Targeting the tumour vasculature: from vessel destruction to promotion.

As angiogenesis was recognized as a core hallmark of cancer growth and survival, several strategies have been implemented to target the tumour vasculature. Yet to date, attempts have rarely been so diverse, ranging from vessel growth inhibition and destruction to vessel normalization, reprogramming and vessel growth promotion. Some of these strategies, combined with standard of care, have translated into improved cancer therapies, but their successes are constrained to certain cancer types. This Review provides an overview of these vascular targeting approaches and puts them into context based on our subsequent improved understanding of the tumour vasculature as an integral part of the tumour microenvironment with which it is functionally interlinked. This new knowledge has already led to dual targeting of the vascular and immune cell compartments and sets the scene for future investigations of possible alternative approaches that consider the vascular link with other tumour microenvironment components for improved cancer therapy.

Physiology of PNS axons relies on glycolytic metabolism in myelinating Schwann cells.

While lactate shuttle theory states that glial cells metabolize glucose into lactate to shuttle it to neurons, how glial cells support axonal metabolism and function remains unclear. Lactate production is a common occurrence following anaerobic glycolysis in muscles. However, several other cell types, including some stem cells, activated macrophages and tumor cells, can produce lactate in presence of oxygen and cellular respiration, using Pyruvate Kinase 2 (PKM2) to divert pyruvate to lactate dehydrogenase. We show here that PKM2 is also upregulated in myelinating Schwann cells (mSC) of mature mouse sciatic nerve versus postnatal immature nerve. Deletion of this isoform in PLP-expressing cells in mice leads to a deficit of lactate in mSC and in peripheral nerves. While the structure of myelin sheath was preserved, mutant mice developed a peripheral neuropathy. Peripheral nerve axons of mutant mice failed to maintain lactate homeostasis upon activity, resulting in an impaired production of mitochondrial ATP. Action potential propagation was not altered but axonal mitochondria transport was slowed down, muscle axon terminals retracted and motor neurons displayed cellular stress. Additional reduction of lactate availability through dichloroacetate treatment, which diverts pyruvate to mitochondrial oxidative phosphorylation, further aggravated motor dysfunction in mutant mice. Thus, lactate production through PKM2 enzyme and aerobic glycolysis is essential in mSC for the long-term maintenance of peripheral nerve axon physiology and function.

Mapping the N-Terminal Hexokinase-I Binding Site onto Voltage-Dependent Anion Channel-1 To Block Peripheral Nerve Demyelination.

The voltage-dependent anion channel (VDAC), the most abundant protein on the outer mitochondrial membrane, is implicated in ATP, ion and metabolite exchange with cell compartments. In particular, the VDAC participates in cytoplasmic and mitochondrial Ca2+ homeostasis. Notably, the Ca2+ efflux out of Schwann cell mitochondria is involved in peripheral nerve demyelination that underlies most peripheral neuropathies. Hexokinase (HK) isoforms I and II, the main ligands of the VDAC, possess a hydrophobic N-terminal structured in α-helix (NHKI) that is necessary for the binding to the VDAC. To gain further insight into the molecular basis of HK binding to the VDAC, we developed and optimized peptides based on the NHKI sequence. These modifications lead to an increase of the peptide hydrophobicity and helical content that enhanced their ability to prevent peripheral nerve demyelination. Our results provide new insights into the molecular basis of VDAC/HK interaction that could lead to the development of therapeutic compounds for demyelinating peripheral neuropathies.

SLUG and Truncated TAL1 Reduce Glioblastoma Stem Cell Growth Downstream of Notch1 and Define Distinct Vascular Subpopulations in Glioblastoma Multiforme.

Glioblastomas (GBM) are high-grade brain tumors, containing cells with distinct phenotypes and tumorigenic potentials, notably aggressive and treatment-resistant multipotent glioblastoma stem cells (GSC). The molecular mechanisms controlling GSC plasticity and growth have only partly been elucidated. Contact with endothelial cells and the Notch1 pathway control GSC proliferation and fate. We used three GSC cultures and glioma resections to examine the expression, regulation, and role of two transcription factors, SLUG (SNAI2) and TAL1 (SCL), involved in epithelial to mesenchymal transition (EMT), hematopoiesis, vascular identity, and treatment resistance in various cancers. In vitro, SLUG and a truncated isoform of TAL1 (TAL1-PP22) were strongly upregulated upon Notch1 activation in GSC, together with LMO2, a known cofactor of TAL1, which formed a complex with truncated TAL1. SLUG was also upregulated by TGF-ß1 treatment and by co-culture with endothelial cells. In patient samples, the full-length isoform TAL1-PP42 was expressed in all glioma grades. In contrast, SLUG and truncated TAL1 were preferentially overexpressed in GBMs. SLUG and TAL1 are expressed in the tumor microenvironment by perivascular and endothelial cells, respectively, and to a minor extent, by a fraction of epidermal growth factor receptor (EGFR) -amplified GBM cells. Mechanistically, both SLUG and truncated TAL1 reduced GSC growth after their respective overexpression. Collectively, this study provides new evidence for the role of SLUG and TAL1 in regulating GSC plasticity and growth.

