiments to assess if VN present in tumor or host microenvironments shows any modification on NB behavior since previous research showed the relationship between VN and metastasis and tumor progression in several human neoplasms.

VN is an attachment glycoprotein that directs cell migration, progression, adhesion and differentiation in many biological and pathological processes [13, 15, 30, 31]. By introducing advanced morphometric methodology, we have been able to demonstrate and robustly quantify VN as an important ECM component in aggressive NB synthetized by undifferentiated neuroblasts. We have found two VN expression patterns which reflect the secretion time and role of this glycoprotein that has typically been described for ECM constituents in cartilage: the territorial VN pattern, where VN is synthetized and recently incorporated to the newly formed matrix facilitating mechanical stresses of tumor cells, and the interterritorial VN pattern, where VN has been integrated some time ago to the matrix contributing to metabolic and biomechanical properties of several tumor tissue elements.

Some contradictory studies of VN expression in NB have been described [12, 27, 32]. Gladson et al. 1997, showed VN expression in differentiating neuroblastic tumors and noted its attachment role in retinoic-acid differentiated neuroblastic cells stimulating in vivo differentiation [12]. Later NB studies detected an overexpression of α_vβ₃ integrin and uPAR, both of which are important VN ligands, in high-risk NB [27, 32]. From a larger cohort of neuroblastic tumors, our immunohistochemical results revealed VN expression in NB and GNB, with a high territorial VN expression in undifferentiated neuroblasts. Our findings are in agreement with the most recent studies mentioned above and the differences from the earlier study [12] could be explained by cohort size. Using digital pathology techniques, we were able to accurately provide a connection between intensity and location. These techniques assure the standardization of all measurements and minimize inter-observer differences [33, 34] and can easily be reproduced in future studies. We are currently carrying out topological analysis on the histopathological images to evaluate non-cellular VN distribution features related to the tumor microenvironment. Our topological approach will capture different aspects of the VN distribution to improve the classification of biopsy samples from NB samples.

The in vitro and vivo preliminary results in the present study mainly focused on human NB, were to emphasize the VN secretion by both NB cell lines and highlight the tumor and/or host microenvironment influence in their VN synthesis. We found that in vitro NB cells expressed a low quantity of VN as a small cytoplasmic dot drops pattern and that the in vivo experiments revealed a high amount of VN as a dense cytoplasmic pattern with no differences in VN staining pattern or tumor growth rate between RAG1^−/− VN^+/+ and RAG1^−/− VN^−/− mice. The increased VN secretion by tumor cells, which produces mechanical stress, generates an initial stiff matrix and results in disrupted cell-cell and cell-ECM interactions, promoting tumor proliferation in both mice strains, as described in ovarian cancer, through the breakdown of  VN bonds to improve metastasis [35]. We confirmed that: a) Fetal bovine serum (FBS) does not contribute to a higher amount of VN in tumor cells as reported by Gladson et al. [36] and b) the importance of VN in NB tumor growth due to its high presence in vivo. These findings reinforce the previously described VN role in improving the migratory ability of tumor cells in NB and suggest that in vivo tumor cells produce VN to achieve greater migratory capacity. Furthermore, given the importance of VN in breast cancer [37], we are developing experiments to clarify the role of VN in vivo in NB tumor growth. Serial tumor passages will allow VN secretion by neuroblasts to be modified in relation to a host microenvironment, affecting tumor growth and genetic instability, as occurs in hormone-dependent tumors [38]. Therefore, although xenotranplanted neuroblastic cells continue to synthetize VN, the absence of liver VN in the host would have a subtle role in the secretory ability of malignant neuroblasts, but some long-term influence on cell proliferation and tumor aggressiveness.

We stress that a high territorial VN expression pattern could contribute to tumor cell adhesion, thus promoting invasion and metastasis in NB, suggesting that VN or its ligands could be used as targets when developing therapeutic strategies for modulating the relationship between tumor cells and the ECM. In fact, the highest presence of territorial VN protein in the present cohort is related to unfavorable independent prognostic INRG variables. The high presence of territorial VN staining in tumors from patients with metastasic stage and unfavorable histology, would lead to higher migration ability of tumor cells by anchorage to fibers and proteoglycans, as well as disrupted cell adhesion and spreading via interactions with specific α_νβ₃ and α_νβ₅ integrins, uPAR and PAI-1 in a stiff matrix. In the case of tumors with genetic instability, a huge presence of territorial VN would generate mechanical alterations in ECM, which would be transmitted to the nuclear matrix and would modulate the response of intracellular signals activating genetic and epigenetic mechanisms of instability in these tumors [39].

The main therapeutic goal would be to focus on decreasing VN expression or to inhibit its joining with α_vβ₃ integrin, uPAR or PAI-1, thus depriving the tumor cells of the mechanical forces necessary to create the appropriate environment for invasion [40‐42]. Via its ligands, VN has a role in biochemical cell-ECM pathways which could be used as therapeutic targets; however, no drug against cell-ECM interactions has yet been approved, although some trials are ongoing as described below.

α_vβ₃ and α_vβ₅ integrins bind to the arginine-glycine-aspartate (RGD) VN sequence and are key factors in angiogenesis. It has been observed that VN activates vascular endothelial growth factor receptor 2 (VEGFR-2) via α_vβ₃, thus contributing to angiogenesis [43]. As anti-α_vβ₃ integrin targeted drugs: Vitaxin, Intetumubab (CNTO 95) and 17E6 (EMD 525797) are in different phases of clinical trials [44], also in vitro melanoma research has displayed metastasis retardation using IH1062, an α_vβ₃ integrin inhibitor that blocks the binding of this integrin to VN [45].

uPAR is a protease that binds to VN in the RGD motif or N-terminal somatomedin B (SMB) domain. It has been observed that this union initiates integrin pathways that promoting migration [46, 47]. In addition, when uPAR binds to urokinase plasminogen activator (uPA), it causes the cleaving of plasminogen to produce plasmin that mediates the degradation of the ECM [48]. An in vivo study into a monoclonal anti-uPAR antibody (ATN-658) reported that this antibody inhibits tumor cell proliferation in prostate cancer [49].

Finally, PAI-1 is a protease inhibitor of the serpins family that binds to the SMB domain of VN where its main function is uPA inhibition. Thus, PAI-1 participates in the uPA/uPAR proteolytic cascade, as well as interfering in the binding of uPAR and α_v integrins family in the SMB domain and RGD motif to VN, respectively [50]. In fact, an in vivo study of NB using PAI-1 deficient mice showed a reduction in tumor size [51]. In vitro and in vivo studies on the inhibition of PAI-1 using compounds such as TM5441, TM5275 and SK-216 have demonstrated toxic effects in cancer cells [52, 53].

In conclusion, NB samples of patients with poor prognostic factors are characterized by the highest territorial VN expression pattern. Our findings suggest the importance of extensive studies on VN as a possible target for inhibiting interactions in NB.