New insights into the role of thrombospondin-1 in glioblastoma development.

Glioblastoma (GB), the most malignant subtype of diffuse glioma, is highly aggressive, invasive and vascularized. Its median survival is still short even with maximum standard care. There is a need to identify potential new molecules and mechanisms, that are involved in the interactions of GB cells with the tumor microenvironment (TME), for therapeutic intervention. Thrombospondin-1 (TSP1) is a multi-faceted matricellular protein which plays a significant role in development, physiology and pathology including cancer. Recent studies have pinpoint an important role of TSP1 in GB development which will be summarized and discussed herein. We will discuss studies, mainly from preclinical research, which should lead to a deeper understanding of TSP1's role in GB development. We will also discuss some issues with regard to the use of this knowledge for the clinic.

Structural Properties of CXCL4L1 and Its Recognition of the CXCR3 N-Terminus.

Chemokine CXCL4L1, a homologue of CXCL4, is a more potent antiangiogenic ligand. Its structural property is correlated with the downstream receptor binding. The two chemokines execute their functions by binding the receptors of CXCR3A and CXCR3B. The receptors differ by an extra 51-residue extension in the CXCR3B N-terminus. To understand the binding specificity, a GB1 protein scaffold was used to carry different CXCR3 extracellular elements, and artificial CXCL4 and CXCL4L1 monomers were engineered for the binding assay. We first characterized the molten globule property of CXCL4L1. The structural property causes the CXCL4L1 tetramer to dissociate into monomers in low concentrations, but native CXCL4 adopts a stable tetramer structure in solution. In the titration experiments, the combination of the CXCR3A N-terminus and receptor extracellular loop 2 provided moderate and comparable binding affinities to CXCL4 and CXCL4L1, while sulfation on the CXCR3A N-terminal tyrosine residues provided binding specificity. However, the CXCR3B N-terminal extension did not show significant enhancement in the binding of CXCL4 or CXCL4L1. This result indicates that the tendency to form a chemokine monomer and the binding affinity together contribute the high antiangiogenic activity of CXCL4L1.

Practical identifiability analysis of a mechanistic model for the time to distant metastatic relapse and its application to renal cell carcinoma.

Distant metastasis-free survival (DMFS) curves are widely used in oncology. They are classically analyzed using the Kaplan-Meier estimator or agnostic statistical models from survival analysis. Here we report on a method to extract more information from DMFS curves using a mathematical model of primary tumor growth and metastatic dissemination. The model depends on two parameters, α and µ, respectively quantifying tumor growth and dissemination. We assumed these to be lognormally distributed in a patient population. We propose a method for identification of the parameters of these distributions based on least-squares minimization between the data and the simulated survival curve. We studied the practical identifiability of these parameters and found that including the percentage of patients with metastasis at diagnosis was critical to ensure robust estimation. We also studied the impact and identifiability of covariates and their coefficients in α and µ, either categorical or continuous, including various functional forms for the latter (threshold, linear or a combination of both). We found that both the functional form and the coefficients could be determined from DMFS curves. We then applied our model to a clinical dataset of metastatic relapse from kidney cancer with individual data of 105 patients. We show that the model was able to describe the data and illustrate our method to disentangle the impact of three covariates on DMFS: a categorical one (Führman grade) and two continuous ones (gene expressions of the macrophage mannose receptor 1 (MMR) and the G Protein-Coupled Receptor Class C Group 5 Member A (GPRC5a) gene). We found that all had an influence in metastasis dissemination (µ), but not on growth (α).

The tumor organismal environment: Role in tumor development and cancer immunotherapy.

Tumor immunotherapy has resulted in dramatic effects in some cancer types, including curing of previously untreatable patients. However, the response rates are typically very heterogenous, with some patients showing dramatic responses whereas others do not or only barely respond. Consequently, there has been an ever-increasing research effort to better understand the factors that govern immunotherapy responsiveness and efficiency in order to identify predictive biomarkers and novel therapeutic targets. Clearly, traits of the tumor cells as well as aspects of the tumor microenvironment (TME) play an important role in this regard. However, a growing tumor not only interacts with cells in its immediate vicinity, but also reciprocally communicates with the entire host organism (and its microbiota). Thus, systemic influences on tumor growth and progression are likely to be similarly important as the microenvironment. In this review, we focus on various aspects of the "tumor organismal environment" (TOE), namely the lymphatic, the hematopoietic, the microbial, the neurogenic and the metabolic environment, and discuss their impact on tumor growth and immunotherapy.