High Endothelial Venules: A Vascular Perspective on Tertiary Lymphoid Structures in Cancer.

High endothelial venules (HEVs) are specialized postcapillary venules composed of cuboidal blood endothelial cells that express high levels of sulfated sialomucins to bind L-Selectin/CD62L on lymphocytes, thereby facilitating their transmigration from the blood into the lymph nodes (LN) and other secondary lymphoid organs (SLO). HEVs have also been identified in human and murine tumors in predominantly CD3+T cell-enriched areas with fewer CD20+B-cell aggregates that are reminiscent of tertiary lymphoid-like structures (TLS). While HEV/TLS areas in human tumors are predominantly associated with increased survival, tumoral HEVs (TU-HEV) in mice have shown to foster lymphocyte-enriched immune centers and boost an immune response combined with different immunotherapies. Here, we discuss the current insight into TU-HEV formation, function, and regulation in tumors and elaborate on the functional implication, opportunities, and challenges of TU-HEV formation for cancer immunotherapy.

A novel 3D nanofibre scaffold conserves the plasticity of glioblastoma stem cell invasion by regulating galectin-3 and integrin-ß1 expression.

Glioblastoma Multiforme (GBM) invasiveness renders complete surgical resection impossible and highly invasive Glioblastoma Initiating Cells (GICs) are responsible for tumour recurrence. Their dissemination occurs along pre-existing fibrillary brain structures comprising the aligned myelinated fibres of the corpus callosum (CC) and the laminin (LN)-rich basal lamina of blood vessels. The extracellular matrix (ECM) of these environments regulates GIC migration, but the underlying mechanisms remain largely unknown. In order to recapitulate the composition and the topographic properties of the cerebral ECM in the migration of GICs, we have set up a new aligned polyacrylonitrile (PAN)-derived nanofiber (NF) scaffold. This system is suitable for drug screening as well as discrimination of the migration potential of different glioblastoma stem cells. Functionalisation with LN increases the spatial anisotropy of migration and modulates its mode from collective to single cell migration. Mechanistically, equally similar to what has been observed for mesenchymal migration of GBM in vivo, is the upregulation of galectin-3 and integrin-ß1 in Gli4 cells migrating on our NF scaffold. Downregulation of Calpain-2 in GICs migrating in vivo along the CC and in vitro on LN-coated NF underlines a difference in the turnover of focal adhesion (FA) molecules between single-cell and collective types of migration.

Tight Junction Protein Claudin-2 Promotes Self-Renewal of Human Colorectal Cancer Stem-like Cells.

Posttreatment recurrence of colorectal cancer, the third most lethal cancer worldwide, is often driven by a subpopulation of cancer stem cells (CSC). The tight junction (TJ) protein claudin-2 is overexpressed in human colorectal cancer, where it enhances cell proliferation, colony formation, and chemoresistance in vitro While several of these biological processes are features of the CSC phenotype, a role for claudin-2 in the regulation of these has not been identified. Here, we report that elevated claudin-2 expression in stage II/III colorectal tumors is associated with poor recurrence-free survival following 5-fluorouracil-based chemotherapy, an outcome in which CSCs play an instrumental role. In patient-derived organoids, primary cells, and cell lines, claudin-2 promoted colorectal cancer self-renewal in vitro and in multiple mouse xenograft models. Claudin-2 enhanced self-renewal of ALDHHigh CSCs and increased their proportion in colorectal cancer cell populations, limiting their differentiation and promoting the phenotypic transition of non-CSCs toward the ALDHHigh phenotype. Next-generation sequencing in ALDHHigh cells revealed that claudin-2 regulated expression of nine miRNAs known to control stem cell signaling. Among these, miR-222-3p was instrumental for the regulation of self-renewal by claudin-2, and enhancement of this self-renewal required activation of YAP, most likely upstream from miR-222-3p. Taken together, our results indicate that overexpression of claudin-2 promotes self-renewal within colorectal cancer stem-like cells, suggesting a potential role for this protein as a therapeutic target in colorectal cancer.Significance: Claudin-2-mediated regulation of YAP activity and miR-222-3p expression drives CSC renewal in colorectal cancer, making it a potential target for therapy. Cancer Res; 78(11); 2925-38. ©2018 AACR.

