GLIOBLASTOM - Anni Hofmann Stiftung

New pericytes in brain tumor blood-vessels originate from a previously unrecognized pericyte progenitor

Project-synopsis: We found that pericytes strongly contribute to neoangiogenesis in glioblastomas (GBM). In physiology pericytes are essential for maintaining the stability of CNS blood-vessels and the blood-brain-barrier. They are closely associated with endothelia and can be identified by a set of markers. New pericytes are required during neo-angiogenesis but how pericytes are generated in CNS neoplasms is unknown. We observed that new, mature pericytes in GBM largely originate from a newly identified precursor cell-type. These precursors are characterized as pericyte marker-negative cells of non-hematopoietic origin, which are distant from blood-vessels and highly proliferative. Lineage ablation of pericyte precursors in brain tumor models showed that they are necessary to generate new, mature pericytes supporting tumor expansion. In our current project funded by the Anni-Hofmann-Stiftung we investigate how pericyte precursors control vascular plasticity and how we can therapeutically target pericyte precursors in GBM.

Project-description: Malignant gliomas, like glioblastoma (GBM), represent the majority of primary CNS neoplasms. GBM are treatment refractory, invasive and highly angiogenic tumours1. To define new prognostic and treatment criteria GBM was systematically studied by “The Cancer Genome Atlas” (TCGA) project and others2. The TCGA dataset indicates aberrations in several key signal transduction pathways driving gliomagenesis and shows that GBM comprises separate tumour-subtypes, which differ in prognosis and treatment characteristics3,4. The most commonly defined genetic subclasses for IDHwt GBM are termed proneural, classical or mesenchymal. The mesenchymal subtype is associated with high therapy-resistance while the proneural subtype appears to be more sensitive to anti-angiogenic therapies5. These trancriptomal subtypes give an explanation for the large inter-individual heterogeneity of GBM patients and provide (relatively broad) criteria for patient-stratification in clinical trials2. The small number of successful GBM trials6,7 (where stratification schemes were applied) and the large number of failed clinical studies (where predictive markers were not available as reviewed by Jue et al. 20158) firmly suggest that stratification of patients into prospective responders and non-responders is a prerequisite for treatment planning in neuro-oncology. Furthermore, since anti-angiogenic treatments are applied subsequent to neurosurgery it will be important to address the impact of anti-angiogenic regimen in preventing tumour-relapse. Most importantly, new targets for anti-angiogenesis are urgently needed which can support clinically established schedules to suppress GBM vascularisation.  
We identified the process of pericyte-formation and expansion as an excellent therapeutic target for anti-angiogenesis in GBM. Pericytes are a vital part of the neurovascular unit and regulate the architecture, plasticity, permeability and tone of brain microvessels 9. It was speculated that mature pericytes in the adult brain may generate new pericytes during vascular remodeling, but this has not been shown 10. Alternatively, it was proposed that progenitor cells from the hematopoietic system contribute new pericytes to the ischemic 11 or neoplastic 12-15 brain. Uncovering the origin of newly established pericytes is of considerable neuroscientific and clinical interest as this can provide ways to modulate pericyte numbers in disease and to improve the outcome of different neuropathologies 16,17 including malignant brain tumors (gliomas; GBM)18,19. Mature pericytes are defined by their anatomical localization in close apposition to endothelial cells and also by expression of a range of pericyte-markers including platelet-derived growth factor-B (PDGFR-B), desmin, neural/glia antigen-2 (NG2) 9,10 or CD146 20. Using a new transgenic model we obtained data suggesting that pericyte precursors generate new pericytes in GBM. Pericyte precursors acquired markers and morphology of mature pericytes only in advanced tumors while the vast majority of these precursor cells in early tumor stages was neither associated with the vasculature nor did they express a set of pericyte markers. Our data from genetic and phenotypic studies on these traced cells suggest that a previously unidentified, pericyte precursor cell population of non-hematopoietic origin is important for angiogenesis in CNS neoplasms and can constitute a new target for anti-angiogenic regimen in GBM. In our project funded by the Anni-Hofmann-Stiftung we will explore: 1. If neoangiogenesis is driven by pericyte precursors in all GBM-subtypes – or if this is a GBM-subset-specific phenomenon (which would require identification of stratification-markers for therapeutic targeting); 2. If pericyte-precursor supported neoangiogenesis is relevant during GBM-relapse; 3. If pericyte precursors are abundant in different human GBM.