Experimental and computational modeling for signature and biomarker discovery of renal cell carcinoma progression.

BACKGROUND: Renal Cell Carcinoma (RCC) is difficult to treat with 5-year survival rate of 10% in metastatic patients. Main reasons of therapy failure are lack of validated biomarkers and scarce knowledge of the biological processes occurring during RCC progression. Thus, the investigation of mechanisms regulating RCC progression is fundamental to improve RCC therapy. METHODS: In order to identify molecular markers and gene processes involved in the steps of RCC progression, we generated several cell lines of higher aggressiveness by serially passaging mouse renal cancer RENCA cells in mice and, concomitantly, performed functional genomics analysis of the cells. Multiple cell lines depicting the major steps of tumor progression (including primary tumor growth, survival in the blood circulation and metastatic spread) were generated and analyzed by large-scale transcriptome, genome and methylome analyses. Furthermore, we performed clinical correlations of our datasets. Finally we conducted a computational analysis for predicting the time to relapse based on our molecular data. RESULTS: Through in vivo passaging, RENCA cells showed increased aggressiveness by reducing mice survival, enhancing primary tumor growth and lung metastases formation. In addition, transcriptome and methylome analyses showed distinct clustering of the cell lines without genomic variation. Distinct signatures of tumor aggressiveness were revealed and validated in different patient cohorts. In particular, we identified SAA2 and CFB as soluble prognostic and predictive biomarkers of the therapeutic response. Machine learning and mathematical modeling confirmed the importance of CFB and SAA2 together, which had the highest impact on distant metastasis-free survival. From these data sets, a computational model predicting tumor progression and relapse was developed and validated. These results are of great translational significance. CONCLUSION: A combination of experimental and mathematical modeling was able to generate meaningful data for the prediction of the clinical evolution of RCC.

Tumor Cell Invasion and the Tumor Microenvironment: Special Focus on Brain Tumors.

Tumor cell invasion is a major issue in oncology since it leads to tumor dissemination and recurrence. In glioblastomas, invasion is an important characteristic, making the disease difficult to treat since tumor recurrence occurs from invasive areas at the borders of the resection cavity. We are discussing herein some of the principal mechanisms at a cellular and molecular level that are involved in glioblastoma invasion. These mechanisms are comprising tumor cell intrinsic factors as well as extrinsic factors and cues produced by the tumor microenvironment. Therapeutically interfering with tumor cell invasion may be useful to improve the clinical outcomes of glioblastoma patients.

The CC and CXC chemokines: major regulators of tumor progression and the tumor microenvironment.

Chemokines are a family of soluble cytokines that act as chemoattractants to guide the migration of cells, in particular of immune cells. However, chemokines are also involved in cell proliferation, differentiation, and survival. Chemokines are associated with a variety of human diseases including chronic inflammation, immune dysfunction, cancer, and metastasis. This review discusses the expression of CC and CXC chemokines in the tumor microenvironment and their supportive and inhibitory roles in tumor progression, angiogenesis, metastasis, and tumor immunity. We also specially focus on the diverse roles of CXC chemokines (CXCL9-11, CXCL4 and its variant CXCL4L1) and their two chemokine receptor CXCR3 isoforms, CXCR3-A and CXCR3-B. These two distinct isoforms have divergent roles in tumors, either promoting (CXCR3-A) or inhibiting (CXCR3-B) tumor progression. Their effects are mediated not only directly in tumor cells but also indirectly via the regulation of angiogenesis and tumor immunity. A full comprehension of their mechanisms of action is critical to further validate these chemokines and their receptors as biomarkers or therapeutic targets in cancer.

Mechanisms of invasion in glioblastoma.