Vascular Transdifferentiation in the CNS: A Focus on Neural and Glioblastoma Stem-Like Cells.

Glioblastomas are devastating and extensively vascularized brain tumors from which glioblastoma stem-like cells (GSCs) have been isolated by many groups. These cells have a high tumorigenic potential and the capacity to generate heterogeneous phenotypes. There is growing evidence to support the possibility that these cells are derived from the accumulation of mutations in adult neural stem cells (NSCs) as well as in oligodendrocyte progenitors. It was recently reported that GSCs could transdifferentiate into endothelial-like and pericyte-like cells both in vitro and in vivo, notably under the influence of Notch and TGFß signaling pathways. Vascular cells derived from GBM cells were also observed directly in patient samples. These results could lead to new directions for designing original therapeutic approaches against GBM neovascularization but this specific reprogramming requires further molecular investigations. Transdifferentiation of nontumoral neural stem cells into vascular cells has also been described and conversely vascular cells may generate neural stem cells. In this review, we present and discuss these recent data. As some of them appear controversial, further validation will be needed using new technical approaches such as high throughput profiling and functional analyses to avoid experimental pitfalls and misinterpretations.

Asymmetric Distribution of GFAP in Glioma Multipotent Cells.

Asymmetric division (AD) is a fundamental mechanism whereby unequal inheritance of various cellular compounds during mitosis generates unequal fate in the two daughter cells. Unequal repartitions of transcription factors, receptors as well as mRNA have been abundantly described in AD. In contrast, the involvement of intermediate filaments in this process is still largely unknown. AD occurs in stem cells during development but was also recently observed in cancer stem cells. Here, we demonstrate the asymmetric distribution of the main astrocytic intermediate filament, namely the glial fibrillary acid protein (GFAP), in mitotic glioma multipotent cells isolated from glioblastoma (GBM), the most frequent type of brain tumor. Unequal mitotic repartition of GFAP was also observed in mice non-tumoral neural stem cells indicating that this process occurs across species and is not restricted to cancerous cells. Immunofluorescence and videomicroscopy were used to capture these rare and transient events. Considering the role of intermediate filaments in cytoplasm organization and cell signaling, we propose that asymmetric distribution of GFAP could possibly participate in the regulation of normal and cancerous neural stem cell fate.

Notch1 stimulation induces a vascularization switch with pericyte-like cell differentiation of glioblastoma stem cells.

Glioblastoma multiforms (GBMs) are highly vascularized brain tumors containing a subpopulation of multipotent cancer stem cells. These cells closely interact with endothelial cells in neurovascular niches. In this study, we have uncovered a close link between the Notch1 pathway and the tumoral vascularization process of GBM stem cells. We observed that although the Notch1 receptor was activated, the typical target proteins (HES5, HEY1, and HEY2) were not or barely expressed in two explored GBM stem cell cultures. Notch1 signaling activation by expression of the intracellular form (NICD) in these cells was found to reduce their growth rate and migration, which was accompanied by the sharp reduction in neural stem cell transcription factor expression (ASCL1, OLIG2, and SOX2), while HEY1/2, KLF9, and SNAI2 transcription factors were upregulated. Expression of OLIG2 and growth were restored after termination of Notch1 stimulation. Remarkably, NICD expression induced the expression of pericyte cell markers (NG2, PDGFRß, and α-smooth muscle actin [αSMA]) in GBM stem cells. This was paralleled with the induction of several angiogenesis-related factors most notably cytokines (heparin binding epidermal growth factor [HB-EGF], IL8, and PLGF), matrix metalloproteinases (MMP9), and adhesion proteins (vascular cell adhesion molecule 1 [VCAM1], intercellular adhesion molecule 1 [ICAM1], and integrin alpha 9 [ITGA9]). In xenotransplantation experiments, contrasting with the infiltrative and poorly vascularized tumors obtained with control GBM stem cells, Notch1 stimulation resulted in poorly disseminating but highly vascularized grafts containing large vessels with lumen. Notch1-stimulated GBM cells expressed pericyte cell markers and closely associated with endothelial cells. These results reveal an important role for the Notch1 pathway in regulating GBM stem cell plasticity and angiogenic properties.