1    Arrillaga-Romany, I. & Norden, A. D. Antiangiogenic therapies for glioblastoma. CNS oncology 3, 349-358, doi:10.2217/cns.14.31 (2014).
2    Aldape, K., Zadeh, G., Mansouri, S., Reifenberger, G. & von Deimling, A. Glioblastoma: pathology, molecular mechanisms and markers. Acta neuropathologica 129, 829-848, doi:10.1007/s00401-015-1432-1 (2015).
3    Verhaak, R. G. et al. Integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17, 98-110, doi:10.1016/j.ccr.2009.12.020 (2010).
4    Brennan, C. W. et al. The somatic genomic landscape of glioblastoma. Cell 155, 462-477, doi:10.1016/j.cell.2013.09.034 (2013).
5    Sandmann, T. et al. Patients With Proneural Glioblastoma May Derive Overall Survival Benefit From the Addition of Bevacizumab to First-Line Radiotherapy and Temozolomide: Retrospective Analysis of the AVAglio Trial. Journal of clinical oncology : official journal of the American Society of Clinical Oncology, doi:10.1200/JCO.2015.61.5005 (2015).
6    Hegi, M. E. et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. The New England journal of medicine 352, 997-1003, doi:10.1056/NEJMoa043331 (2005).
7    Stupp, R. et al. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. The New England journal of medicine 352, 987-996, doi:10.1056/NEJMoa043330 (2005).
8    Jue, T. R., Hovey, E., Davis, S., Carleton, O. & McDonald, K. L. Incorporation of biomarkers in phase II studies of recurrent glioblastoma. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 36, 153-162, doi:10.1007/s13277-014-2960-3 (2015).
9    Armulik, A., Genove, G. & Betsholtz, C. Pericytes: developmental, physiological, and pathological perspectives, problems, and promises. Dev Cell 21, 193-215, doi:10.1016/j.devcel.2011.07.001 (2011).
10    Winkler, E. A., Bell, R. D. & Zlokovic, B. V. Central nervous system pericytes in health and disease. Nature neuroscience 14, 1398-1405, doi:10.1038/nn.2946 (2011).
11    Kokovay, E., Li, L. & Cunningham, L. A. Angiogenic recruitment of pericytes from bone marrow after stroke. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism 26, 545-555, doi:10.1038/sj.jcbfm.9600214 (2006).
12    Song, S., Ewald, A. J., Stallcup, W., Werb, Z. & Bergers, G. PDGFRbeta+ perivascular progenitor cells in tumours regulate pericyte differentiation and vascular survival. Nature cell biology 7, 870-879, doi:10.1038/ncb1288 (2005).
13    Lamagna, C. & Bergers, G. The bone marrow constitutes a reservoir of pericyte progenitors. J Leukoc Biol 80, 677-681, doi:10.1189/jlb.0506309 (2006).
14    Du, R. et al. HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell 13, 206-220, doi:10.1016/j.ccr.2008.01.034 (2008).
15    De Palma, M. et al. Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors. Cancer Cell 8, 211-226, doi:10.1016/j.ccr.2005.08.002 (2005).
16    Zhao, Z., Nelson, A. R., Betsholtz, C. & Zlokovic, B. V. Establishment and Dysfunction of the Blood-Brain Barrier. Cell 163, 1064-1078, doi:10.1016/j.cell.2015.10.067 (2015).
17    Tarasoff-Conway, J. M. et al. Clearance systems in the brain--implications for Alzheimer diseaser. Nat Rev Neurol 12, 248, doi:10.1038/nrneurol.2016.36 (2016).
18    Sun, H. et al. Hyperplasia of pericytes is one of the main characteristics of microvascular architecture in malignant glioma. PloS one 9, e114246, doi:10.1371/journal.pone.0114246 (2014).
19    Svensson, A., Ozen, I., Genove, G., Paul, G. & Bengzon, J. Endogenous brain pericytes are widely activated and contribute to mouse glioma microvasculature. PloS one 10, e0123553, doi:10.1371/journal.pone.0123553 (2015).
20    Crisan, M. et al. A perivascular origin for mesenchymal stem cells in multiple human organs. Cell stem cell 3, 301-313, doi:10.1016/j.stem.2008.07.003 (2008).