PURPOSE OF REVIEW: This review provides an overview of recent updates in understanding the mechanisms by which glioblastoma cells interact with their cellular and molecular partners within the microenvironment. RECENT FINDINGS: We have now a better knowledge of the cell populations involved in Glioblastoma (GBM) invasion. Recent works discovered the role of new molecular players in GBM invasion, and, most importantly, better models are emerging which better recapitulate GBM invasion. SUMMARY: Invasive properties of glioblastoma make complete surgical resection impossible and highly invasive cells are responsible for tumor recurrence. In this review, we focus on recent updates describing how invasive cells progress in the surrounding tissue along brain structures. We also provide an overview of the current knowledge on key cells and molecular players within the microenvironment that contribute to the invasive process. VIDEO ABSTRACT.

Beyond the tumour microenvironment.

In contrast to the once dominant tumour-centric view of cancer, increasing attention is now being paid to the tumour microenvironment (TME), generally understood as the elements spatially located in the vicinity of the tumour. Thinking in terms of TME has proven extremely useful, in particular because it has helped identify and comprehend the role of nongenetic and noncell-intrinsic factors in cancer development. Yet some current approaches have led to a TME-centric view, which is no less problematic than the former tumour-centric vision of cancer, insofar as it tends to overlook the role of components located beyond the TME, in the 'tumour organismal environment' (TOE). In this minireview, we highlight the explanatory and therapeutic shortcomings of the TME-centric view and insist on the crucial importance of the TOE in cancer progression.

Consensus guidelines for the use and interpretation of angiogenesis assays.

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference.

Inference of Low and High-Grade Glioma Gene Regulatory Networks Delineates the Role of Rnd3 in Establishing Multiple Hallmarks of Cancer.

Gliomas are a highly heterogeneous group of brain tumours that are refractory to treatment, highly invasive and pro-angiogenic. Glioblastoma patients have an average survival time of less than 15 months. Understanding the molecular basis of different grades of glioma, from well differentiated, low-grade tumours to high-grade tumours, is a key step in defining new therapeutic targets. Here we use a data-driven approach to learn the structure of gene regulatory networks from observational data and use the resulting models to formulate hypothesis on the molecular determinants of glioma stage. Remarkably, integration of available knowledge with functional genomics datasets representing clinical and pre-clinical studies reveals important properties within the regulatory circuits controlling low and high-grade glioma. Our analyses first show that low and high-grade gliomas are characterised by a switch in activity of two subsets of Rho GTPases. The first one is involved in maintaining normal glial cell function, while the second is linked to the establishment of multiple hallmarks of cancer. Next, the development and application of a novel data integration methodology reveals novel functions of RND3 in controlling glioma cell migration, invasion, proliferation, angiogenesis and clinical outcome.

History and conceptual developments in vascular biology and angiogenesis research: a personal view.

Vascular biology is an important scientific domain that has gradually penetrated many medical and scientific fields. Scientists are most often focused on present problems in their daily scientific work and lack awareness regarding the evolution of their domain throughout history and of how philosophical issues are related to their research field. In this article, I provide a personal view with an attempt to conceptualize vascular development research that articulates lessons taken from history, philosophy, biology and medicine. I discuss selected aspects related to the history and the philosophy of sciences that can be extracted from the study of vascular development and how conceptual progress in this research field has been made. I will analyze paradigm shifts, cross-fertilization of different fields, technological advances and its impact on angiogenesis and discuss issues related to evolutionary biology, proximity of different molecular systems and scientific methodologies. Finally, I discuss briefly my views where the field is heading in the future.

CXCL7 is a predictive marker of sunitinib efficacy in clear cell renal cell carcinomas.

BACKGROUND: Sunitinib is one of the first-line standard treatments for metastatic clear cell renal cell carcinoma (ccRCC) with a median time to progression shorter than 1 year. The objective is to discover predictive markers of response to adapt the treatment at diagnosis. METHODS: Prospective phase 2 multi-centre trials were conducted in ccRCC patients initiating sunitinib (54 patients) or bevacizumab (45 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials). The plasmatic level of CXCL7 at baseline was correlated with progression-free survival (PFS). RESULTS: The cut-off value of CXCL7 for PFS was 250 ng ml-1. Patients with CXCL7 plasmatic levels above the cut-off at baseline (250 ng ml-1) had a significantly longer PFS (hazard ratio 0.323 (95% confidence interval 0.147-0.707), P=0.001). These results were confirmed in a retrospective validation cohort. The levels of CXCL7 did not influence PFS of the bevacizumab-treated patients. CONCLUSIONS: CXCL7 may be considered as a predictive marker of sunitinib efficacy for ccRCC patients.

