A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

co-culture-glioblastoma-stem-like-cells-on-patterned-neurons-to-study

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

University Bordeaux, LAMC

INSERM U1029, University Bordeaux

University Bordeaux, INSERM, LAMC, U1029, 33600, Pessac, France

Gorham's syndrome is a rare illness of unknown etiology. It is characterized by a local proliferation of blood or lymphatic vessels that in bones leads to progressive resorption and destruction. The cause of the disease is not elucidated, and therapeutic options remain limited.

The therapeutic response in Gorham's syndrome to the beta-blocking agent propranolol is correlated to VEGF-A, but not to VEGF-C or FLT1 expression.

The CXCL4 paralog CXCL4L1 is a less studied chemokine that has been suggested to exert an antiangiogenic function. However, CXCL4L1 is also expressed in patient tumors, tumor cell lines, and murine xenografts, prompting a more detailed analysis of its role in cancer pathogenesis. We used genetic and antibody-based approaches to attenuate CXCL4L1 in models of pancreatic ductal adenocarcinoma (PDAC). Mechanisms of expression were assessed in cell coculture experiments, murine, and avian xenotransplants, including through an evaluation of CpG methylation and mutation of critical CpG residues. CXCL4L1 gene expression was increased greatly in primary and metastatic PDAC. We found that myofibroblasts triggered cues in the tumor microenvironment, which led to induction of CXCL4L1 in tumor cells. CXCL4L1 expression was also controlled by epigenetic modifications at critical CpG islands, which were mapped. CXCL4L1 inhibited angiogenesis but also affected tumor development more directly, depending on the tumor cell type. In vivo administration of an mAb against CXCL4L1 demonstrated a blockade in the growth of tumors positive for CXCR3, a critical receptor for CXCL4 ligands. Our findings define a protumorigenic role in PDAC development for endogenous CXCL4L1, which is independent of its antiangiogenic function. Cancer Res; 76(22); 6507-19. ©2016 AACR.

Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer.

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.

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

The chemokine receptor CXCR3 plays important roles in angiogenesis, inflammation and cancer. Activation studies and biological functions of CXCR3 are complex due to the presence of spliced isoforms. CXCR3-A is known as a pro-tumor receptor whereas CXCR3-B exhibits anti-tumor properties. Here, we focused on the conformational change of CXCR3-A and CXCR3-B after agonist or antagonist binding using Plasmon Waveguide Resonance (PWR). Agonist stimulation induced an anisotropic response with very distinct conformational changes for the two isoforms. The CXCR3 agonist bound CXCR3-A with higher affinity than CXCR3-B. Using various concentrations of SCH546738, a CXCR3 specific inhibitor, we demonstrated that low SCH546738 concentrations (≤1 nM) efficiently inhibited CXCR3-A but not CXCR3-B's conformational change and activation. This was confirmed by both, biophysical and biological methods. Taken together, our study demonstrates differences in the behavior of CXCR3-A and CXCR3-B upon ligand activation and antagonist inhibition which may be of relevance for further studies aimed at specifically inhibiting the CXCR3A isoform.

Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR).

CXCR3 plays important roles in angiogenesis, inflammation, and cancer. However, the precise mechanism of regulation and activity in tumors is not well known. We focused on CXCR3-A conformation and on the mechanisms controlling its activity and trafficking and investigated the role of CXCR3/LRP1 cross talk in tumor cell invasion. Here we report that agonist stimulation induces an anisotropic response with conformational changes of CXCR3-A along its longitudinal axis. CXCR3-A is internalized via clathrin-coated vesicles and recycled by retrograde trafficking. We demonstrate that CXCR3-A interacts with LRP1. Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to a sustained receptor activity and an increase in tumor cell migration. This was validated in patient-derived glioma cells and patient samples. Our study defines LRP1 as a regulator of CXCR3, which may have important consequences for tumor biology.

The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors.

We present a protocol for the specific labeling and isolation of proteins from the membrane surface of endothelial cells and the surrounding extracellular matrix of organs, experimental wounds and tumors using chicken embryos. Proteins are deglycosylated on streptavidin resin and purified after gentle elution and trypsin digestion. Peptides are analyzed by spectroscopy and reverse proteomic fingerprinting. The major advantages of this protocol include reductions in both the background and overrepresentation of single proteins that would otherwise mask less well-represented proteins in the mass spectroscopy analysis. We also present methods to identify putative vascular and endothelial cell targets from isolated chicken membranes and extracellular proteins. The use of human genome and transcriptome data facilitates this analysis. Human orthologs of isolated chicken proteins are identified using best hit BLAST searches against the Human Reference Sequence Database. The expression of Human orthologs is then assessed for endothelial and non-endothelial cell enrichment using second generation RNA-seq sequenced libraries. Scanning of the published literature then provides a ranking score of those genes most likely involved in cancer or having a link to angiogenesis.