Computational Modelling of Metastasis Development in Renal Cell Carcinoma.

The biology of the metastatic colonization process remains a poorly understood phenomenon. To improve our knowledge of its dynamics, we conducted a modelling study based on multi-modal data from an orthotopic murine experimental system of metastatic renal cell carcinoma. The standard theory of metastatic colonization usually assumes that secondary tumours, once established at a distant site, grow independently from each other and from the primary tumour. Using a mathematical model that translates this assumption into equations, we challenged this theory against our data that included: 1) dynamics of primary tumour cells in the kidney and metastatic cells in the lungs, retrieved by green fluorescent protein tracking, and 2) magnetic resonance images (MRI) informing on the number and size of macroscopic lesions. Critically, when calibrated on the growth of the primary tumour and total metastatic burden, the predicted theoretical size distributions were not in agreement with the MRI observations. Moreover, tumour expansion only based on proliferation was not able to explain the volume increase of the metastatic lesions. These findings strongly suggested rejection of the standard theory, demonstrating that the time development of the size distribution of metastases could not be explained by independent growth of metastatic foci. This led us to investigate the effect of spatial interactions between merging metastatic tumours on the dynamics of the global metastatic burden. We derived a mathematical model of spatial tumour growth, confronted it with experimental data of single metastatic tumour growth, and used it to provide insights on the dynamics of multiple tumours growing in close vicinity. Together, our results have implications for theories of the metastatic process and suggest that global dynamics of metastasis development is dependent on spatial interactions between metastatic lesions.

Evidence for the interaction of fibroblast growth factor-2 with the lymphatic endothelial cell marker LYVE-1.

LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a homolog of the hyaluronan receptor CD44, and one of the most widely used markers of lymphatic endothelial cells in normal and tumor tissues. However, the physiologic role of LYVE-1 in the lymphatic system still remains unclear. It is well established that fibroblast growth factor 2 (FGF2) induces lymphangiogenesis. Based on the known interaction between FGF2 and CD44 and based on the structural similarity of CD44 and LYVE-1, we investigated whether FGF2 might interact with LYVE-1. We found that FGF2 is able to bind LYVE-1 using AlphaScreen, or after surface-immobilization or in solution. FGF2 binds to LYVE-1 with a higher affinity than any other known LYVE-1­binding molecules, such as hyaluronan or PDGF-BB. Glycosylation of LYVE-1 is important for FGF2 binding. Furthermore, FGF2 interacts with LYVE-1 when overexpressed in CHO cells. Soluble LYVE-1 and knockdown of LYVE-1 in lymphatic endothelial cells impaired FGF2 signaling and functions. In addition, FGF2 but not VEGF-C-induced in vivo lymphangiogenesis, was also inhibited. Conversely, FGF2 also modulates LYVE-1 expression in cells and ex vivo. Thus, our data demonstrate a functional relationship to the interaction between FGF2 and LYVE-1.

Mapping the extracellular and membrane proteome associated with the vasculature and the stroma in the embryo.

In order to map the extracellular or membrane proteome associated with the vasculature and the stroma in an embryonic organism in vivo, we developed a biotinylation technique for chicken embryo and combined it with mass spectrometry and bioinformatic analysis. We also applied this procedure to implanted tumors growing on the chorioallantoic membrane or after the induction of granulation tissue. Membrane and extracellular matrix proteins were the most abundant components identified. Relative quantitative analysis revealed differential protein expression patterns in several tissues. Through a bioinformatic approach, we determined endothelial cell protein expression signatures, which allowed us to identify several proteins not yet reported to be associated with endothelial cells or the vasculature. This is the first study reported so far that applies in vivo biotinylation, in combination with robust label-free quantitative proteomics approaches and bioinformatic analysis, to an embryonic organism. It also provides the first description of the vascular and matrix proteome of the embryo that might constitute the starting point for further developments.

Alternative C-terminal helix orientation alters chemokine function: structure of the anti-angiogenic chemokine, CXCL4L1.