Methods for Mapping the Extracellular and Membrane Proteome in the Avian Embryo, and Identification of Putative Vascular Targets or Endothelial Genes.

The notion of tumor microenvironment (TME) has been brought to the forefront of recent scientific literature on cancer. However, there is no consensus on how to define and spatially delineate the TME. We propose that the time is ripe to go beyond an all-encompassing list of the components of the TME, and to construct a multilayered view of cancer. We distinguish six layers of environmental interactions with the tumor and show that they are associated with distinct mechanisms, and ultimately with distinct therapeutic approaches.

The Multiple Layers of the Tumor Environment.

We undertook a systematic study focused on the matricellular protein Thrombospondin-1 (THBS1) to uncover molecular mechanisms underlying the role of THBS1 in glioblastoma (GBM) development. THBS1 was found to be increased with glioma grades. Mechanistically, we show that the TGFβ canonical pathway transcriptionally regulates THBS1, through SMAD3 binding to the THBS1 gene promoter. THBS1 silencing inhibits tumour cell invasion and growth, alone and in combination with anti-angiogenic therapy. Specific inhibition of the THBS1/CD47 interaction using an antagonist peptide decreases cell invasion. This is confirmed by CD47 knock-down experiments. RNA sequencing of patient-derived xenograft tissue from laser capture micro-dissected peripheral and central tumour areas demonstrates that THBS1 is one of the gene with the highest connectivity at the tumour borders. All in all, these data show that TGFβ1 induces THBS1 expression via Smad3 which contributes to the invasive behaviour during GBM expansion. Furthermore, tumour cell-bound CD47 is implicated in this process.

Deciphering the complex role of thrombospondin-1 in glioblastoma development.

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.

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

Protein tyrosine phosphatases are essential modulators of angiogenesis and have been identified as novel therapeutic targets in cancer and anti-angiogenesis. The roles of atypical Phosphatase of Regenerative Liver (PRL) phosphatases in this context remain poorly understood. Here, we investigate the biological function of PRL phosphatases in developmental angiogenesis in the postnatal mouse retina and in cell culture. We show that endothelial cells in the retina express PRL-2 encoded by the Ptp4a2 gene, and that inducible endothelial and global Ptp4a2 mutant mice exhibit defective retinal vascular outgrowth, arteriovenous differentiation, and sprouting angiogenesis. Mechanistically, PTP4A2 deletion limits angiogenesis by inhibiting endothelial cell migration and the VEGF-A, DLL-4/NOTCH-1 signaling pathway. This study reveals the importance of PRL-2 as a modulator of vascular development.

ArticleTotal : 2	Year
Publication title Cited by 49	2020
Publication title Cited by 2	2021

Article	Year
The therapeutic response in Gorham's syndrome to the beta-blocking agent propranolol is correlated to VEGF-A, but not to VEGF-C or FLT1 expression. BMC research notes\| PubMed ID: 26238450	2015
Dual Roles for CXCL4 Chemokines and CXCR3 in Angiogenesis and Invasion of Pancreatic Cancer. Cancer research\| PubMed ID: 27634764	2016
History and conceptual developments in vascular biology and angiogenesis research: a personal view. Angiogenesis\| PubMed ID: 28741165	2017
Ligand activation induces different conformational changes in CXCR3 receptor isoforms as evidenced by plasmon waveguide resonance (PWR). Scientific reports\| PubMed ID: 28878333	2017
The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors. Nature communications\| PubMed ID: 29146996	2017
Methods for Mapping the Extracellular and Membrane Proteome in the Avian Embryo, and Identification of Putative Vascular Targets or Endothelial Genes. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 29264797	2018
The Multiple Layers of the Tumor Environment. Trends in cancer\| PubMed ID: 30470302	2018
Deciphering the complex role of thrombospondin-1 in glioblastoma development. Nature communications\| PubMed ID: 30850588	2019
The tumor organismal environment: Role in tumor development and cancer immunotherapy. Seminars in cancer biology\| PubMed ID: 31891779	2020
PRL-2 phosphatase is required for vascular morphogenesis and angiogenic signaling. Communications biology\| PubMed ID: 33097786	2020

Andreas Bikfalvi

Biography

JoVE Journal Publications

Other Publications