BACKGROUND: CXCL4L1 is a highly potent anti-angiogenic and anti-tumor chemokine, and its structural information is unknown. RESULTS: CXCL4L1 x-ray structure is determined, and it reveals a previously unrecognized chemokine structure adopting a novel C-terminal helix conformation. CONCLUSION: The alternative helix conformation enhances the anti-angiogenic activity of CXCL4L1 by reducing the glycosaminoglycan binding ability. SIGNIFICANCE: Chemokine C-terminal helix orientation is critical in regulating their functions. Chemokines, a subfamily of cytokines, are small, secreted proteins that mediate a variety of biological processes. Various chemokines adopt remarkable conserved tertiary structure comprising an anti-parallel ß-sheet core domain followed by a C-terminal helix that packs onto the ß-sheet. The conserved structural feature has been considered critical for chemokine function, including binding to cell surface receptor. The recently isolated variant, CXCL4L1, is a homologue of CXCL4 chemokine (or platelet factor 4) with potent anti-angiogenic activity and differed only in three amino acid residues of P58L, K66E, and L67H. In this study we show by x-ray structural determination that CXCL4L1 adopts a previously unrecognized structure at its C terminus. The orientation of the C-terminal helix protrudes into the aqueous space to expose the entire helix. The alternative helix orientation modifies the overall chemokine shape and surface properties. The L67H mutation is mainly responsible for the swing-out effect of the helix, whereas mutations of P58L and K66E only act secondarily. This is the first observation that reports an open conformation of the C-terminal helix in a chemokine. This change leads to a decrease of its glycosaminoglycan binding properties and to an enhancement of its anti-angiogenic and anti-tumor effects. This unique structure is recent in evolution and has allowed CXCL4L1 to gain novel functional properties.

Regulation of the Src kinase-associated phosphoprotein 55 homologue by the protein tyrosine phosphatase PTP-PEST in the control of cell motility.

PTP-PEST is a cytosolic ubiquitous protein tyrosine phosphatase (PTP) that contains, in addition to its catalytic domain, several protein-protein interaction domains that allow it to interface with several signaling pathways. Among others, PTP-PEST is a key regulator of cellular motility and cytoskeleton dynamics. The complexity of the PTP-PEST interactome underscores the necessity to identify its interacting partners and physiological substrates in order to further understand its role in focal adhesion complex turnover and actin organization. Using a modified yeast substrate trapping two-hybrid system, we identified a cytosolic adaptor protein named Src kinase-associated phosphoprotein 55 homologue (SKAP-Hom) as a novel substrate of PTP-PEST. To confirm PTP-PEST interaction with SKAP-Hom, in vitro pull down assays were performed demonstrating that the PTP catalytic domain and Proline-rich 1 (P1) domain are respectively binding to the SKAP-Hom Y260 and Y297 residues and its SH3 domain. Subsequently, we generated and rescued SKAP-Hom-deficient mouse embryonic fibroblasts (MEFs) with WT SKAP-Hom, SKAP-Hom tyrosine mutants (Y260F, Y260F/Y297F), or SKAP-Hom SH3 domain mutant (W335K). Given the role of PTP-PEST, wound-healing and trans-well migration assays were performed using the generated lines. Indeed, SKAP-Hom-deficient MEFs showed a defect in migration compared with WT-rescued MEFs. Interestingly, the SH3 domain mutant-rescued MEFs showed an enhanced cell migration corresponding potentially with higher tyrosine phosphorylation levels of SKAP-Hom. These findings suggest a novel role of SKAP-Hom and its phosphorylation in the regulation of cellular motility. Moreover, these results open new avenues by which PTP-PEST regulates cellular migration, a hallmark of metastasis.

CXCR3, a double-edged sword in tumor progression and angiogenesis.

CXC chemokines are involved in chemotaxis, regulation of cell growth, induction of apoptosis and modulation of angiostatic effects. CXCL9, CXCL10, CXCL11, CXCL4 and its variant CXCL4L1 are members of the CXC chemokine family, which bind to the CXCR3 receptor to exert their biological effects. These chemokines are associated with a variety of human diseases including chronic inflammation, immune dysfunction, cancer and metastasis. In this review, we focus on accumulating evidence demonstrating the pivotal role of CXCR3 in tumor progression. Its effects are mediated directly in tumor cells or indirectly through the regulation of angiogenesis and tumor immunity. Understanding the emerging role of CXCR3 and its signaling mechanisms further validates this receptor as a biomarker and therapeutic target for tumor progression and tumor angiogenesis.